WATKR POLLUTION CONTROL RESEARCH SERIES • 17070 DRP 12/70
   DEVELOPMENT OF  TECHNIQUES
 FOR  ESTIMATING THE BACTERIAL
 POPULATION OF SEWAGE SLUDGE
U.S. ENVIRONMENTAL PROTECTION AGENCY

-------
          WATER POLLUTION CONTROL RESEARCH SERIES
The Water Pollution Control Research Series describes the
results and progress in the control and abatement of pollution
in our Nation's waters.  They provide a central source of
information on the research, development, and demonstration
activities in the water research program of the Environmental
Protection Agency, through inhouse research and grants and
contracts with Federal, State, and local agenices, research
institutions, and industrial organizations.

Inquiries pertaining to Water Pollution Control Research
Reports should be directed to the Chief, Publications Branch
(Water), Research Information Division, R&M, Environmental '
Protection Agency, Washington, D.C. 20460.

-------
          DEVELOPMENT  OF TECHNIQUES  FOR ESTIMATING
          THE BACTERIAL POPULATION OF SEWAGE SLUDGE
                              by
                       William Spangler
                       Walter Langston
                 Midwest Research  Institute
                Kansas City, Missouri  64110
                           for the

             OFFICE OF  RESEARCH AND MONITORING

              ENVIRONMENTAL PROTECTION AGENCY
                     Project #17070 DRP
                    Contract #14-12-569
                        December 1970
For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402 - Price $1.00

-------
                 EPA Review Notice
This report has been reviewed by the Environmental
Protection Agency and approved for publication.
Approval does not signify that the contents necessarily
reflect the views and policies of the Environmental
Protection Agency, nor does mention of trade names or
commercial products constitute endorsement or recommenda-
tion for use.
                          11

-------
                                  ABSTRACT
           This research program was initiated to develop practical methods
 for evaluation of the biomass in anaerobic sewage sludge and to determine
 if  predictions could be made concerning digester performance.   Sampling and
 handling methods were improved and standardized to give maximum anaerobic
 counts.   A simplified technique for growing obligate anaerobes that can be
 safely performed by technicians with minimum training in bacteriology was
 developed.  Anaerobic media were improved to yield as high or  higher counts
 of  methanogenic bacteria than heretofore reported.  A simple freeze-dry
 technique was developed for preparation of consistent batches  of sludge
 supernatant used in media as a supplement for growth of obligate sludge
 anaerobes.  The possible relationship between concentration of a growth
 factor required by Methanobacterium ruminantium (used to evaluate potency
 of  growth factor extracted) and digester efficiency could have important
 practical implications.  Limited data obtained indicated that  growth factor
 concentrations were much higher in "normal" digesters than in  unbalanced
 or  "upset" digesters.  Practical applications of the methods developed can
 have considerable impact upon future research and development  in anaerobic
 sludge digestion and could lead to improvements in our ability to predict
 impending digester failure and control of digester performance.

           This report was submitted in fulfillment of Project  No. 17070DRP,
Contract 14-12-569, under the sponsorship of the Water Quality Office, En-
vironmental Protection Agency.
                                    iii

-------
                                CONTENTS
Conclusions 	   1

Recommendations 	   3

Introduction  	   5

Phase I - Development of Techniques for Sampling and Handling of
            Anaerobic Sewage Sludge 	   7

Phase II - Development of a Simplified Technique for Enumeration of
             the Obligate Anerobes in Sewage Sludge 	  25

Phase III - Development of a Culture Medium for Growth of Micro-
              organisms Representative of the Total Biomass in
              Sewage Sludge 	  35

Phase IV - Determination of Growth Substance(s) in Sewage Sludge
             that Enhances Growth of Sludge Microorganisms  	  69

Acknowledgements	79

References	81

Glossary	83

-------
                       FIGURES
                                                            Page

Anaerobic Sludge Sampler Designed for Collection of
  Samples Under Anaerobic Conditions 	   10

Overall View of Inoculating Press and Roll-Tube Jig   ...   28

Closeup of Roll-Tube Jig with Needle in Position for
  Inoculation	   29

Clamps, Manifold and Copper Column Used in the Preparation
  of Media	   31

Scheme for Purification and Concentration of Sewage Sludge
  Factor	   74
                           VI

-------
                                 TABLES
 1       Effect of Sampling Method on Total Colony Count 	  12

 2       Effect of Anaerobic Dilution Solution Composition on
           Total Colony Count	14

 3       Effect of Dioctyl Sodium Sulfosuccinate (DSS) on Total
           Colony Count	  16

 4       Effect of Blending Time on Total Colony Count	 .  18

 5       Effect of Method of Initial Mixing on Total Colony Count  .  19

 6       Comparison of Total Colony Counts/Milliliter on Samples
           on Samples Blended Undiluted with Samples Blended as
           1/10 Dilutions	20

 7       Effect of Catalase on Total Colony Count  	  22

 8       Effect of Sludge Supernatant Concentration on Total Colony
           Count . „	36

 9       Effect of Rumen Fluid Supernatant Concentration on Total
           Colony Count  	  37

10       Comparison of Normal Sludge Supernatant with Aqueous
           Extract of Lyophilized Sludge and Filter Clarified
           Sludge Supernatant  	  39

11       Effect of Treatment of CSS on Total Colony Count	41

12       Comparison of Various Modifications of HSM to Control
           Medium with a 50-50 C02/H2 Atmosphere	44

13       Comparison of Various Modifications of HSM to Control
           Medium with a 100% CC>2 Atmosphere	45

14       Evaluation of Dithiothreitol (DTT) as a Replacement for
           Sodium Sulfide		  47

15       Evaluation of Dithiothreitol (DTT) as a Replacement for
           Sodium Sulfide and Cysteine	  .  .  .  48

                                    vii

-------
                           TABLES (Continued)
16       Effect of Sodium Formaldehyde Sulfoxylate (NaFS) on
           Total Anaerobic Colony Count  	  49

17       Effect of Sodium Deletion on Total Colony Count 	  50

18       Effect of Cellobiose on Total Colony Count Obtained with
           HSM	.o	52

19       Effect of Volatile Fatty Acid Mixture (VFA) and Trypticase.  53

20       Effect of Yeast Extract and Trypticase Deletion on Total
           Colony Counts with HSM Using a 50-50 H2/C02 Atmosphere   .  54

21       Effect of Yeast Extract and Trypticase Deletion on Total
           Colony Counts with HSM Using a 100% C02 Atmosphere  ...  55

22       Comparison of Complete HSM With Basal HSM   	56

23       Effect of Gaseous Atmosphere on Total Colony Count with
           Basal HSM	58

24       Effect of Gaseous Atmosphere on Total Colony Count with
           Basal HSM + 0.1% Acetate	59

25       Effect of Gaseous Atmosphere on Total Colony Count with
           Basal HSM + 0.1% Formate	  60

26       Effect of Gaseous Atmosphere on Total Colony Count with
           Basal HSM + 0.1% Acetate + 0.1% Formate	61

27       Comparison of Total Colony Counts of Sludge Samples from
           Four Different Digesters with an N2/C02 Atmosphere  ...  64

28       Comparison of Total Colony Counts of Sludge Samples from
           Four Different Digesters with an H2/C02 Atmosphere  ...  65

29       Results of Aerobic Plate Counts for Anaerobic Sludge from
           Four Different Digesters  	  66

30       Total Estimated Methanogens Based Upon Methane Determin-
           ations on Roll Tubes with an N2/C02 Atmosphere	66

                                    viii

-------
                           TABLES (Concluded)
                                                                      Page
31       Total Estimated Methanogens Based Upon Methane Determin-
           ations on Roll Tubes with an H2/C02 Atmosphere  . . . . «   67

32       Effect of Sludge Treatment on Factor Recovery 	   76

33       Factor Recovery at Various Steps in Extraction Procedure  .   76

34       Comparison of Factor Levels in Sludge Supernatants from
           Four Sewage Plants	   77
                                    IX

-------
                               CONCLUSIONS
          1.  Sampling of digester contents was done with a sampler which
preserved the anaerobic condition of the sample.  Handling methods ensuring
continuity of anaerobic conditions were standardized to give maximum total
counts by the Hungate procedure.

          2.  A nutrient medium containing 20% of an aqueous extract of
lyophilized sludge supernatant and sodium sulfide plus cysteine-HCl to reduce
residual traces of oxygen was used in combination with appropriate gaseous
atmospheres (oxygen-free C02, H2, N£ mixture depending on type of organism
to be grown) to maintain anaerobic conditions and low oxidation-reduction
potential.  Nutrients were added to or deleted from the medium to yield maxi-
mum total anaerobic counts.

          3.  A growth factor present in sludge supernatant was assayed and
found to occur at approximately the same concentration in "normal" sludge
supernatant as in rumen fluid.

          4.  The method of preparation of sludge supernatant was improved
and simplified by using batch-lyophilized sludge.  Aqueous extract of lyoph-
ized sludge (AELS) is prepared from this material by placing a weighed quan-
tity on a filter mat of hyflosupercel and washing the mat with sufficient
hot water (with suction) to restore the original supernatant volume.  Large
amounts of sludge can be lyophilized and stored indefinitely to yield a
more consistent sludge supernatant as required.

          5.  The roll-tube technique of Hungate has been simplified and
made much safer to use.  A permanent butyl rubber stopper is used with a
retaining clamp where necessary.  All additions to the culture tube are
made through syringe needles from a mechanically mounted syringe.  Direct
colony counts are made in the cylindrical inside agar surface of the tube.

          6.  Media preparation has been simplified by dispensing gas-
flushed, prereduced medium in multiples of five tubes which are simultan-
eously flushed from a manifold.  After sealing with a butyl rubber .stopper,
the medium is sterilized in clamps.  All subsequent operations are performed
without removing the stoppers from the tubes.  Shortly before use, the
medium is reduced further by syringe addition of sodium sulfide—cysteine-HCl
reducing solution through the rubber stoppers.

          7.  A device consisting of a syringe mounted on a modified drill
press and an aluminum roll-tube jig in a water bath was used to make all
additions, dilutions, and inoculation of roll-tubes.  The technique provides
a simplified and safer means of manipulating roll-tubes remotely without

-------
the necessity of removing rubber stoppers from tubes.  The technique re-
quires much less manual dexterity than the conventional roll-tube technique
and can be performed by technicians with little or no prior experience in
microbiology.

          8.  The possible relationship between concentration of growth
factor required for growth of Methanobacterium ruminantium (used in this
study to evaluate potency of growth factor extracted) and digester effi-
ciency could have important practical implications.  Limited data obtained
indicated that growth factor concentrations were much higher in "normal"
digesters than in unbalanced or "upset" digesters.  These trends were ob-
served later in the final phase of the study and more work is needed to
validate and establish the significance of these preliminary trends.

-------
                             RECOMMENDATIONS
          On the basis of progress and observations made during the course
of this work, several recommendations for further studies can be made:

          1.  Further experiments should be carried out to microbiologically
monitor several digesters over a period of time to determine what changes
in types and numbers of microorganisms occur as a digester approaches failure.

          2.  Media should be modified such that cultural conditions more
closely simulate the prevailing condition of a digester being monitored.
The modified media should be directly compared to media which closely sim-
ulate a "normal" digester.  Various modifications should be evaluated for
samples taken from poorly functioning digesters—e.g., inclusion of sludge
supernatant from each such digester, addition of purified sludge factor to
sludge supernatant from the digester in question, and comparison of the
supernatant from each digester to normal control supernatant with respect
to methanogenesis and types and number of organisms grown.

          3.  Further studies are needed to identify the unknown sludge
factor and to subsequently develop a microbiological or chemical assay which
can be quantitatively compared to standard curves, prepared with the known
compound, and used to monitor factor levels more accurately.

          4.  Studies using laboratory digesters are also needed.  The
digesters could be monitored for total anaerobic counts, aerobic plate
counts (for facultatives), methane production, and factor level when the
digesters are functioning normally and when induced toward failure.  The
effect of supplemental addition of purified factor should also be studied,
as well as any role for the factor in normal digestion and the possibility
of reversing an upset condition with purified factor.

          The studies presented herein have laid the groundwork for those
studies which now can be undertaken.  Through continuation of these studies,
a great deal of information could be gained on changes in biomass, factor
levels, importance of factor and corrective steps which might be taken to
prevent failure of a digester.

-------
                                INTRODUCTION
           The anaerobic treatment of sewage waste is  widely used by muni-
 cipal plants in the United States and elsewhere.   Despite the extensive
 employment of the system,  little is known about the fundamental nature
 of the process.  Because of this lack of understanding,  few control meas-
 ures are available for correcting or explaining process  upsets.

           The anaerobic digestion of organic wastes generally proceeds in
 two stages.   In the first  stage, complex compounds are broken down to
 simple organic materials.   Fats, proteins,  and carbohydrates are converted,
 for the most part, to organic fatty acids.   A group of bacteria termed
 "acid-formers" carry out the primary conversions.   These saprophytic
 bacteria are abundant in sewage, and normally reproduce  rapidly during
 the initial stage of the digestion process  with the production of large
 amounts of volatile acids .is.?./   No waste stabilization  occurs during  the
 first stage of the process, but substrates  are produced  that can be uti-
 lized by the bacteria in the second stage.

           During the second stage, the organic acids  are ultimately con-
 verted to methane and carbon dioxide,  resulting in waste stabilization.
 The methane-producing bacteria utilize the  volatile acids produced by
 the acid-formers in a symbiotic relationship.   They ferment only very  se-
 lect substrates, and do not utilize carbohydrates  and amino acids as do
 most saprophytes.3,47

           The methane bacteria are obligate anaerobes which require very
 low oxidation-reduction potentials for growth.   They  cannot be grown by
 classical anaerobic techniques.   Consequently few data,  concerning their
 population in sewage, physiology,  and ecology,  are available.   To date,
 few species  of methane bacteria have been cultured and identified from
 anaerobic sludge,  rumen contents,  and anaerobic bottom sediments.   Only
 six species  have  been isolated in pure culture.3,5/   While there are  a
 number of references to methane-forming bacteria in sewage sludge,  only
 three species have been isolated in pure culture and  identified,  namely
 Methanobacterium formicum,^./ Methanobacterium omelianskii,]_/ and
 Methanobacterium ruminantium.A/   Methanobacterium omelianskii has since
 been shown to be a symbiotic relationship between a methanogenic and a
 non-methanogenic species .JL/

          Much of  the investigation on anaerobic sludge digestion has been
concerned with the chemical and biochemical aspects of the problem.  Con-
siderable data have been obtained on end-products of processes, which
resulted in some control over the digestion process by noting  certain
changes, e.g., pH, volatile acids, temperature, trace metals,  etc.  How-
ever, little is known  about the organisms which cause initial digestion

-------
and secondary stabilization.  A knowledge of these organisms is essential
for proper control and understanding of sludge digestion.

          The research described in this report has been designed to simplify
the methods and media used in growing sludge (in this report, it should be
assumed, unless otherwise stated, that any reference to sludge or sewage
sludge is taken to mean anaerobic sewage sludge as opposed to aerobic or
"activated" sludge) anaerobes and to obtain useful information concerning
the biomass in sewage sludge.  The experiments were carried out in four
phases, as follows:

          Phase I - Development of Techniques for Sampling arid Handling of
Anaerobic Sewage Sludge.

          Phase II - Development of a Simplified Technique for Enumeration
of the Obligate Anaerobes in Sewage Sludge.

          Phase III - Development of a Culture Medium for Growth of
Microorganisms Representative of the Total Biomass in Sewage Sludge.

          Phase IV - Determination of Growth Substance(s) in Sewage Sludge
that Enhances Growth of Sludge Microorganisms.

          Efforts were directed toward development of simple and inexpensive
techniques and equipment that are reliable for cultivation and isolation
of the microorganisms responsible for anaerobic sludge digestion.  Data
obtained will be important in developing a more systematic and stand-
ardized approach to cultivation of the sewage biomass which is currently
lacking.  (For a fuller statement of the principal conclusions and
recommendations based upon the work in this research program, see page
1-3.)

-------
           PHASE I - DEVELOPMENT OF TECHNIQUES FOR SAMPLING AND
                   HANDLING OF ANAEROBIC SEWAGE SLUDGE
          The bacteria of importance in anaerobic sludge digestion are
obligately anaerobic  (particularly the methane bacteria), and require a
very low oxidation-reduction potential for growth.  Even brief exposure
of these organisms to oxygen results in a rapid decrease in numbers of
viable organisms present..i/  it is, therefore, mandatory that samples to
be studied in the laboratory be collected under anaerobic conditions at
a low Eh (oxidation-reduction potential), and be maintained in this con-
dition during transport to the laboratory.  The literature reveals that
little attention has been given to the collection and handling of sludge
samples in the absence of oxygen.

          Work during the early stage of this program involved basic labo-
ratory studies to evaluate the sampling and handling procedures for sewage
sludge.  Since most experiments carried out in this program involved the
growth and enumeration of the obligate anaerobes in sewage sludge, it was
necessary to initially establish a reliable procedure for collection,
suspension, dilution, and counting of samples which would result in a
maximum viable count of the obligate anaerobes present in sewage sludge
digests.
Preparation of Medium and Roll-Tube Procedure

          The medium and roll-tube procedure to be described were used
throughout the initial phase of this project (Phase I).  The roll-tube
procedure described is a modification of that originally developed by
Hungate for growth of anaerobic, mesophilic, cellulolytic, rumen bacteria.10/
The medium selected is a rather simple but complete medium designed to
contain essential nutrients and gaseous atmosphere for concomitant growth
of both methanogenic and non-methanogenie species.

          Roll-Tube Medium (100 ml)

          Clarified sludge supernatant (CSS)    30.000 ml
          Mineral phosphate, S-l                 5.000 ml
          Phosphate supplement, S-2              5.000 ml
          Sodium formate                         0.100 gm
          Potassium acetate                      0.100 gm
          Trypticase                             0.200 gm
          Yeast extract                          0.050 gm
          Agar                                   2.000 gm
          Cysteine -HCl-I^O                      0.025 gm
          Resazurin (0.1% solution)              0.100 ml
          Distilled water                       56.000 ml
                                    7

-------
          Ingredients were placed in a 500-ml round-bottom flask and brought
to a boil under oxygen-free C02.  Oxygen removal was accomplished by passing
the gas through a hot copper column.  Boiling was continued with gassing
until the resazurin became reduced (colorless).   The flask was then
stoppered and wired to prevent loss of the stopper during sterilization.
After autoclaving at 15 lb/15 min, the medium was cooled to 47 to48°C, in a
water bath, and opened aseptically under 50-50 H2/C02.  Sterile 8% CC>2
equilibrated Na2C03 was added to give a final pH of 6.8 (2.5 ml/100 ml
medium).  The medium was tubed in 9 ml amounts by aseptic anaerobic
transfer to sterile 18 x 150 mm tubes, and tightly stoppered.  (Sterile
tubes had been previously gassed with C02, stoppered and stored for future
use.)  Tubes were aseptically opened and gassed with (X>2/H2 during transfer
of medium using a gas-flushed, sterile, 10-ml pipette with a rubber tubing
mouthpiece.  Gassing was continued for an additional 10 to 15 sec before
the gassing needle was removed and the stopper securely seated.  The tubed
medium was stored for future use.

          Final reduction of the medium was accomplished by adding 0.2 ml
of sterile Na2S-9H20 (1.257. stored under N2 in stoppered tubes) 2 to 24 hr
prior to use.  Tubed medium was melted just before use by placing in a
jig which securely held the stoppers in place during melting in an Arnold
steamer (5-10 min).  Tubes were held in a water bath at 47 to 48°C until used.

          Mineral solution S-l contained the following in g/liter: NaCl,
16.0; MgS04«7H20, 1.8; CaCl2, 0.4; (NH^SO^, 3.0; MnS04'H20, 0.2;
CaCl2'6H20, 0.02; NaMoO^l^O, 0.01; and FeS04'7H20, 0.02.

          Mineral solution S-2 contained 6.0 g/liter each of K^HPO^ and
KH2P04.  Mineral solutions were dispensed in 100-ml quantities, auto-
claved at 15 lb/15 min and stored at room temperature.

          Clarified sludge supernatant (CSS), used in preparation of media
and anaerobic dilution solution (ADS), was prepared as follows.  A 6 to 8
liter sample of sludge was collected and sterilized.  Large particles were
removed by filtration through a double thickness of cheesecloth in a
Buchner funnel under vacuum.  Several changes of cheesecloth were neces-
sary, since the sediment had a tendency to cake and impede filtration.
The filtrate was clarified by passage through a Sharpies continuous centri-
fuge at approximately 40,000 rpm.  Two or more passages were often neces-
ary to achieve final clarification.  The clarified sludge supernatant was
bottled in 100-ml quantities, sterilized (15 lb/15 min), and stored in the
refrigerator.  In the initial experiments, CSS was added to media and
anaerobic dilution solution (ADS) at an arbitrary concentration of 30% (V/V).

-------
          Anaerobic dilution solution used in preparation of serial dilutions
for roll-tube inoculation was prepared in the same manner as the roll-tube
medium, except that no substrates and agar were included:  i.e., no sodium
formate, potassium acetate, trypticase or yeast extract.  The ADS was tubed
aseptically in 8.8-ml amounts.  Prior (2-24 hr) to an experiment, 0.2 ml
of Na2S-9H20 solution was added to achieve final reduction and give a final
volume of 9 ml/tube.
Roll-Tube Procedure

          Preparation of roll tubes varied with the number of tubes prepared
from each dilution and the number of conditions tested per dilution; how-
ever, the basic dilution procedure and method of inoculation did not vary
throughout Phase I.  Decimal dilutions of the initial 1/10 dilution of
sewage sludge were prepared by anaerobic transfer of 1 ml of the dilution
to a 9-ml dilution blank.  A gas-flushed 1-ml pipette with a rubber tubing
mouthpiece attached was used.  Each dilution blank was first opened and a
gassing needle inserted into the tube to maintain the gaseous atmosphere
and prevent entry of oxygen.  Following transfer of 1 ml of material, the
gassing needle was removed, the stoppers quickly replaced and seated, and
the contents of the tube were mixed thoroughly by vigorous shaking.  When
all dilutions had been prepared in this manner, 0.5-ml aliquots were trans-
ferred aseptically and anaerobically from the desired dilutions, in tripli-
cate, to roll-tube medium.  The contents of each roll tube were mixed by
gentle agitation and the medium was solidified to a uniform layer in the
tubes by horizontal rolling in a flat pan containing ice water.  Roll tubes
thus prepared constitute an oxygen-free, closed system at low oxidation-
reduction potential.

          Periodic colony counts were performed on the roll tubes by ro-
tating each tube under a Quebec colony counter and carefully counting the
colonies which appeared during each successive incubation period.  Each
colony was marked by a felt pen.  A color coding system was established
by marking all colonies appearing at different time intervals with a dif-
ferent color.  This enabled us to determine when a particular colony type
might appear, and eliminated the need to recount all colonies at each
counting period.
Evaluation of Sampling Methods

          A sampler, shown in Figure 1, was devised and constructed which
could be lowered into an anaerobic digester and opened at any desired depth.
The sampler is then closed by means of a spring-loaded lid, the sample with-
drawn, and a portion removed under anaerobic conditions for transport back
to the laboratory.  The sample is removed by placing the tubing at the bot-
tom of the sampler to the bottom of the nitrogen-flushed thermos bottle.

-------
Figure 1 - Anaerobic Sludge Sampler Designed for Collection
                of Samples Under Anaerobic Conditions
                               10

-------
The valve is then opened and the stopper at the top of the sampler is
"cracked" to allow flow into the thermos bottle.  The uppermost level of
material in the sampler was never collected due to the possibility of oxygen
diffusion into the upper few inches of sample.  The sample thus obtained is
homogeneous, free of surface scum and floating debris, and representative
of digester contents at the depth sampled.  The tubing is flushed with a
portion of the sampler contents before the sample itself is taken.

          The thermos bottles used for transporting the samples from the
sewage plant to the laboratory are nitrogen-flushed to displace air and
fitted with gas-tight lids equipped with Bunsen valves to relieve gas
pressure while simultaneously excluding entry of oxygen.  The thermos
bottles were "sanitized" with boiling water for 15 min before flushing
with nitrogen.

          Sampling was evaluated under the following conditions:  (1) sample
collection under nitrogen only; (2) sample collection under nitrogen with
0.025% each of cysteine-HCl and Na2S added at the time of sampling to
determine the effect of reducing agents on total counts; and (3) sample
collection under nitrogen using 0.25% sodium pyruvate as a protective
agent.  Pyruvate was added on the assumption that it might protect the
obligate anaerobes from oxidative effects by destruction of peroxides
which could be present in the samples, or could form as a result of entry
of small amounts of oxygen during the sampling process.ii/

          Samples transported to the laboratory under the above conditions
were diluted 1/10 by transferring 20 ml of sample to a stainless steel
Waring blender cup containing 180 ml anaerobic dilution solution consisting
of 30% (v/v) clarified sludge supernatant, 10% mineral salts solution,
2.5% C02 equilibrated Na2C03 (8% solution), 0.025% cysteine'HCl-Na2S re-
ducing solution, and 0.1% of a 0.1% solution of resazurin as an 0/R indi-
cator.  Samples were blended 1 min at high speed while being flushed vigor-
ously with C02-H2  (50-50) to maintain anaerobic conditions.  The 10~1
dilution was then serially diluted to 10~8 in duplicate and triplicate
0.5-ml samples of 10~6, 10-7 and 10~8 dilutions transferred to roll-tube
medium (a total of six roll tubes from each dilution of sample).  All tubes
were incubated at 37°C and weekly counts made for a total- of five weeks.
Due to the very slow growth of these organisms, more frequent counting inter-
vals were not necessary.

          The results of sampling studies shown in Table 1 are  the averages
of three experiments for each sampling condition.  Each value represents
the average of 17 to 18 roll-tube counts which have been evaluated statis-
tically to determine the standard deviation of the means and the signi-
ficance between the means.   In this, and subsequent tables,, all counts
presented are total anaerobic colony counts per milliliter of orginal sludge
sample unless specifically designated otherwise in tables or text.  The


                                    11

-------
                                                   TABLE 1

                               EFFECT OF SAMPLING METHOD ON TOTAL COLONY COUNT

                                	Days Incubation at 37°C	
                                Z                 l^.               21_               28.              35.

Sample collected          6.2 x 107  xySj    9.8 x 107  x     2.5 x 108  x     3.6 x 108  x     4.5 x 108  x
  under N2               (2.3 x 10?) 17b/   (4.6 x 107) 17   (1.1 x 108) 17   (2.4 x 108) 17   (2.4 x 108) 17

N2 plus 0.025% Na2S-      8.2 x 107  y       1.3 x 108  y     3.0 x 108  x     4.4 x 108  x     6.1 x 108  x
  9H20-cystcine-HCl      (2.5 x 107) 18     (0.4 x 108) 18   (1.6 x 108) 18   (2.4 x 108) 18   (2.9 x 108) 17

N2 plus 0.25% sodium      5.4 x 107  x       9.7 x 107  x     2.4 x 108  x     3.9 x 108  x     5.1 x 108  x
  pyruvate               (3.6 x 107) 17     (4.6 x 107) 18   (1.5 x 108) 18   (2.5 x 108) 18   (2.6 x 108) 17
_a/  Mean colony counts of replicate tubes.  Means followed by one letter:  means followed by the same letter
      are not statistically different (5% level of probability).  Other pairs are statistically different.
      Means followed by two letters:  consider that pairs of means followed by one matching letter are not
      statistically different.
b/  Values in parentheses represent the standard deviation of the mean.  Numbers following the parentheses
      correspond to the number of replicate tubes counted.

-------
colony counts for samples taken under nitrogen and under nitrogen with
pyruvate are in good agreement, and no advantage to added pyruvate is in-
dicated.  The addition of reducing solution gave consistently higher counts,
although the large standard deviation of the mean indicates an insignificant
difference.  Samples containing reducing solution did, however, result in
better handling conditions (less tendency to oxidize during roll-tube prep-
aration) , it was decided to include this procedure in subsequent samples.
Evaluation of Handling Methods

          The literature contains no methods which are used consistently
for the handling of sewage sludge samples.  We therefore devoted consider-
able effort to developing handling techniques which would yield a maximum
survival rate during the period required for blending and subsequent dilu-
tion of samples to be counted.  The method which proved most effective
in our hands was adopted for use throughout the remainder of this program.
The following parameters were evaluated:  (1) composition of anaerobic
dilution solution; (2) blending time; (3) use of a surfactant to aid in
breaking of clumps and suspension of cells for subsequent dilution and
counting; (4) effect of initial mixing; and (5) effect of catalase addition
on total colony count.

          Anaerobic dilution solution was evaluated by a basal dilution
solution consisting of mineral salts solution, 10% (v/v); resazurin (0.1%),
0.1% (v/v); cysteine.HCl«H20, 0.025%; Na2S,  0.025%; Na2C03, 0.2%; and C02-H2
gas phase (50-50).  A second solution was compared, which contained basal
anaerobic dilution solution plus 30% clarified sludge supernatant (CSS).
CSS apparently contains unknown factors that exert a protective effect.
The addition of sludge supernatant, however, results in considerable foam-
ing during shaking which can lead to inconsistencies in pipetting.  A third
comparison was therefore made to determine the value of adding Dow-Corning
Antifoam-C to reduce foaming caused by the sludge supernatant.

          Data on the effect of the composition of the anaerobic dilution
solution are summarized in Table 2.  It is apparent that the addition of
CSS to the ADS results in consistently higher counts than ADS alone.  The
addition of Antifoam-C to retard foaming negates the increased count due
to sludge supernatant; in fact, the data indicate a lower count than ADS
alone and that Antifoam-C is toxic to sludge anaerobes.  We found that the
foaming resulting from added CSS is of little consequence if the pipette
was lowered to the bottom of the dilution tube before solution was allowed
to enter.  Any foam adhering as the pipette was withdrawn could be elim-
inated by touching the pipette to the tube during withdrawal.  In subse-
quent experiments, the CSS was included in the ADS with no Antifoam-C added.
                                     13

-------
                                               TABLE  2
                          EFFECT  OF  ANAEROBIC DILUTION SOLUTION COMPOSITION ON
                                          TOTAL COLONY COUNT
                                                     Days Incubated at 37°C
 Basal  anaerobic
   dilution  solu-
   tion  (ADS)

 ADS  plus  30%
   clarified sludge
   supernatant  (CSS)

 ADS  + CSS +
   Antifoam-C
                                              14
                                         21
                                        28
 4.7 x 107  x£/    7.4 x 107   x      8.8  x  107   x      1.4  x  108   x
(3.7 x 107) ll-/  (4.1 x 107)  11    (4.2  x  107)  12    (0.9  x  108)  12
 1.3 x 108  y
(0.5 x 108) 11
 7.1  x 107   x
(4.4  x 107)  7
 1.5 x 108   y
(0.5 x 108)  11
 1.0 x  108   x
(0.4 x  108)  7
 1.7 x 108  y
(0.7 x 108)  11
 1.1  x 108  x
(0.4  x 108) 7
 2.1  x 108  y
(1.1  x 108) 11
 1.2 x 108  x
(0.4 x 108) 7
                                        3_5

                                   1.8 x 108  x
                                  (1.0 x 108) 12
 2.7 x 108  y
(1.0 x 108) 11
 1.4 x 108  x
(0.4 x 108)  7
a.b/  See Table 1.

-------
          The surfactant Dioctyl sodium sulfosuccinate (DSS) was compared
at levels of 0, 0.1% and 0.5% (w/v) in anaerobic dilution solution to deter-
mine the effect of reduced surface tension on dispersal of bacterial clumps
in the initial 1/10 dilution.  Dilutions were prepared to 10~^, and roll
tubes were inoculated from the 10~6, 10"? and 10~8 dilutions.  A control was
included in which the original 1/10 dilution was blended without DSS.  Anti-
foam-C was included in the dilution solution, although Table 2 indicates
that this compound exhibits toxicity to the sludge bacteria.  An overlap
of experiments was necessary throughout the program.  Because of very slow
growth rate of some species; roll tubes in experiments were counted after
varying incubation periods up to a maximum of 35 days.  Without overlap,
only one experiment could be initiated per month.  Obviously, it was neces-
sary to begin many experiments without having completed data from concurrent
experiments which might directly affect new work.

          Table 3 shows the results of addition of surfactant to the ADS.
The counts are obviously much lower when DSS is used and it would appear
that a significant portion of the anaerobic flora is sensitive to this
compound.  Dioctyl sodium sulfosuccinate was not used in other experiments.

          Bile salts were also tested as a possible aid to suspension of
bacteria and/or dispersal of clumps.  Anaerobic dilution solution and
roll-tube media were prepared with bile salts added at levels of 0, 0.05%,
and 0.15%.  The raw sludge sample was preblended for 1 min, under anaer-
obic conditions, without prior dilution to insure a homogeneous inoculum
for all subsequent dilutions.  Following the preblend, subsamples were
diluted 1/10 with ADS containing the indicated levels of bile salts and
blended an additional 1 min under anaerobic conditions.  Suitable dilu-
tions of each were inoculated into three sets of roll tubes, each contain-
ing one level of bile salts.

          The results of this experimental set are not shown since growth
was obtained only in control sets (no bile salts).  The lowest bile salt
level tested was almost completely inhibitory at the lowest dilution
inoculated (10~6).  It appears probable that, unlike the coliforms, the
sludge bacteria as a group are rather sensitive to reduced surface tension,
and such agents should not be used in preparation of media used for their
growth.

          The literature reveals no agreement as to blending times or
blending conditions used to prepare sludge samples for dilution.  Several
papers have appeared, however, in which samples were blended as 1/10 dilu-
tions, for no more than 30 sec to 1 min, under anaerobic conditions in a
Waring blender .if
                                     15

-------
                                                  TABLE 3

                     EFFECT OF DIOCTYL SODIUM SULFOSUCCINATE (DSS) ON TOTAL COLONY COUNT

                                 	DaysJEncubation at 3_7°C_
                                 1                I4

 "Normal ADS" (ADS
   with CSS and Anti-
   foam-C)

 Normal ADS + 0.1%
  DSS                     (0.4 x 107) 4

 Normal ADS + 0.5%         1.2 x 107  y      1.9 x 107  y     2.7 x 107  y     3.6 x 107  y     4.0 x 107  y
   DSS                     (0.2 x 107) 6     (0.6 x 107) 6    (0.9 x 107) 6    (1.0 x 107) 6    (1.1 x 107) 6
7_
107 x-2/
107) 6^7
107 y

1.0
(0.2
4.6
i4.
x 108 x
x 108) 6
x 107 y

2.2
(0.8
5.7
2J_
x 108
x 108)
x 107

x
6
y

3.
(0.
6.

3
7
5
28
x 108
x 108)
x 107

x
6
y

3.
(0.
7.
35.
8x1
6x1
1 x 1
a,b/  See Table 1.

-------
          To determine the optimum blending time for anaerobic sludge sam-
ples, 20 ml of sludge was blended with 180 ml of anaerobic dilution solu-
tion as previously described.  Samples were taken for further dilution and
roll-tube inoculation after 30 sec, 1, 2, 3, 5, and 10 min blending at high
speed in the Waring blender.  Blending for periods of greater than 2 min
duration resulted in heat generation due to friction.  Samples were either
blended for no longer than 2-min intervals followed by a 2-min cooling
period, or the blender cup was wrapped in a cool towel.  Both methods were
equally effective in preventing loss of viability due to excessive heat.

          The results of these experiments are shown in Table 4.  It is
apparent that sludge samples can be blended up to 10 min without great loss
in viability of sewage anaerobes.  There appears to be no advantage, how-
ever, to blending longer than 3 min.  The 30 sec to 1 min blending period
often found in the literature would appear to be too short for maximum colony
counts.  The most significant blending times appear to be either 2 or 3
min.

          The effect of initial mixing on total colony count was tested by
comparing 1 min blending time with samples diluted 1/10 before blending,
with hand-shaken samples diluted 1/10, and with undiluted samples blended
directly.

          The results shown in Table 5 indicate that significantly higher
counts are obtained when samples are blended undiluted as opposed to
blending of the initial 1/10 dilution or shaking by hand.  In fact, a
1-min blending of a 1/10 dilution does not give significantly better dis-
persion than hand shaking of a 1/10 dilution.  Apparently blending of a
more concentrated sample results in more clumps being broken up per unit
time, resulting in a more uniform mixture and better release of cells from
clumps.  This was further resolved by comparing samples blended for longer
periods of time.

          The results presented in Table 6 further indicate the need for
blending longer than 1 min.  In contrast to 1-min blending comparisons,
when blending is carried out for 2 min to compare undiluted samples and
samples which have been diluted 1/10 before blending, there is not signi-
ficant difference in total count.  There is, nevertheless, a distinct ad-
vantage to blending the sample undiluted in that reduced dilution solution
is not required in the blender cups.  The 1/10 dilution is a rather diffi-
cult and time-consuming step which, when eliminated, simplifies the han-
dling procedure considerably.  It is still necessary to maintain strict
anaerobic conditions, however, which is accomplished by measuring a
200-ml sludge sample (containing 0.025% each of cysteine-HCl-t^O and
Na2S-9H20) into a gas-flushed, sterile graduated cylinder and quickly
pouring this, with gassing, into a gas-flushed stainless steel blender
cup which is then tightly closed.  The sample is blended 2 min at high
                                   17

-------
                                                    TABLE  4
                                 EFFECT OF BLENDING TIME  ON  TOTAL  COLONY COUNT
oo
     Blending Time for
     1/10 Dilution of
       Sludge Sample

          30 sec
           1 min
           2 min
           3 min
           5  min
          10  rnin
                               Days  Incubation  at  37°C
                        14
                       21
      28
 4.8 x 107  x-/    6.6 x 107   x     7.6  x  107   x      1.2 x  108  x
(0.6 x 107) 12^  (0.9 x 107)  12    (0.9  x  107)  12    (0.3 x  108) 12
                                        c/
 6.9 x 107  y£
(2.6 x 107) 12

 6.9 x 107  y
(2.4 x 107) 14

 6.6 x 107  y
(2.2 x 107) 17

 5.4 x 107  xy
(1.0 X 107) 14

 4.0 x 107  x
(0.8 x 107) 8
 3.5 x 107  xz
(2.8 x 107)  12

 9.7 x 107  z
(2.3 x 107)  14

 8.8 x 107  z
(2.8 x 107)  17

 8.4 x 107  xz
(1.3 x 107)  14

 5.4 x 107  xy
(1.4 x 107)  8
 9.6  x 107   xy
(2.5  x 107)  12

 1.5  x 108   z
(0.4  x 108-)  14

 1.2  x 108   y
(0.3  x 108)  17

 1.1  x 108   y
(0.4  x 108)  14

 6.4  x 107   xy
(1.9  x 107)  8
 1.2  x 108  x
(0.3  x 108)  12

 2.0  x 108  z
(0.5  x 108)  14

 1.9  x 108  z
(0.6  x 108)  17

 1.7  x 108  yz
(0.6  x 108)  14

 1.3  x 108,  xy
(0.5  x 108)  8
      35.

 1.6 x 108  xy
(0.4 x 108) 12

 1.6 x 108  x
(0.4 x 108) 12

 2.2 x 108  z
(0.5 x 108) 14

 2.5 x 108  z
(0.7 x 108) 17

 2.1 x 108  yz
(0.6 x 108) 11

 1.6 x 108  xy
(0.4 x 108) 8
     a,b/  See Table 1.
     £/    Total counts corrected for initial 1/10 dilution.

-------
                                                  TABLE 5
                          EFFECT OF METHOD OF INITIAL MIXING ON TOTAL COLONY COUNT
                                                        Days Incubation at  37°C
                                                 14
                                        21
1/10 dilution of sample   3.9 x 107  x-/    6.4 x 107  x£/   1.5 x 108   x
  shaken by hand for     (1.9 x 10?) 16-/  (1.6 x 10?) 16   (0.3 x 108)  16
  1 rain

Sample blended un-
  diluted for 1 min

1/10 dilution blended
  1 min
 5.3  x 107   y
(2.1  x 107)  16
 7.3  x 107  x
(2.2  x 107) 16
 4.5  x 107   xy£/    6.5 x 107  x
(1.3  x 107)  16     (2.0 x 107) 16
 1.8 x 108   xy
(0.5 x 108)  16

 1.0 x 108   y
(0.3 x 108)  15
                                       28
                                                    2.2 x 108  x
                                                   (0.5 x 108) 16
 3.3  x 108  y
(1.6  x 108)  16

 1.9  x 108  x
(0.9  x 108)  15
                                       11

                                   2.8 x  108  x
                                  (0.8 x  108) 15
 4.8 x 108  y
(2.3 x 108) 16

 2.6 x 108  x
(1.1 x 108) 15
a,b/  See Table 1.
£/    Total counts corrected for initial 1/10 dilution.

-------
                                                    TABLE 6

                          COMPARISON OF TOTAL COLONY COUNTS/MILLILITER ON SAMPLES BLENDED
                                 UNDILUTED WITH SAMPLES BLENDED AS 1/10 DILUTIONS
                                                            Days Incubation at 37°C
NO
o
     Samples blended un-
       diluted for 2 min

     Samples blended as
       1/10 dilutions
       for 2 min
                                                     14
 6.1 x 107 xa/    9.2 x 107  x
(0.7 x 10?) 20k/ (1.2 x 10?) 20
 6.2 x 107  x£/
(2.0 x 107)  20
 8.9 x 107  x
(2.2 x 107)  20
     a,b/    See Table 1.
     c/      Total counts  corrected for initial 1/10 dilution.
      11

 3.1 x 108  x
(1.1 x 108)  20

 2.2 x 108  y
(0.9 x 108)  20
      28.

 4.3 x 108  x
(1.2 x 108) 20

 3.5 x 108  y
(1.4 x 108) 20
      35.

 4.8 x 108  x
(1.3 x 108) 20

 4.4 x 108  x
(1.1 x 108) 20

-------
speed with gas flushing.  Subsequent dilutions are then prepared in di-
lution tubes.  The 2-min blending period is more convenient than a 3-min
period since neither a waiting period between blends nor external cooling
is required.

          Many of the obligate anaerobes lack catalase and are extremely
sensitive to hydrogen peroxide which can accumulate in the presence of
oxygen.  If even trace amounts of oxygen are present in the medium, perox-
ide toxicity could conceivably result.

          To explore the possibility of protection against possible perox-
ide formation, the effect of adding catalase to the sample container, anaer-
obic dilution solution, and medium was studied.  Duplicate sludge samples
were taken in two thermos, each without reductants.  One thermos bottle
contained 1,000 sigma units of catalase per milliliter of sample.  From the
sample with catalase, dilutions were prepared in ADS with catalase and medium
with and without catalase.  Dilutions were prepared from the sample with-
out catalase, and medium with and without catalase was inoculated.

          Results of catalase experiments are shown in Table 7.   It is
apparent that catalase yields a higher count when added to the sample con-
tainer, ADS, or medium.  Although the highest counts were obtained when
catalase was added at all stages of roll-tube preparation, it would appear
that the greatest benefit is derived only when catalase is added at the
time of sample collections—more so than addition to ADS or medium only.
Since catalase is heat sensitive, it must be filter-sterilized and added
to individual tubes of ADS and/or medium after sterilization.  In the in-
terest of simplicity it would perhaps be best used only in the sample con-
tainer and added at the time of collection.

          Because of the broad scope of work and the large number of exper-
iments which were performed throughout this program,.no single aspect of a
problem could be thoroughly studied.  We were thus unable to devote suffi-
cient time to further testing of the effect of catalase in particular,
or sampling and handling techniques in general.  It would be of particular
interest to determine the effect of both reductants and catalase on total
colony counts; the effect of catalase on samples taken- in open containers
without reductants or N£ atmosphere; and the effect of catalase on viable
counts when samples are held for extended periods of time before prepar-
ation for roll-tube inoculation.
Recommended Method for Sampling and Handling

          On the basis of work accomplished in Phase I, we make the following
recommendations regarding the sampling and handling of sewage sludge samples
to be used for roll-tube inoculation by the Hungate technique.

                                    21

-------
                                                       TABLE 7
                                       EFFECT OF CATALASE ON TOTAL COLONY COUNT
                                                              Days Incubated  at  37°C
                                                       14
                                        21
                                       28
                                        35
NJ
     Catalase  in  sample
     c ontaincr :

       ADS with catalase,
         medium without

       ADS with catalase,
         medium with
 6.6 x 107
(1.1 x 10?) 8k/

 6.6 x 107  x
(0.9 x 107) 8
 1.0 x  108   x
(0.1 x  108)  8

 1.1 x  108   x
(0.1 x  108)  8
 4.5  x 108   y
(0.5  x 108)  8

 5.7  x 108   x
(0.9  x 108)  8
 6.3 x 108  x
(0.9 x 108)  8

 6.7 x 108  x
(1.0 x 108)  8
 7.0 x 108  x
(0.6 x 108) 8

 7.4 x 108  x
(0.7 x 108) 8
     No catalase in sample
     container:

       ADS without catalase,
         medium without

       ADS without catalase,
         medium with
 4.2 x 107  x
(0.9 x 107)  8

 6.9 x 107  y
(1.3 x 107)  8
 8.0 x 107   x
(2.0 x 107)  8

 1.2 x 108   y
(0.2 x 108)  8
 3.3 x 108  x
(0.7 x 108)  8

 4.4 x 108  y
(0.8 x 108)  8
 4.6 x 108  x
(0.9 x 106) 8

 5.6 x 108  x
(1.0 x 108) 8
 5.5 x 108  x
(0.8 x 108) 8

 6.2 x 108  x
(0.8 x 108) 8
     a,b/  See Table 1.

-------
          It is well established that minute amounts of oxygen are extremely
toxic to the obligately anaerobic population of sewage.  It is, therefore,
mandatory that all precautions possible be taken to exclude oxygen during
all phases of sampling and subsequent handling.

          Samples should be taken from the digester as directly as possible
with minimum exposure to air, and collected in thermos bottles flushed with
oxygen-free nitrogen.  These containers should have a tight seal which ex-
cludes entry of air but provides for simultaneous release of gas (Bunsen
valves) which is inevitably produced by any active sludge sample.  At the
time of sampling, we recommend addition of sufficient reduced Na2S-cysteine
•HC1 solution to give a final concentration 0.025% of each reductant in the
sample.

          After transport to the laboratory, the thermos bottles should be
inverted several times to insure mixing of contents.  The sample is then
opened, blanketed with oxygen-free C02-H2 (50-50) or N2 and a 200-ml sample
is measured into a sterile stainless steel blender cup which has been freed
of oxygen by gassing prior to addition of sample.  The sample is blended
for a 2-min interval, with gassing.  The blended sample is then serially
diluted in anaerobic dilution solution containing clarified sludge superna-
tant, and suitable dilutions are inoculated into anaerobic media for enumer-
ation of obligate anaerobes.  The blendor container should be one which can
be tightly closed to exclude entry of air, and should have an opening at the
top just sufficient to allow entry of the gassing needle.  Gas flow during
blending should be sufficient to maintain positive pressure in the blender
cup and exclude entry of air.

          Regardless of improvements in techniques, any method involving
enumeration of the obligate anaerobes in sewage sludge will involve sample
collection, homogenization, and subsequent dilution of samples for ino-
culation into media for counting.  We therefore believe that effort along
the lines of establishing methods which will result in obtaining higher
and more consistent total counts has been worthwhile.
                                    23

-------
     PHASE II - DEVELOPMENT OF A SIMPLIFIED TECHNIQUE FOR ENUMERATION
                OF THE OBLIGATE ANAEROBES IN SEWAGE SLUDGE
          The techniques most commonly used for the enumeration of obligate
anaerobes generally involve some modification of the anaerobic jar technique,
glove box procedures or the roll-tube technique.  These techniques all suffer
one or more of the following disadvantages:  they are slow; they require a
high degree of manual dexterity; they require large enclosures in which oxygen-
free conditions are difficult and expensive to maintain for long periods of
time; the mechanics of sample preparation and plating procedures are diffi-
cult, and require long training periods for proficiency; they cannot be
handled routinely by unskilled personnel; or the hazard in their use is too
great for routine usage.

          The anaerobic jar technique involves the growth of anaerobes on
agar plates in a closed container (usually a cylindrical jar in which plates
are stacked) in which oxygen is removed by alternate evacuation and gas flush-
ing, combustion, or by chemical agents.  Although anaerobic conditions can be
maintained for long periods of time, once established, the method is only
of value if plates to be placed in the jars, and the dilutions used in the
plating procedure, are prepared in an oxygen-free atmosphere.i-L/  Even if
this were accomplished, the jars would need to be opened and closed several
times to perform plate counts.  The plate counts themselves would have to
be performed in an inert atmosphere to avoid contact with air during the count-
ing procedure.  These limitations make the anaerobic-jar technique of .little
value in enumerating obligately anaerobic bacteria.

          Anaerobic glove box procedures utilize various types and sizes
of enclosures, a common characteristic of which is that they contain an
oxygen-free environment accessible from the outside by one or more pairs
of flexible gloves.  All manipulations are performed in the oxygen-free
environment, from outside the chamber, by use of these gloves.

          Various glove boxes which have been designed specifically for the
culture of anaerobes are described in the literature.i^zi^./  With the excep-
tion of the most recent design by Aranki, et al.,.UL/ these glove boxes suffer
from the disadvantages of complexity, expense, elaborate setup procedures,
and failure during long-term operations, or are beyond the technical com-
petence of the average bacteriological technician.  The glove box described
by Aranki is an exception in that it is inexpensive to build, simpler to
operate, and can be maintained over long periods of time.  This is, however,
a design which is best suited to the isolation and study of organisms with
generation times sufficiently short that discrete colonies can be detected
(a week or less) before the plates evaporate to the point that they will no
longer support growth.  A decided advantage of the method is that plates do

                                     25

-------
not become vet, due to condensation in a high humidity,  causing the severe
swarming encountered with other methods.  Although this  method should defi-
nitely be considered in future isolation and nutritional studies of the ob-
ligate anaerobes from sewage sludge, we could not consider use of this tech-
nique because of the large number of different experiments which had to be
performed, the space required for large numbers of plates which would have
to be stored in these chambers for extended periods of time,  the necessity
of long incubation periods (up to 35 days), and the need to use various gas
mixtures without the need for additional sets of equipment for each atmosphere.

          The roll-tube technique as originally described by Hungate is
the anaerobic equivalent of the "pour plate" method of enumerating bacteria.
The name is derived from the method of preparing "plates" by rolling inoc-
ulated, liquified agar medium in a rubber-stoppered test tube until solidi-
fication occurs on the walls of the tube.  Exclusion of  air during prepara-
tion and tubing of media or transfer of media and specimens is accomplished
by introducing a stream of sterile, oxygen-free gas whenever a stopper is
removed.  The method is simple in principle, but very complex in operation
compared to conventional bacteriological procedures.  The complexity of the
method arises from the necessity of a simultaneous handling of gassing needles,
tubes, pipettes, and rubber stoppers during transfer of  materials.

          The most severe disadvantage to the roll-tube  technique is the
hazard involved in the numerous manipulations which must be performed by
hand in the preparation of media, ADS, and the inoculation of the roll-tubes
themselves.  From the time of initial sterilization and  gassing of the tubes
to the final inoculation and rolling of the tube, each tube is opened no less
than four times with gassing under aseptic conditions.  In addition, each
tube of the dilution series is opened and closed a minimum of five times.
At each interval, the danger of breakage is encountered.  Due to the pres-
sure and twisting motion which must be applied to seat the rubber stopper
sufficiently to prevent entry of oxygen and the danger of tube breakage
when the gassing needle is withdrawn, there is severe cumulative danger
involved in the technique.  The hazard is not only very  real, but an ac-
cident also becomes more likely as one becomes more proficient and at ease
with the method.

          In spite of this, we adopted the roll-tube method for the initial
studies because of its flexibility and proven reliability for growing fas-
tidious anaerobic bacteria.  We envisioned that, after completion of Phase
I, we would attempt to improve the method to a point that it could be simply
and safely handled by personnel with a minimum of bacteriological training.
It is unfortunate that these attempts were not begun before an accident
occurred involving the roll-tube technique.

          The accident, which involved a rather serious  laceration of fingers
during seating of rubber stoppers, resulted in immediate cessation of use of
the technique and initiation of work to eliminate direct handling of tubes.

                                    26

-------
          The method finally adopted is a modification of that developed by
Dr. Paul Smith after a similar accident, of a more serious nature, which
occurred in his laboratory during the use of the Hungate technique.  The
method consists of preparation of media in stoppered tubes which are steri-
lized in clamps to prevent blowing of stoppers when the autoclave is exhausted.
All subsequent operations are performed by syringe inoculation through the
rubber stopper rather than opening and closing of tubes several times dur-
ing the course of an experiment.  The inoculating press shown in Figures 2
and 3 was assembled and is used to hold the test tubes, while the syringe
needle is forced through the stopper by means of mechnical pressure.  This
technique eliminates the opening and closing of tubes, eliminates the handling
of tubes during manipulation, results in greater safety for laboratory per-
sonnel without sacrificing accuracy and flexibility, and very significantly
simplifies the procedure for media preparations and roll-tube inoculation.
A complete description of the method follows.
Modified Roll-Tube Procedures

          Media and dilution solutions are prepared in basically the same
manner as for the Hungate technique, except that the medium or ADS is tubed
directly from the flask before sterilization.  All ingredients except cys-
teine'HCl-H20, NaHC03, and Na2S-9H20 are brought into solution in a round-
bottom flask.  (NaHC03 was substituted for Na2CC>3 because equilibration with
C02 is much more rapid.)  Cysteine is added and the mixture is heated with
gassing until reduction is complete (resazurin	>• colorless).  The flask
is cooled (only to 48°C if agar is included), and the proper amount of NaHC03
is added to buffer the mediam at pH 6.8.  The amount of NaHC03 required is
0.9% by weight for a 100% C02 atmosphere, and 0.5% for 50-50 C02/H2.

          After reduction and gas equilibration are complete, the medium is
dispensed directly into tubes by means of a 10-ml Cornwall continuous pipette
which is washed with several volumes of boiling water to flush oxygen from
the system.  A few syringefuls of medium or ADS are wasted in washing the
syringe to insure proper flushing and oxygen removal.  While the main res-
ervoir is being continuously flushed with oxygen-free gas, the medium or ADS
is tubed in multiples of five tubes (each of which is being simultaneously
flushed during filling).  A series of manifolds were built for this purpose
containing five hoses and gassing needles each for use in gassing several
tubes or flasks simultaneously.  After each series of tubes is gassed for a
few seconds, a moistened butyl rubber stopper is inserted as the gassing
needle is withdrawn.  The procedure is continued in multiples of five until
a sufficient number of tubes have been prepared.  The prepared medium is
then clamped in a jig which serves to give the tubes a final seating and
holds them securely during sterilization and exhausting of the autoclave.
Since butyl rubber has an extremely low permeability for oxygen, the medium
                                    27

-------
Figure 2 - Overall View of Inoculating Press and Roll-Tube Jig
                               28

-------
Figure 3 - Closeup of Roll-Tube Jig with Needle in Position for Inoculation
                                    29

-------
can be stored in a reduced condition for extended periods of time without
fear of oxidation.  In fact, if only one medium-and-gas atmosphere is used
at any one time, as many tubes may be prepared in one operation as is con-
venient for future experiments.

          The jigs are constructed from two pieces of plate aluminum large
enough to cover a 72-place test tube rack.  The jigs are tightened by means
of wing nuts on stainless steel, threaded rods.  A 1/4-in. rubber pad is
laid over the stoppers before tightening to act as a shock absorber and to
prevent stoppers from sticking to the aluminum plate.  A view of a jig and
gassing manifold used in preparation of media is shown in Figure 4.

          Roller culture tubes (Bellco 16 x 150 mm for size 0 stoppers) were
found to be quite suitable for the technique.  These tubes have a reinforced
lip and a slight constriction at the top which serves to hold the stoppers
tightly seated after autoclaving.  Very little pressure is required to seal
the butyl rubber stoppers in these tubes.  The stoppers have a tendency to
conform to the shape of the tube and thus form a very effective seal (Figure 2)

          The mineral salt mixtures were modified for this procedure to be
added as a 10% solution of each of the following:
        Salts A

KH2P04         10.0 g
NaCl           20.0
NH4C1          10.0
MgS04.7H20      1.0
CaCl2           1.0
Distilled H20     2 liters
                  Salts B

          K2HP04-3H20
          Distilled H20
10.0 mg
 2 liters
                                 Salts C
                       CaCl2-6H20
                       FeS04-7H20
                       Hemin
                       Distilled H20
 5.0 mg
40.0
20.0
20.0
 5.0
 2 liters
The solutions were made up and stored at room temperature in 500-ml bottles
without sterilization.
                                     30

-------
Figure 4 - Clamps, Manifold and Copper Column Used
                in the Preparation of Media
                          31
                                                    U.S.

-------
Inoculation Press and Roll-Tube Jig

          The inoculation press and roll-tube jig, built for use in the
modified roll-tube technique, is shown in Figure 1.  The press consists of
a Dayton Drill Stand (No. 2Z041 for 1/2-in. drill), modified by rigid attach-
ment of a pair of 6-in. vise grips to the spindle.  The vise grips are used
to hold the hub of a 19 gauge x 1-1/2-in. Huber point needle while it is
forced through the rubber stopper during inoculation or transfer.  The jig
designed to hold the roll tubes was machined from a solid block of aluminum.
The jig is attached to the post of the drill press and rests on an adjustable
collar so that it may be swiveled out of the way while changing tubes.  The
collar has a stainless-steel stop pin which is adjusted so that when the
jig is swiveled against the stop, the tube held in the jig is centered ex-
actly under the inoculating needle.  A machined cover is attached to the jig
on a swivel so that it can be swung over the stopper before the needle is
withdrawn.  The cover securely holds the stopper to prevent accidental removal
from the tube during withdrawal of the needle (Figure 2).   Sufficient clear-
ance is provided so that the cover will not touch the needle or the sterile
area of the stopper at any time.  The need to sterilize any portion of the
press or jig is thus prevented.  The jig is designed to be used in a water
bath and is drilled so that warm water surrounds the tube at all times and
prevents premature solidification of agar medium.  The aluminum block becomes
heated to water-bath temperature, and is quite effective in maintaining the
medium at the desired temperature during inoculation or transfer.

          All inoculation or transfers are made using a syringe and needle.
The syringe holders are of the Cornwall type and contain a 2-ml Luer-lock
syringe which has been greased with Fisher brand Cello-Seal (nontoxic) to
prevent leakage of oxygen between the barrel and plunger of the syringe.
Syringe holders are used so that the volume injected is precisely controlled.
The lock nut is removed from all syringe holders so that the adjustment screw
may be turned freely.  Materials are thus injected by turning the screw rather
than by pushing the plunger of the syringe holder.  The holders are indexed
on the knurled nut and the top of the holder barrel so that as little as  1/2
or one turn can be injected if desired (0.04 ml/turn).

          The protocol for roll-tube preparation using the modified Hungate
technique follows:

          Sampling is handled as before as is melting of solid media and
labeling of tubes.  Final reduction of the medium is obtained by injection
of 0.22 ml of a sterile Na2S-9H20 solution per 5 ml of medium.  The sterile
stock solution of  Na2S-9H20 contains 62.5 mg/ml to give a final concentration
of 0.025% w/v in the medium or ADS.  Aseptic-anaerobic injection of sodium
sulfide is accomplished by flushing the syringe several times with sterile,
oxygen-free nitrogen by means of a gassing needle in a sterile test tube.
                                    32

-------
Just prior to injection, the area of the rubber stopper to be contacted by
the needle is flamed by means of a C02/H2 gassing needle with a pinpoint flame.

          The initial 1/10 dilution in ADS is prepared by transfer of 1 ml
of sludge to the first 9 ml blank of the dilution series, using a large bore,
gassed pipette.  This is the only tube which is opened or handled directly
throughout the course of an experiment as compared to handling each tube
four or five times by the original method.  Mixing is accomplished by in-
serting a sterile gas-flushed syringe, using the modified drill press.  The
tube is inverted, the syringe is filled and the contents expressed through
the needle several times (a minimum of 10 times for the first two dilutions—
five or six for subsequent dilutions).  The shear type mixing attained in
this manner is surprisingly violent; enough so that the initial blending
step can be eliminated by using this method if the sample is well shaken
before the initial 1-ml aliquot is taken.  Subsequent dilutions are prepared
in the same manner by transfering 1 ml from the previous dilution to a fresh
blank.  Syringes are changed between tubes, and a fresh gas-flushed syringe
is used to transfer material to the next higher dilution.  Roll tubes are
prepared in triplicate from appropriate dilutions by transfer of 0.5 ml. of
inocula/tube.  Duplicate experiments were usually performed giving a mini-
mun of six roll tubes/dilution/condition tested.

          We believe that the technique described is a very significant
improvement over that originally used.  It is not only much faster (our work
output more than doubled with one less technician required), but also is a
highly reliable and safe technique requiring very little training for some-
one with no prior experience with the method.  .With the exception of" the
necessity to gas each tube during preparation of media, this technique now
compares favorably with the aerobic plate count method with respect to ease
of operation and degree of manual skill required.
                                     33

-------
 PHASE III - DEVELOPMENT OF A CULTURE MEDIUM FOR GROWTH OF MICROORGANISMS
          REPRESENTATIVE OF THE TOTAL BIOMASS IN SEWAGE SLUDGE
          All work during this phase was carried out to improve existing
media, media supplements, reducing compounds, and gaseous atmospheres such
that a maximum total count could be obtained with a simplified medium repre-
sentative of the sludge environment.

          The major problem encountered in development of a medium which
will yield a maximum total count of organisms typical of the biomass in
sewage sludge is the evaluation of the efficacy of the medium once the medium
has been developed.  This problem is complicated by the fact that very few
organisms have been isolated from anaerobic sludge in pure culture, and the
significance of such isolates to the digestion process per s_e has never been
established.  A true means for evaluation is thus not at hand, and will not
be until the medium in question is developed and used to isolate, describe,
and assign a role to the major species present in sewage sludge.  The solu-
tion to this paradox is thus a compromise between the real and the ideal,
i.e., to develop a simplified medium which yields a maximum total count and
which probably supports the growth of a significant number of predominant
species.

          Unidentified growth factors are known to exist in rumen fluid
which are necessary for the growth of the methane-producing bacteria.  Since
these factors have never been successfully replaced by other ingredients,
rumen fluid is routinely added to media used in the study of rumen micro-
flora.  Because of the likelihood that the same, or similar, factor(s) occurs
in sewage sludge, sludge supernatant is likewise added to media used in the
culture of sludge anaerobes.  Various workers do not consistently agree as
to whether rumen fluid or sludge supernatant should be used for growth of
the obligate anaerobes from sewage sludge.  There is also no consistent
agreement to the amount of either to be incorporated into media.

          The initial experiments in this series were therefore designed to
determine which supernatant gives the maximum yield of sludge anaerobes and
to determine the optimum amount to be added to culture medium.  Experiments
were carried out by direct comparison of various percentages of rumen fluid
and sludge supernatant using the same basal medium and ADS discussed in Phase
I.  Rumen fluid or sludge supernatant was added to media to give the following
final percentages:  0, 10, 20, 30, and 50.

          The results shown in Tables 8 and 9 indicate that there is no
significant difference in total count when the optimum concentration of
either rumen fluid or sludge supernatant is used.  The optimum concentra-
tion does, however, vary with 20% indicated as optimum for rumen fluid and
10% for sludge supernatant.  These data also confirm the necessity of either

                                    35

-------
                                               TABLE  8

                   EFFECT OF SLUDGE SUPERNATANT CONCENTRATION  ON TOTAL COLONY COUNT

Percent Clarified
Sludge Supernatant           	Days  Incubated  at 37°C
    in Medium	

        0
       10
       20
       30
       50
;?
4.4 x 107 x-/
(0.9 x 107) 8-'
5.9 x 107 xy
(1.3 x 107) 8
6.6 x 107 y
(1.0 x 107) 8
4.5 x 107 x
(0.6 x 107) 8
4.6 x 107 x
(2.4 x 107) 8
14.
9.6 x 107 x
(1.1 x 107) 8
1.2 x 108 y
(0.2 x 108) 8
1.1 x 108 xy
(1.0 x 108) 8
8.0 x 107 x
(0.8 x 107) 8
6.9 x 107 z
(2.6 x 107) 8
21
2.9 x 108 xy
(0.9 x 108) 7
4.2 x 108 y
(1.1 x 108) 6
3.0 x 108 xy
(1.2 x 108) 8
2.1 x 108 xz
(0.7 x 108) 8
9.6 x 107 z
(3.6 x 107) 8
28.
4.2 x 108 x
(1.5 x 108) 8
5.7 x 108 x
(1.2 x 108) 6
4.8 x 108 x
(1.2 x 108) 8
4.1 x 108 x
(1.4 x 108) 8
1.6 x 108 y
(0.3 x 108) 8
35_
4.7 x 108 x
(1.5 x 108) 8
6.5 x 108 z
(1.1 x 108) 6
5.9 x 108 xz
(1.1 x 108) 8
4.7 x 108 x
(1.6 x 108) 8
2.6 x 108 y
(0.9 x 108) 8
a.b/  See Table 1.

-------
                                               TABLE 9

                EFFECT  OF RUMEN FLUID  SUPERNATANT CONCENTRATION ON TOTAL COLONY COUNT

Percent Clarified
Rumen Fluid Super-           	Days Incubated  at 37°C	
natant in Medium             1_                14               21                28               35

        0              2.1 x 107  x^    7.0 x 107  x     2.5 x 108   x     3.9  x  108  xy     4.6 x 108   x
                      (1.4 x 107) 8-    (2.8 x 107)  8    (1.2 x 108)  8    (1.4  x  108) 8     (1.2 x 108)  8

       10              3.2 x 107  x      7.4 x 107  x     2.9 x 108   x     4.1  x  108  xy     5.1 x 108   xy
                      (1.6 x 107) 8     (1.1 x 107)  8    (0.9 x 108)  8    (1.3  x  108) 8     (1.0 x 108)  8

       20              2.9 x 107  x      7.0 x 107  x     3.0 x 108   x     5,3  x  108  x      6.4 x 108   y
                      (1.8 x 107) 8     (2.8 x 107)  8    (1.9 x 108)  7    (2.1  x  108) 7     (1.8 x 108)  7

       30              3.5 x 107  x      6.3 x 107  x     2.0 x 108   x     3.1  x  108  y      4.5 x 108   x
                      (1.5 x 108) 8     (1.5 x 107)  8    (1.0 x 108)  8    (1.3  x  108) 8     (1.3 x 108)  8

       50              3.1 x 107  x      6.0 x 107  x     2.2 x 108   x     3.7  x  108  xy     4.5 x 108   x
                      (1.4 x 108) 8     (1.5 x 107)  8    (0.6 x 108)  8    (1.3  x  108) 8     (1.1 x 108)  7
  a,b/   See Table  1.

-------
rumen fluid or sludge supernatant for maximum total counts.  Although con-
centrations over 50% were not used because of the tendency toward excessive
foaming of the ADS, it appears that concentrations above the optimum are
inhibitory.  The significance of this observation remains to be determined.

          The method originally used to obtain clarified sludge supernatant,
for use as a supplement in media, was an awkward process sometimes requiring
up to 3 man-days to process an 8-liter batch.  The process involved auto-
claving the sludge sample, followed by a filtration step to remove larger
particles.  Final clarification was achieved by centrifuging in the Sharpies
until clear.  The initial filtration step was often prolonged and messy in
that sludge has a tendency to form a solid mat, even on a very porous back-
ing material such as cheesecloth.  Several changes of filter were often nec-
essary to filter a batch sufficiently to pass the orifice of the Sharpies.
In addition, several passes through the Sharpies were often necessary to
achieve sufficient clarity for use in culture media.  In order to improve
this procedure, the following method was adopted:  freshly collected sludge
was autoclaved and a measured quantity was batch-lyophilized (up to 16 liters/
batch).  The dried product was weighed, mixed thoroughly and stored for
future use.  As required, small amounts (usually 2-liter batches) of clari-
fied supernatant were prepared by placing a weighed quantity of this material
on a filter mat of hyflosupercel and washing the mat with sufficient hot water
to restore the original supernatant volume.  This supernatant was bottled in
100-ml quantities, autoclaved and stored in the refrigerator for future
use.  The result is a more consistent CSS which can be prepared, as needed,
in a fraction of the time previously required.  For convenience and batch
consistency, large amounts can be processed at one time and stored in a
small space for future use.

          Experiments were carried out to compare sludge supernatant pre-
pared as above (aqueous extract of lyophilized sludge, or AELS) with that
prepared by filter clarification alone and by filtration plus centrifuga-
tion.  All supernatants were added to media at the 20% level.

          The results of these comparisons are shown in Table 10.  It is
apparent that both filter-clarified sludge supernatant and AELS give higher
counts than "normal" CSS with AELS being significantly higher statistically.
It is probable that the difference in the three methods is due to incon-
sistency in recovery of sludge supernatant by Celite filtration or centrif-
ugation:  i.e., the recovery of supplemental ingredients is more complete
and consistent when a weighed quantity of well-mixed lyophilized sludge is
used to prepare sludge supernatant.  This improved method for preparation
of sludge supernatant was, therefore, adopted for use in media prepared
throughout the remainder of Phase III experiments.
                                    38

-------
                                                 TABLE 10

                      COMPARISON OF NORMAL SLUDGE SUPERNATANT WITH AQUEOUS EXTRACT OF
                        LYOPHILIZED SLUDGE AND FILTER CLARIFIED SLUDGE SUPERNATANT
                                                        Days Incubated at 37°C
"Normal" CSS
Aqueous extract of
  lyophilized sludge

Filter clarified
  sludge supernatant
 8.7 x 10?   xj/
(3.4 x 10?) 8-7

 6.4 x 10?   y
(2.6 x 10?) 8

 5.6 x 10?   y
(0.8 x 10?) 8
      14

 1.2  x 108   x
(0.2  x 108)  8

 2.1  x 108   y
(0.3  x 108)  8

 1.3  x 108   x
(0.2  x 108)  8
      21.

 4.6 x 108  x
(0.8 x 108)  8

 6.9 x 108  y
(1.2 x 108)  8

 5.5 x 108  xy
(1.3 x 108)  8
                                                                                         x
      28.

 7.2 x 108
(1.8 x 108) 8

 9.1 x 108  y
(1.0 x 108) 8

 8.0 x 108  xy
(1.6 x 108) 8
      35.

 8.4 x 108  x
(1.3 x 108) 8

 9.8 x 108  y
(0.8 x 108) 8

 9.0 x 108  xy
(1.3 x 108) 8
a,b/  See Table 1.

-------
          Concurrently with the AELS experiments, tests were run to determine
the feasibility of using supernatant fractions or media supplements rather
than crude sludge supernatant.  The following comparisons were made:  medium
with no clarified sludge supernatant (CSS);  medium with 30% CSS; medium with
30% Norit-treated sludge supernatant; medium with 30% Norit extract and
medium with 30% CSS prepared from sludge preincubated with 0.4% yeast extract.

          Norit is reported to adsorb the factor(s) present in rumen fluid
and can be eluted with 0.1 M ethanolic ammonium hydroxide following a hot
water wash (see Figure 3, page 29).   The Norit supernatant used in these
experiments was prepared by treating CSS with Norit followed by filtration.
This filtrate was incorporated directedly into the medium.

          Norit extract was prepared by extracting the washed residue with
0.1 M ethanolic ammonium hydroxide.   This solution was evaporated to a small
volume under vacuum to remove ethanol and ammonia.  Following reconstitu-
tion to the original CSS volume, the extract was added to the medium at a
final concentration of 30%.

          Preliminary work by Dr. Bryant indicates that preincubation of
rumen fluid with 0.4% yeast extract results  in increased rumen fluid factor
concentration.

          To test this effect with sludge supernatant, sludge was preincu-
bated 40 hr under nitrogen with 0.4% yeast extract.  The sludge was then
sterilized and CSS was prepared and added to the culture medium to give a
final concentration of 30%.

          The results of these comparisons are shown in Table 11.  Lower
colony counts are again indicated for medium without CSS, than for medium
containing CSS.  Although counts were higher for Norit-treated supernatant
than for the control, there is an indication that something has been removed
by the Norit.  The Norit extract yields counts significantly lower than the
control (no CSS).  This is rather difficult  to explain unless some toxic
material was present in the Norit which was  eluted with the ethanolic ammo-
nium hydroxide or other nutrients present in the crude supernatant are miss-
ing.  The latter is a distinct possibility.

          Another possible explanation might be that certain species of
sludge bacteria are selectively inhibited by the eluted material.  The
present state of knowledge of the species involved would not allow further
clarification of this observation.  It is possible that in the future when
the sludge factor(s) is identified and the dominant species of sewage sludge
bacteria have been classified, it will be possible to design media and
select organisms to study such problems further.  Further studies of super-
natant fractions at this time, however, would be of questionable value.
                                    40

-------
                                               TABLE 11
                           EFFECT OF TREATMENT OF CSS ON TOTAL COLONY COUNT
                                                     Days Incubated at 37°C
                                              14
                                        21
                                       28
                                        35
ADS and Medium with:
  No CSS
  Filter clarified
    CSS  (30%)

  Norit—treated su-
    pernatant  (30%)
 3.4  x 107   x,
(0.9  x 107)  8-

 5.2  x 107   y
(1.5  x 107)  15

 4.1  x 107   xy
(1.3  x 107)  8
  Norit extract  (30%)  3.9 x 107  xy
                       (1.0 x 107) 8

  CSS  prepared from    5.2 x 107  y
  sludge  (30%) pre-    (1.1 x 107) 8
  incubated with 0.4%
  yeast extract
 7.5  x 107   x
(0.8  x 107)  8

 1.1  x 108   y
(0.1  x 108)  16

 8.0  x 107   x
(1.6  x 107)  8

 6.9  x 107   x
(1.1  x 107)  7

 7.1  x 107   x
(0.9  x 107)  8
 3.9  x 108   x
(1.4  x 108)  8

 4.8  x 108   x
(1.1  x 108)  16

 4.2  x 108   x
(1.3  x 108)  8

 1.3  x 108   y
(0.3  x 108)  7

 1.7  x 108   y
(0.2  x 108)  8
 4.7 x 108  x
(1.3 x 108) 8

 6.1 x 108  x
(1.3 x 108) 16

 5.5 x 108  x
(1.2 x 108) 7

 2.8 x 108  y
(0.4 x 108) 8

 4.5 x 108  x
(1.4 x 108) 8
 5.0 x 108  x
(1.3 x 108) 8

 6.8 x 108  y
(1.2 x 108) 16

 6.0 x 108  xy
(1.0 x 108) 7

 3.3 x 108  z
(0.4 x 108) 8

 5.4 x 108  xy
(1.4 x 108) 8
a,b/  See Table 1.

-------
Development of a Habitat Simulating Medium for Sludge Anaerobes

          A series of experiments was carried out to develop and evaluate
a medium which would, hopefully, simulate the sludge environment, as closely
as possible in solid medium.  Such a medium would yield a maximum total
count representative of the biomass in sewage sludge.  It must, of course,
be realized that any medium developed for a colony count cannot completely
duplicate the sludge environment for three primary reasons.  First, the
sludge environment is essentially a continuous liquid culture, whereas the
medium used for total count is by necessity a solid medium in which discrete
colonies must form.  Because of the inability of organisms to migrate through-
out the medium, there is much less opportunity for the numerous symbiotic
relationships to occur which must surely develop in the natural environment;
hence the possibility of not growing some species which require relationships
with other species.  Second, a medium used for enumeration must be rather
transparent and free of particulate matter.  This is another strict imposi-
tion in that sewage sludge is quite turbid with much more insoluble mate-
rial available for surface attack and surface adsorption.  Finally, the
gaseous atmosphere is difficult to simulate in the closed system used for
enumeration.  Once the system is closed, it cannot be reopened to replenish
the gaseous atmosphere; therefore any required gases must be initially pres-
ent in sufficient quantity to sustain growth.  In the case of the methanogens,
gaseous hydrogen must be supplied for growth to compensate for metabolic hy-
drogen which would be scavenged by these organisms in the sludge environment.
A higher than normal t^ concentration is thus obligatory, which could be
toxic to certain nonmethanogenic species .A/

          The approach used was to begin with a rather complete medium, then
to add or delete ingredients until a medium was found which supported the most
growth.  As mentioned above, there is a definite limitation to those nutri-
ents which are water soluble so that a transparent medium is obtained with-
out floccules which would interfere with colony counts.  Gelatin was selected
as a protein source; soluble starch as a source of polysaccharide and cel-
lobiose (repeating unit of cellulose) for cellulose.  Sewage sludge is gen-
erally high in suspended lipids which would be difficult to simulate under
culture conditions.  Glycerol and a mixture of short chain, volatile fatty
acids (acetate, propionate, butyrate, isobutyrate, valerate, isovalerate,
2-methylbutyrate, and formate) were, therefore, substituted as a soluble
"lipid" source.  Trypticase and yeast extract was included as a source of
peptides, amino acids and vitamins and AELS was included as a source of
supplementary nutrients.

          The final ingredients selected for the initial habitat, simulating
medium, consisted of the following:  (designated HSM-1) CSS, 20% v/v; min-
eral solution, 10% v/v each of salts A, B, and C; gelatin, 0.1% w/v; starch,
0.1%; glycerol, 0.1%; cellobiose, 0.05%; trypticase, 0.05%; yeast extract,
0.05%; volatile fatty acid mixture, 0.32%; 0.025%, cysteine-Na2S as reduc-
tants; and C02 equilibrated NaHCC03 as buffer.

                                    42

-------
           For  initial  comparisons  roll  tubes were prepared in triplicate
using  suitable dilutions  of  anaerobic sludge in  the following media:  normal
 (medium previously  used in Phase I), HSM, HSM without volatile fatty acids
plus acetate and  formate,  HSM-1 without glycerol, HSM without gelatin, and
HSM without starch.  Two  sets  of each medium were prepared—one with 100%
CC>2 gas phase  and the  other  with 50-50  C02/H2 gas phase.

           Tables  12 and 13 show the results of total colony counts obtained
  from  the  initial various  modifications of HSM.  All experiments were run
using  "normal" medium  as  the control.   Except for replacement of casamino
acids  with trypticase  and  reduction of  CSS from  30% to  20%, this medium is
the same as that  described in  Phase I which was  used for sampling and han-
dling  studies. The counts obtained with the basic HSM  were the highest ob-
tained to  this time.  Of  all individual ingredients of  this medium tested,
.starch seems to be  most important.  Single deletion of  VFA (volatile fatty
acids), glycerol, and  gelatin  did  not markedly affect the total count.
Higher counts  were  obtained  with 100% C02 than with C02/H2 mixture—a con-
sistent observation in subsequent  experiments.

           In addition  to  testing various nutrients which might affect total
colony counts, we also tested  essential nonnutrient factors in the medium
which  might have  direct relationship to growth such as  reducing agents and
buffer systems.

           With the  exception of oxygen  exclusion, perhaps the most important
parameter  for  growth of the  obligate anaerobes is a low oxidation-reduction
 (0-R)  potential.  It has  been  shown that an 0-R  potential of -520 to -530
mv  is  optimum  for anaerobic  sludge digestion.±i/ It is important to note
that methane production decreases  significantly  and the digester approaches
failure as the 0-R  potential is increased to -430 mv.   Only with extreme care
is  it  possible to obtain  an  0-R potential, in culture media, of -350 mv or
below.   It follows  that if the 0-R potential could be reduced routinely
beyond that obtained with reductants currently in use,  which exhibit little
or  no  toxicity to sludge  anaerobes, colony counts in excess of those pre-
viously reported  might be obtained.  It is also  possible that many of the
more fastidious anaerobes  have never been grown  in routine roll-tube cultures
and that more  highly reduced media would give a  more representative picture
of  the obligate anaerobic flora in sewage sludge.

           Four reductants  were selected for evaluation: formamidine sul-
finic  acid, formamidine disulfide  dihydrochloride, dithiothreitol, and
formaldehyde sulfoxylate.

           Preliminary  tests  showed that formamidine disulfide was unstable
to  heating and exhibited  considerable toxicity to dilute broth cultures of
                                     43

-------
                                                     TABLE 12
                               COMPARISON OF VARIOUS MODIFICATIONS OF HSM  TO  CONTROL
                                       MEDIUM WITH A 50-50 C02/H2 ATMOSPHERE
                                                           Days Incubated at  37°C
-p-
-P-
     Control
     HSM
I ISM with 0.1% ace-
  tate and 0.1%
  formate, without
  VFA

HSM with acetate
  and formate

HSM without
  glycerol

HSM without
  gelatin

HSM without
  starch
 6.5 x 107  x-§/
(3.0 x 107)

 8.8 x 107  xz
(0.8 x 107)  6

 1.3 x 108  z
(0.1 x 108)  3
                             7.8 x 107  x
                            (1.1 x 107)  3

                             6.1 x 107  x
                            (4.1 x 107)  6

                             6.4 x 107  x
                            (4.3 x 107)  6

                             4.3 x 107  y
                            (2.7 x 107)  6
      14

 1.3 x 108  x
(0.5 x 108)  12

 1.3 x 108  x
(0.1 x 108)  6

 1.7 x 108  x
(0.3 x 108)  3
 1.2 x 10e  x
(0.1 x 108) 3

 1.1 x 108  x
(0.3 x 108) 6
 1.1 x
(0.2 x
108  x
108)  6
 1.1.x 108  x
(0.5 x 108) 6
           3.9 x 108  x
          (2.6 x 108) 12

           4.0 x 108  x
          (2.4 x 108) 6

           8.6 x 108  y
          (1.7 x 108) 3
 3.1 x 108  x
(0.2 x 108)  3

 2.0 x 108  x
(0.9 x 108)  6

 2.1 x 108  x
(0.8 x 108)  6

 1.6 x 108  x
(0.6 x 108)  6
      i8

 7.5 x 108  x
(2.2 x 108)  12

 8.6 x 108  xy
(1.5 x 108)  6

 1.1 x 109  xy
(0.2 x 109)  3
                                                      7.5 x 10°  x
                                                     (1.7 x 108) 3

                                                      5.6 x 108  x
                                                     (5.2 x 108) 6

                                                      5.7 x 108  x
                                                     (4.4 x 108) 6

                                                      2.8 x 108  y
                                                     (1.4 x 108) 6
                                          35_

                                     9.1  x 108  x
                                    (2.1  x 108)  12

                                     1.1  x 109  x
                                    (0.1  x 109)  6

                                     1.2  x 109  x
                                    (0.1  x 109)  3
                    1.0  x  109  x
                   (0.1  x  109)  3

                    8.9  x  108  x
                   (4.2  x  108)  5

                    8.6  x  108  x
                   (4.3  x  108)  6

                    4.9  x  108  y
                   (1.3  x  108)  6
     a,b/  See Table  1.

-------
                                                TABLE  13

                              COMPARISON OF VARIOUS MODIFICATIONS OF HSM TO
                                CONTROL MEDIUM WITH A  100%  C02  ATMOSPHERE

                              	Days Incubated at 37°C
                              7.                14                21               28                 35_

Control                 6.4 x 107  xy-^   3.1 x  108  x      8.4 x 108  x     1.0 x 109  y       1.3  x 109  xy
                       (1.9 x 107) 12-    (2.2 x  108) 12    (2.5 x 108)  12   (0.2 x 109) 12     (0.2  x 109)  12

HSM                     1.0 x 108  x      5.0 x  108  x      1.3 x 109  z     1.6 x 109  x       1.7  x 109  z
                       (0.1 x 108) 6      (4.0 x  108) 6     (0.2 x 109)  6    (0.2 x 109) 6      (0.2  x 109)  6
                                                                                                           xz
HSM with 0.1%  acetate  1.1 x 108  xz     1.1 x  109  y      1.4  x 109  z     1.5 x 109  x      1.5 x  109
  and 0.1% formate,    (0.2 x 108) 3      (0.1 x  109) 3     (0.2  x 109)  3    (0.2 x 109) 3      (0.2 x  109)  3
  without VFA

HSM with acetate        1.2 x 108  z      1.5 x  108  x      1.2  x 109  xz    1.4 x 109  x      1.5 x  109   xz
  and formate          (0.1 x 108) 3      (0.1 x  108) 3     (0.0  x 109)  3    (0.1 x 109) 3      (0.1 x  109)  3

HSM without             5.9 x 107  x      3.5 x  108  x      1.0  x 109  xz    1.4 x 109  x      1.5 x  109   xz
  glycerol             (3.8 x 107) 6      (3.4 x  108) 3     (0.5  x 109)  5    (0.4 x 109) 5      (0.3 x  109)  5

HSM without             6.9 x 107  x      4.8 x  108  x      1.1  x 109  xz    1.4 x 109  x      1.6 x  109   z
  gelatin              (3.6 x 107) 6      (4.0 x  1Q8) 6     (0.4  x 109)  6    (0.2 x 109) 6      (0.2 x  109)  6

HSM without             3.9 x 107  y      1.3 x  108  x      7.0  x 108  x     9.9 x 108  y      1.1 x  109   y
  starch               (2.4 x 107) 6      (0.7 x  108) 6     (3.2  x 108)  6    (1.8 x 108) 6      (0.1 x  109)  5
a.b/   See  Table 1.

-------
sludge organisms incubated under anaerobic conditions.  Formamidine di-
sulfide dihydrochloride was ineffective at physiological pH's.  Of the re-
maining reductants, dithiothreitol (DTT) appeared most promising because of
its rapid reaction time, stability to oxygen, and the very low 0-R potentials
possible with this compound.  At concentrations of 0.025% to 0.05% (w/v),
the 0-R potential obtained in culture media was sufficiently low to easily
reduce benzyl viologen  (lower than -359 mv).   With the addition of 0.025%
cysteine and 0.025% to 0.05% DTT, methyl viologen was easily reduced (lower
than -446 mv).  DTT was evaluated for toxicity by comparison with normal
reductants (cysteine-sulfide) using the roll-tube technique.  Levels of 0.01,
0.025, and 0.05% DTT alone and 0.01,  0.025, and 0.05% DTT with 0.025% cysteine
were tested using the same medium with normal reductants as controls.

          The results of evaluation of dithiothreitol as a reductant are
shown in Tables 14 and 15.  The results indicate that this compound is too
toxic to be of value in increasing colony counts by replacement of either
or both Na2S-9H20-cysteine-HCl as reductants.  This is not to say that
organisms requiring a very low 0-R potential are not enriched to the exclu-
sion of other species; however, any differences simply are not expressed in
terms of increased total counts.  Further elucidation would be beyond the
scope of this work.

          Table 16 shows the effect of sodium formaldehyde sulfoxylate
(NaFS) on total anaerobic counts when NaFS is used as the final reductant
(as a replacement for Na2S-9H20).  This compound was tested because,  unlike
Na2S.9H20 solution, it is stable in air and can be added directly to the
medium at the time of preparation.  Basal HSM was used in these experiments
with basal HSM plus cysteine-sulfide as the control.  NaFS was tested under
both N2/C02 and H2/C02 atmospheres.

          The results indicate that at 0.1% w/v NaFS compares quite favor-
ably with sulfide, but does not yield higher total counts.  We, therefore,
chose to continue using Na2S-9H20 as the final reductant although NaFS might
serve as well.  A definite advantage in the use of NaFS (0.1%) would be the
elimination of addition of Na2S after media preparation.  When the point is
reached that a dehydrated synthetic medium is possible, then NaFS should be
considered further as a replacement for sulfide.
Effect of Sodium Deletion on Total Colony Counts

          Some workers have indicated that high sodium ion concentration
may be toxic to sludge anaerobes.  Although effects have been shown using
abnormally high concentrations of sodium, we were concerned that the levels
of NaHC03 used to buffer the medium, in addition to the salt added to the
mineral salts mixture, might lead to some reduction in total counts.  As
shown in Table 17, the levels of sodium used do not appear to be a problem
in normal medium buffered with NaHC03.

                                    46

-------
                                                TABLE 14
                               EVALUATION OF DITHIOTHREITOL (DTT) AS A
Normal medium with
  0.025% cysteine,
  0.025% Na2S'9H20

Normal medium with
  0.025% cysteine,
  0.01% DTT

Normal medium with
  0.025% cysteine,
  0.025% DTT

Normal medium with
  0.025% cysteine,
  0.05% DTT
REPLACEMENT FOR

7.
106 x^7
io6) &y

14
2.2 x IO7
(0.9 x IO7)
SODIUM SULFIDE
Days Incubated
21
x 4.6 x IO7
6 (1.2 x IO7)
at 37°C

x 5.3
6 (1.3

28
x IO7 xy
x IO7) 6

35
1.9 x 1(
(0.4 x 1(
 1.2 x 107  y
(0.7 x 107)  6
 5.7 x 106  x
(3.2 x 106)  6
 7.7 x 10b  xy
(3.7 x 106) 6
 3.4 x 107  y     5.7 x 107  xy    7.0 x 107  x
(1.0 x 107)  6    (1.9 x 107)  6    (1.7 x 107)  6
 2.1 x 107  x
(0.5 x 107)  6
 3.0 x 107  xy
(0.6 x 107)  6
 4.6 x 107   x
(0.5 x 107)  6
 6.7 x 107  y
(1.4 x 107)  6
 5.0 x 107  y
(0.7 x 107)  6
 7.0 x 107  x
(1.4 x 107)  6
                                    1.8 x 108  x
                                   (0.6 x 108) 6
 6.2 x 107  y
(1.5 x 107)  6
 7.8 x 107  y
(1.9 x 107) 6
a,b/  See Table  1.

-------
                                                      TABLE 15

                                EVALUATION  OF  DITHIOTHREITOL (DTT)  AS A REPLACEMENT
                                          FOR  SODIUM SULFIDE AND CYSTEINE
                                                             Days  Incubated at 37 °C
-F-
CD
Normal medium with
  0.025% cysteinc,
  0.0257o Na2S.9H20

Normal medium
  + 0.01% DTT,
  no cysteinc

Normal medium
  + 0.025% DTT
  no cysteine

Normal medium
  + 0.057o DTT,
  no cysteine
                                                     14
                             1.8 x  10
                                     7
                             (0.3 x  107)  6±
b/
 4.6 x 107  x
(0.4 x 107)  6
      2,1

 6.9 x 107  x
(0.5 x 107)  6
                             3.5 x  10'  y
                             (0.6 x  107) 6
                             3.2 x 10'  y
                            (0.6 x 107) 6
      6.7 x 107  y
     (0.8 x 107)  6
                  7.4 x 107  x
                 (0.7 x 107) 6
      I8

 9.2 x 107  x
(0.7 x 107)  6
                             2.0 x  107  x       6.0  x 107   y     7.3 x 107  x     8.6 x 107  x
                             (0.6 x  107) 6      (0.5  x 107)  6    (1.0 x 107) 6    (0.8 x 107) 6
      6.2  x 107  y     7.0 x 107  x     8.7 x  107  x
     (0.6  x 107)  6    (0.6 x 107) 6     (0.7 x  107) 6
                  8.5 x 10'  x
                 (1.4 x 107) 6
                                                                                                    35
                                                                                                             8
 2.9 x 10C  x
(0.2 x 108) 6
                                                           1.9 x 108  y
                                                          (0.4 x 108) 6
                                                      1.7 x 108  yz
                                                     (0.2 x 108) 6
                   9.1 x  107   z
                   (1.4 x  107)  6
      a,b/   See Table 1.

-------
                                               TABLE  16
                          EFFECT OF  SODIUM  FORMALDEHYDE  SULFOXYLATE (NaFS)
ON TOTAL ANAEROBIC COLONY COUNT

Gas Medium
H2/C02 Basal with cys-
50-50 teine-Na2S


With-
out <
NaoS
£•
Basal + 0.01%
NaFS
Basal + 0.02%
NaFS

Basal + 0.04%
—
N2/C02 Basal with cy-
50-50 teins-Na2S


With-
out '
Na2S


Basal + 0.01%
NaFS
Basal + 0.02%
NaFS

Basal + 0.04%
NaFS


4.4
(1.1
4.2
(1.1
2.5
(0.9

1.5
(0.7
5.7
(1.6
5.1
(1.9
3.8
(1.5

2.7
(0.5

7
x 107
x 107)
x 107
x 107)
x 107
x 107)

x 107
x 107)
x 107
x 108)
x 107
x 107)
x 107
x 107)

x 107
x 107)


Days
14
a/
f "^/
0 ~~"
xy
6
yz
6

z
6
x
6
x
6
xy
6

y
6
7.5 x
(3.7 x
6.4 x
(1.6 x
4.1 x
(1.7 x

2.9 x
(1.2 x
2.1 x
(1.5 x
1.8 x
(1.3 x
7.0 x
(3.1 x

4.5 x
(0.9 x
107 x
107) 6
10 7 xy
107) 6
107 yz
107) 6

107 z
107) 6
108 x
108) 6
108 xy
108) 6
10 7 xy
107) 7

107 y
107) 6
Incubation at 37
21
2.8 x
(0.9 x
1.6 x
(0.9 x
5.2 x
(1.7 x

4.0 x
(1.5 x
8.0 x
(1.8 x
6.9 x
(1.7 x
6.2 x
(2.1 x

5.0 x
(1.8 x
108 x
108) 6
108 xy
108) 6
107 y
107) 6

107 y
107) 6
108 x
108) 6
108 xy
108) 6
10 8 xy
108) 6

108 y
108) 5
°C

28
6.8 x
(0.7 x
4.1 x
(0.9 x
6.1 x
(2.0 x

5.1 x
(0.4 x
9.5 x
(1.6 x
8.6 x
(2.1 x
8.3 x
(1.6 x

8.5 x
(1.5 x
108 x
108) 6
108 x
108) 6
107 y
107) 6

107 y
107) 2
108 x
108) 6
108 x
108) 6
108 x
108) 6

108 x
108) 5

35
8.9 x 108 x
(0.8 x 108) 6
5.8 x 108 x
(0.6 x 108) 6
7.2 x 107 y
(2.2 x 107) 6

5.5 x 107 y
(0.9 x 107) 2
1.0 x 109 x
(0.1 x 109) 8
9.2 x 108 x
(2.3 x 108) 8
9.2 x 108 x
(1.6 x 108) 6

9.5 x 108 x
(1.6 x 108) 5
a,b/  See Table 1.

-------
                                                      TABLE 17
                                  EFFECT OF  SODIUM DELETION ON TOTAL COLONY COUNT
                                                                Days Incubated at 37°C
Ln
O
Buffer       Medium

NaHC03     IISM-1 without
            minerals

           HSM-1 with  low
            sodium

           HSM-1 with  nor-
            mal minerals

KHC03      HSM-1 without
            minerals

           HSM-1 with  low
            sodium

           HSM-1 with  nor-
            mal minerals
 2.6 x 107  -x.-
(1.1 x 10?) 6-7

 2.2 x 107  x
(0.8 x 107) 6

 2.2 x 107  x
(0.5 x 107) 6

 2.6 x 107  x
(0.9 x 107) 6

 2.6 x 107  x
(1.1 x 10 ) 6

 2.0 x 107  x
(1.2 x 107) 6
      14

 5.3 x 107  x
(1.2 x 107) 6

 5.8 x 107  x
(1.1 x 107) 6

 4.9 x 107  x
(0.6 x 107) 6

 4.6 x 107  x
(0.6 x 107) 6

 5.5 x 107  x
(1.8 x 107) 6

 5.4 x 107  x
(2.3 x 107) 6
                                                                      6.4 x 107  x
                                                                     (1.8 x 107) 6

                                                                      7.7 x 107  x
                                                                     (1.0 x 107) 6
                                                                      6.4 x 107
                                                                     (0.8 x 107)
                                                                      6.2 x 107  x
                                                                     (1.4 x 107) 6

                                                                      9.4 x 107  y
                                                                     (2.1 x 10 ) 5

                                                                      7.3 x 107  xy
                                                                     (1.9 x 107) 6
      2J3

 1.9 x 108  x
(1.3 x 108)  6

 3.8 x 108  y
(3.1 x 108)  5

 4.2 x 108  y
(2.4 x 108)  6

 1.5 x 108  x
(0.7 x 108)  6

 3.2 x 108  y
(2.2 x 108)  5

 2.2 x 108  xy
(1.1 x 108)  6
      3_5

 4.1 x 108  x
(2.1 x 108) 6

 7.2 x 108  y
(1.5 x 108) 5

 7.6 x 108  y
(1.8 x 108) 6

 5.8 x 108  x
(1.3 x 108) 6

 7.5 x 108  *
(3.2 x 108) 6

 5.9 x 108  x
(1.2 x 108) 6
     a.b/  See Table  1.

-------
The use of KHC03 buffer with low sodium indicated no change in counts as
compared to the same condition with NaHCC>3 buffer.  There may, however, be
some antagonism due to K+ if KHC03 is used to buffer the HSM when normal
minerals are used.
Effect of Cellobiose on Total Colony Counts Obtained with HSM

          Table 18 shows the effect of deletion of cellobiose and the ef-
fect of increased concentration of cellobiose when compared to the normal
level (0.05%) in HSM.  The results indicate that there is no significant
change in total colony counts (a) with or without cellobiose and (b) either
a C02 or C02/H2 atmosphere.  Cellobiose was therefore excluded from media
prepared in subsequent testing of HSM.

          The effect of deletion of the volatile fatty acid mixture from
HSM is shown in Table 19.  There appears to be no significant change in
colony count for any condition tested for either gaseous atmosphere.  For
these experiments, the complete HSM, as previously described, was used
(page 42).

          Tables 20 and 21 show the results of deletion of both yeast ex-
tract and trypticase from the complete HSM medium.  As indicated, the dele-
tion of yeast extract and trypticase had no significant effect on total
colony counts.  There was, however, a significant decrease in total colony
count, with an H2/C02 atmosphere, when trypticase was deleted and yeast
extract concentration was doubled.  This effect was not exhibited with a
100% C02 atmosphere.

          To this point it appeared that with the exception of starch, de-
letion of various ingredients from HSM had very little effect on the total
growth obtained.  It thus appears that a rather simple medium containing AELS
might yield higher counts for methane bacteria than a complex medium such
as complete HSM.  In addition to obtaining higher counts for the methanogenic
species, it also appeared that a simpler medium might support the growth of
nonmethanogens as well as complete HSM.  Table 22 shows the results of ex-
periments designed to test this assumption.

          Complete HSM plus 0.1% acetate was compared to basal HSM plus
acetate with an N2/C02 (50-50) atmosphere and an H2/C02 (50-50) atmosphere.
Basal medium consisted of the following:  AELS, 20%; soluble starch, 0.1%;
trypticase, 0.2%; yeast extract, 0.1%; resazurin, 0.001%; mineral solution,
10% each of salts A, B, and C; agar, 2%; pH adjusted to 6.8 with NaHC03.
Because of the importance indicated for acetate in sludge ,j20/ it was added
to both media at a level of 0.1%.  The results presented in Table 21 indi-
cate that basal HSM is as effective as complete HSM for total growth of
sludge organisms with a C02~containing atmosphere.

                                    51

-------
                                                  TABLE 18
                        EFFECT OF CELLOBIOSE  ON  TOTAL  COLONY  COUNT  OBTAINED  WITH  HSM
Atmos-
phere Medium
C02/H2 HSM
HSM without
cellobiose
HSM with
2x (0.1%)
cellobiose
C02 HSM
HSM without
cellobiose
HSM with
2x (0.1%)
cellobiose
Days Incubated at 37°C
7 14
3.2 x
(1.9 x
4.6 x
(2.0 x
3.0 x
(0.9 x
4.1 x
(3.5 x
3.1 x
(2.1 x
3.1 x
(2.5 x
107
107)
107
107)
107
107)
107
107)
lO7
107)
107
107)
f b /
6 —
x
6
x
6
x
6
x
6
x
6
6.1 x
(2.7 x
7.6 x
(3.6 x
7.6 x
(3.6 x
7.4 x
(4.4 x
7.8 x
(4.1 x
7.5 x
(3.8 x
107 x
107) 6
107 x
107) 6
107 x
107) 6
107 x
107) 6
107 x
107) 6
107 x
107) 6
21
7.8 x
(3.9 x
9.1 x
(4.3 x
8.9 x
(4.5 x
5.9 x
(0.9 x
5.9 x
(2.0 x
2.7 x
(0.5 x
107
107)
107
107)
107
107)
108
108)
108
108)
108
108)
x
6
x
6
x
6
x
6
x
6
y
6
28.
3.2 x 108 x
(2.5 x 108) 6
1.8 x
(0.9 x
1.9 x
(1.3 x
9.2 x
(1.3 x
8.4 x
(2.1 x
7.4 x
(2.1 x
108 x
108) 6
108 x
108) 6
108 x
108) 6
108 x
108) 6
108 x
108) 6
35
7.0 x
(2.4 x
6.7 x
(2.6 x
4.8 x
(1.7 x
9.5 x
(1.0 x
1.0 x
(0.2 x
8.6 x
(2.1 x
108 x
108) 6
108 x
108) 6
108 x
108) 6
109 x
108) 6
109 x
109) 6
108 x
108) 6
a.b/  See Table 1.

-------
                                                       TABLE  19
                             EFFECT OF VOLATILE  FATTY ACID MIXTURE (VFA) AND TRYPTICASE
Ul
u>
Atmos-
phere
C02/H2



C02




Medium
HSM
HSM without
VFA
HSM without
trypticase
HSM with 0.2%
trypticase
HSM
HSM without
VFA
HSM without
trypticase
HSM with 0.2%
trypticase-

7
4.1 x IO7 x-7
6.3 x IO7 y
(1.3 x 107) 6
4.4 x IO7 x
(1.4 x IO7) 6
2.7 x IO7 x
(1.9 x IO7) 6
4.4 x IO7 x
(0.8 x IO7) 6
3.2 x IO7 x
(1.5 x IO7) 6
3.0 x IO7 x
(1.4 x IO7) 6
4.0 x IO7 x
(1.4 x IO7) 6
Days
14
1.2 x IO8 x
(0.5 x IO8) 6
1.2 x IO8 x
(0.4 x IO8) 6
1.0 x IO8 x
(0.3 x IO8) 6
1.5 x IO8 x
(0.3 x IO8) 6
1.2 x IO8 x
(0.3 x IO8) 6
1.2 x IO8 x
(0.3 x IO8) 6
9.9 x IO7 x
(4.6 x IO7) 6
9.3 x IO7 x
(3.3 x IO7) 6
Incubated at
21
1.4 x IO8 x
(0.5 x IO8) 6
1.4 x IO8 x
(0.4 x IO8) 6
1.2 x IO8 x
(0.2 x IO8) 6
1.6 x IO8 x
(0.4 x IO8) 6
3.6 x IO8 x
(2.7 x IO8) 5
5.5 x IO8 x
(4.1 x IO8) 6
2.8 x IO8 x
(1.8 x IO8) 5
4.1 x IO8 x
(2.8 x IO8) 6
37°C
28
2.4 x IO8 xy
(0.5 x IO8) 6
3.8 x IO8 y
(2.1 x IO8) 6
1.7 x IO8 x
(0.1 x IO8) 6
2.0 x IO8 x
(0.3 x IO8) 6
7.4 x IO8 x
(2.4 x IO8) 5
9.4 x IO8 x
(3.4 x IO8) 6
6.5 x IO8 x
(2.5 x IO8) 4
6.3 x IO8 x
(4.6 x IO8 6

35
3.7 x IO8 x
(1.6 x IO8) 6
5.0 x IO8 x
(3.1 x IO8) 6
3.0 x IO8 x
(1.1 x IO8) 6
3.2 x IO8 x
(1.5 x IO8) 6
1.2 x IO9 x
(0.1 x IO9) 5
1.2 x IO9 x
(0.1 x IO9) 6
9.9 x IO8 x
(2.3 x IO8) 5
8.2 x IO8 x
(5.3 x IO8) 5
      a.b/  See Table 1.

-------
                                               TABLE 20
                   EFFECT OF YEAST EXTRACT AND  TRYPTICASE  DELETION  ON TOTAL COLONY
COUNTS
WITH HSM USING A 50-50 H2/C02 ATMOSPHERE
Days Incubation at 37°C
Gas Medium
H2/C02 HSM
HSM without
yeast ex-
tract
trypticase
3
(0
4
(0


.4 x
.5 x
.0 x
.7 x


7
107 K-
107) 6^
107 x
107) 5


14
4.7 x
CO. 5 x
6.2 x
(0.8 x


107 x
107) 6
107 y
107) 5


21
28
5.3 x 107 x
(0.6 x 10?) 6
6.6 x 10
(0.9 x 10


7 x
7) 5


2
(1
1
(1


.6 x
.9 x
.9 x
.2 x


108 x
108) 6
108 x
108) 6


35
6.1 x
(1.9 x
4.8 x
(1.2 x


           HSM with 4 x
             trypticase
             without yeast
             extract

           HSM with 2 x
             yeast ex-
             tract with-
             out trypticase
 3.7 x 107  x
(0.8 x 10?)  6
 3.4 x 10'   x
(0.6 x 107)  6
 4.6 x 107  x
(0.6 x 107)  6
 4.9 x 107  x
(0.6 x 107)  6
 5.1 x 107  x
(0.6 x 107)  6
 5.5 x 107  x
(1.0 x 107)  6
 2.1 x
            x
(1.6 x 108)  6
 1.5 x 10a  x
(1.0 x 108)  6
 5.7 x 108  xy
(1.1 x 108)  6
 4.3 x 108  y
(0.8 x 108)  6
a,b/  See Table 1.

-------
                                                     TABLE 21
                         EFFECT  OF YEAST EXTRACT AND TRYPTICASE DELETION ON TOTAL COLONY
      Gas
      C02
Ul
Ln
HSM
COUNTS WITH HSM USING A 100% C02 ATMOSPHERE
Days Incubation at 37
Medium
2.4
(1.0
without yeast 1.8
:ract and (0.9
7
x
x
X
X
7 a/
IO7 x-
107) 6k/
IO7 x
IO7) 6
14
4.7 x
(1.3 x
3.9 x
(1.6 x
IO7 x
IO7) 6
IO7 x
IO7) 6
5.3
(1.1
5.0
(1.3
21
x IO8
x 108)
x IO8
x IO8)
°C


28
x
6
x
6
8.7 x
(1.7 x
8.5 x
(1.5 x
IO8
ID8)
108
IO8)
x
6
x
6


35
9.5 x
(2.0 x
9.4 x
(1.6 x
108 x
108) 6
IO8 x
io8) 6
  trypticase

HSM with 4 x
  trypticase
  without yeast
  extract
 2.6  x IO7  x
(0.7  x IO7)  6
 5.3 x IO7  x
(0.9 x IO7)  6
 3.7  x 108  x     7.2 x IO8  x
(0.9  x IO8)  6    (1.3 x IO8)  6
              HSM with  2  x yeast  2.8 x IO7  x
                extract without  (0.5 x IO7)  6
                trypticase
                                       4.9 x IO7  x      5.0 x IO8  x     8.4 x IO8  x
                                      (1.4 x IO7)  6     (1.7 x IO8) 6    (3.5 x IO8) 6
 7.7 x IO8  x
(2.1 x 108) 6
                                                                       9.4 x  IO8   x
                                                                       (2.0 x  108)  6
      a,b/   See  Table  1.

-------
                                                   TABLE 22
                                   COMPARISON OF COMPLETE HSM WITH BASAL HSM
                                                         Days Incubation at 37°C

N2/C02
50-50

50-50


HSM 4- 0.1%
acetate
Basal HSM +
0.1% acetate
HSM + 0.1%
acetate
Basal HSM +
0. 1% acetate

5.3 x
(0.7 x
1.4 x
(0.1 x
5.6 x
(0.8 x
1.3 x
(0.1. x
1_
107 x^7
107) 6^7
108 y
108) 6
107 x
107) 6
108 y
108) 6

9.4 x
(1.3 x
1.7 x
(0.1 x
9.0 x
(1.2 x
1.5 x
(0.1 x
14
107 x
107) 6
108 y
108) 6
107 x
107) 6
108 y
108) 6

3.2 x
(1.9 x
5.0 x
(0.8 x
9.6 x
(1.2 x
1.5 x
(0.1 x
2!
108 x
108) 6
108 y
108) 6
107 x
107) 6
108 y
108) 6

6.6
(1.9
7.1
(1.3
1.0
(0.1
3.6
(0.3
?JL
x 108 x
x 108) 6
x 108 x
x 108) 6
x 108 x
x 108) 6
x 108 y
x 108) 6

7.5
(1.9
7.5
(1.2
1.4
(0.2
5.3
(0.7
35.
x 108 x
x 108) 6
x 108 x
x 108) 6
x 108 x
x 108) 6
x 108 y
x 108) 6
a.b/  See Table 1.

-------
Further, a simplified medium appears obligatory for maximum total colony
count in an H2/C02 atmosphere.

          To further test the total growth obtained with basal medium as
well as the effect of gaseous atmosphere on total colony count, the follow-
ing experiments were carried out.  Four modifications of basal HSM were
compared with each of six gaseous atmospheres.  Media tested consisted of:
basal HSM, basal HSM +0.1% acetate, basal HSM +0.1% formate, and basal
HSM +0.1% each of acetate and formate.  The gaseous atmospheres compared
with each of these media were:  N2/C02, 50-50;  N£, 100%; C02, 100%; H2/C02,
50-50; H2/C02, 80-20; and N2/C02/H2, 60-30-10.  Triplicate tubes of 10-5,
10~6 and 10~7 dilutions were inoculated for each combination of gas and
medium.  Duplicate experiments were run using an arbitrary control of N2/C02-
At the end of 35 days' incubation (termination of experiment), all tubes
were analyzed for methane.  Methane was determined by gas chromatography
of high dilution tubes containing significant colonies (20-200) .  Any tube
containing significant amounts of methane was considered to contain at
least one methanogenic colony.  The highest dilution tube containing methane
was therefore used for total estimated methanogens (Example:  if the 10~6
dilution was positive for methane and the 10~7 dilution was negative, then
the total number of methanogens per milliliter of original sample would
range from 1 x 10^ to 9 x 106 and the methanogens per milliliter would thus
be reported as
          The results of these experiments are depicted in Tables 23 to
26.  Of all combinations tested, highest total colony counts were obtained
with basal medium plus 0.1% acetate in an N2/C02 (50-50) atmosphere and with
basal medium plus 0.1% formate in an N2/C02/H2 (60-30-10) atmosphere.  Of
the two combinations, however, the latter would appear to be the combination
of choice for maximum total numbers of both methanogens and nonmethanogens,
whereas the former most likely yields the maximum number of nonmethanogens
with reduced numbers of methanogens .  For growth of the maximum numbers of
methanogens, basal medium plus formate with an H2/C02 (50-50) atmosphere
appears best.  It is worthy of note that H2 appears to be quite toxic to
nonmethanogens as reflected by the reduction in total colony and increase in
estimated methanogenic counts as H2 concentration increases.

          If H2 concentration is lowered to 10% and formate is added, a
distinct increase in total counts is obtained with a concomitant increase
in total methanogens.  It would thus appear that addition of formate spares
the H2 requirement for the methanogens and thereby reduces the effect of H2
toxicity (less H2 required) .
                                     57

-------
                                                   TABLE 23
                      EFFECT OF GASEOUS ATMOSPHERE ON TOTAL COLONY COUNT  WITH  BASAL
Days Incubation
Gas
N2/C02
50-50
N2
co2
H2/C02
50-50
H2 /C02
80-20
N2/C02/H2
60-30-10
4.6 x
(1.0 x
2.5 x
(1.5 x
2.9 x
(0.6 x
5.0 x
(0.6 x
3.9 x
(0.6 x
4.5 x
(1.0 x
7
107 x^
107) 30-/
107 y
107) 6
107 y
107) 6
107 x
107) 6
107 x
107) 6
107 x
107) 5
2.4
(0.8
3.8
(1.3
1.1
(0.2
7.2
(0.6
6.3
(0.6
1.4
(0.6
14
x 108 x
x 108) 29
x 108 y
x 108) 6
X 108 2
x 108) 6
X 107 2
x 107) 6
X 107 2
x 107) 6
x 108 xz
x 108) 5
6.8 x
(1.6 x
6.8 x
(1.0 x
6.7 x
(1.0 x
1.4 x
(0.2 x
1.7 x
(0.8 x
5.5 x
(0.7 x
21
108 x
108) 29
108 x
108) 6
108 x
108) 6
108 y
108) 6
108 y
108) 6
108 x
108) 6
7.9
(1.5
8.6
(2.1
8.2
(1.8
6.5
(1.5
5.3
(1.1
7.1
(0.5
at 37°C
23
x 108 x
x 108) 30
x 108 x
x 108) 6
x 108 x
x 108) 6
x 108 y
x 108) 6
x 108 y
x 108) 6
x 108 xy
x 108) 6

9.0
(1.6
9.0
(2.2
8.6
(1.9
8.7
(1.5
7.4
(0.7
9.0
(0.9

35
x 108 x
x 108) 30
x 108 x
x 108) 6
x 108 x
x 10S) 6
x 108 x
x 108) 6
X 108 y
x 108) 6
x 108 x
x 108) 6
Methanoger
> 106
< 105
> 105
> 106
> 106
> 105
al    Basal medium:  AELS 20%; sol. starch 0.1%; trypticase 0.2%; yeast extract 0.1%; resazurin 0.0001%;
                       minerals A,B,C, 30% (10% each); and agar 2%.
b,c/  Same as a,b/ Table 1.
d/    Estimated methanogens per milliliter based upon highest dilution tube which yields significant methane by
        gas chromatographic analysis.

-------
Ui
       Gas
                                                       TABLE 24

                   EFFECT OF  GASEOUS ATMOSPHERE  ON TOTAL COLONY COUNT WITH BASAL HSM  + 0.1% ACETATE

                                                            Days Incubation at 37°C
                                     14                21                28                35          Methanogens/ml£/
N2/C02        4.5 x 107  x-     2.6 x 108  x      7.1 x 108  x      8.4 x 108  x      9.5 x 108  x         > 105
80-50        (0.7 x 107) 30-'  (0.7 x 108) 30    (1.7 x 108) 28    (1.4 x 108) 29    (1.5 x 108) 29

N2            2.4 x 10?  y      4.0 x 108  y      6.9 x 108  x      8.5 x 108  x      8.9 x 108  xy        > 105
             (0.7 x 107) 6     (1.1 x 108) 6     (1.5 x 108  5     (1.6 x 108) 6     (1.4 x 108) 6

C02           2.8 x 107  y      9.3 x 107  z      5.8 x 108  x      7.1 x 108  x      7.2 x 108  yz        > 1Q5
             (1.0 x 107) 6     (4.6 x 107) 6     (0.8 x 108) 5     (1.2 x 108) 5     (102 x 108) 5

H2/C02        4.8 x 107  x      7.3 x 107  z      1.1 x 108  y      3.9 x 108  y      6.1 x 108  z         > 107
50-50        (0.6 x 107) 6     (1.6 x 107) 6     (0.2 x 108) 6     (0.9 x 108) 6     (1.2 x 108) 6

H2/C02        4.7 x 107  x      8.2 x 107  z      1.4 x 108  y      3.1 x 108  y      5.3 x 108  z         > 107
80-20        (0.4 x 107) 6     (1.0 x 107) 6     (0.3 x 108) 6     (1.5 x 108) 6     (0.7 x 108) 6

N2/C02/H2     5.3 x 107  x      1.3 x 108  z      5.0 x 108  x      6.3 x 108  x      8.1 x 108  xy        > 105
60-30-10     (0.5 x 107) 6     (0.3 x 108) 6     (0.3 x 108) 6     (1.1 x 108) 6     (0.8 x 108) 6
    a,b/   See Table 1.
    £/     See d./,  Table 23.

-------
    Gas

 N2/C02
 50-50

 N2
 H2/C02
 50-50

 H2 /C02
 80-20

 N2/C02/H2
 60-30-10
                                                    TABLE 25

                 EFFECT OF  GASEOUS  ATMOSPHERE  ON  TOTAL COLONY COUNT WITH BASAL HSM + 0.1% FORMATE

                     	Days Incubation at 37°C	
 4.4 x 107  x-§/
(1.2 x 107) 30^

 2.6 x 107  y
(0.8 x 107) 6

 2.2 x 107  y
(1.1 x 107) 5

 4.3 x 107  x
(0.3 x 107) 6

 4.0 x 107  x
(0.6 x 107) 6

 5.0 x 107  x
(0.6 x 107) 6
      14.

 2.7 x 108  x
(1.3x  108)  29

 2.8 x 108  x
(0.5 x 108)  5

 1.1 x 108  y
(0.2 x 108)  5

 6.4 x 107  y
(1.1 x 107)  6

 6.5 x 107  y
(1.3 x 107)  6

 1.2 x 108  y
(0.5 x 108)  6
      2_L

 6.8 x 108   x
(1.2 x 108)  29

 6.2 x 108   x
(1.1 x 108)  6

 6.7 x 108   x
(1.7 x 108)  6

 1.1 x 108   y
(0.2 x 108)  6

 1.5 x 108   y
(0.6 x 108)  6

 6.4 x 108   x
(1.9 x 108)  6
      28

 8.1  x 108  x
(1.1  x 108)  29

 6.2  x 108  xy
(1.1  x 108)  6

 6.9  x 108  x
(1.9  x 108)  6

 4.9  x 108  yz
(0.8  x 108)  6

 3.4  x 108  z
(2.4  x 108)  6

 8.0  x 108  x
(2.4  x 108)  6
      35.

 9.3  x 108  x
(1.3  x 108)  29

 7.3  x 108  xy
(1.1  x 108)  6

 7.6  x 108  y
(2.0  x 108)  6

 8.2  x 108  xy
(2.3  x 108)  6

 6.3  x 108  y
(1.2  x 108)  6

 9.5  x 108  x
(2.0  x 108)  6
Me th anogen s/ml~

    > 106


    < 105


    > 105


    > 107


    > 107


    > 107
a.b/  See Table 1.
£/    See d/, Table 23.

-------
                                                   TABLE 26
                               EFFECT OF GASEOUS ATMOSPHERE ON TOTAL COLONY COUNT
   Gas
50-50

N2


C02
H2/C02
50-50

H2/C02
80-20

N2/C02/H2
60-30-10

4.4
(1.1

7.
x 107 ^
x 107) 30-'
WITH BASAL HSM
Days
li
2.3 x 108 x
(0.8 x 108) 28
+ 0.1% ACETATE + 0.1% FORMATE
Incubation at 37°C
21
6.3 x 108 x 7.1
(3.9 x 108) 27 (1.8

28.
x 108 x
x 108) 28

35.
8.9 x 1(
(1.7 x K
 2.9 x 107  y
(1.6 x 107) 6

 2.0 x 107  y
(1.4 x 107) 6

 4.8 x 107  x
(0.5 x 107) 6

 4.1 x 107  x
(0.8 x 107) 6

 4.2 x 107  x
(0.7 x 107) 5
 3.3 x 108  x
(0.6 x 108)  5

 3.8 x 107  y
(2.5 x 107)  6

 7.1 x 107  y
(0.8 x 107)  6

 6.9 x 107  y
(0.8 x 107)  6

 7.2 s 107  y
(1.8 x 107)  5
 7.4 x 108  x
(0.9 x 108)  6

 5.8 x 108  x
(0.9 x 108)  6

 9.7 x 107  y
(1.4 x 107)  6

 1.3 x 108  y
(0.2 x 108)  6

 5.8 x 108  x
(1.0 x 108)  6
 9.1  x 108  x
(1.5  x 108)  6

 7.4  x 108  x
(1.1  x 108)  6

 3.4  x 108  y
(0.7  x 108)  6

 4.0  x 108  y
(2.1  x 108)  6

 7.2  x 108  x
(1.7  x 108)  6
 9.4 x 108  x
(1.4 x 108)  6

 8.5 x 108  xz
(0.7 x 108)  6

 6.5 x 108  yz
(1.3 x 108)  6

 5.8 x 108  y
(1.3 x 108)  6

 8.1 x 108  xz
(1.4 x 108)  6
Methanogens/ml—

    > 106


    > 105


    > 105


    > 107


    > 106


    > 106
                                                                                                                     —
 a,b/  See  Table  1.
 c/    See  d/,  Table 23.

-------
          On the basis of the foregoing observations, the following con-
clusions were reached concerning media and gaseous atmosphere.  For isolation
of maximum total numbers of mixed methanogens and nonmethanogens, basal
medium plus 0.1% formate with an N2/C02/H2 (60-30-10) atmosphere is recom-
mended.  For maximum total count of methanogenic species with reduced numbers
of nonmethanogens, basal medium plus 0.1% formate in an H2/C02 (50-50)
atmosphere is recommended.  An N2/C02 (50-50) atmosphere and basal medium
plus 0.1% acetate is recommended for maximum total count of nonmethanogens
with reduced numbers of methanogens.
Variation in Total Anaerobic Counts in Digesters at Various Performance Levels

          As a practical application of the media and techniques developed
during the course of this work, four digesters at various performance levels
were sampled to determine if digester condition would be reflected by dif-
ferences in total anaerobic counts.  These digesters were selected on the
basis of current and past performance records.

          Indian Creek:  This digester has an excellent performance record.
Methane production is good, pH has not fluctuated significantly in the past
2 to 3 years of constant operation, and volatile acids have remained within
the normal range.  It is moderately fed with municipal sludge.

          4800 Nail:  This digester is fed only on weekends when the sludge
incinerators at this plant are not in operation.  The digester receives only
municipal wastes and laboratory records are not kept on performance.  An
appreciable amount of methane is produced, and no upsets have occurred in
the past 2 to 3 years.

          Kansas City, Kansas:  This plant was built to receive only indus-
trial wastes (packing plant and soap manufacturing wastes).  In nearly 2
years' operation, normal digestion has not been achieved.  The system has
been constantly plagued by excessive foaming, high volatile acids, low pH
and low methane production.

          Olathe, Kansas:  This digester is quite old and has a consistently
poor performance record due primarily to overloading.  Although pH is not low
(volatile acids are not determined), digestion is incomplete and very little
methane is produced.

          Each of these digesters was sampled twice, and six roll tubes
from each sample were inoculated from the 10~2, 10~3, 10~4, 10~5, 10~6 and
10-7 dilutions.  Two media were inoculated; basal HSM plus 0.1% acetate
with an N2/C02 atmosphere and basal HSM plus formate with an H2/C02 atmos-
phere.  Aerobic pour plates were also prepared from each sample, using basal
                                    62

-------
HSM plus 0.1% acetate,  to obtain aerobic and/or  facultative counts.  These
plates were  incubated at 37°C, and  colony  counts were made at  24-hr intervals
for 4 days.  Roll  tubes were  counted  after 4,  7, 14, 21, 28 and 35 days'
incubation at 37°C.  Methane  was determined at each counting interval.  Low
dilution uncountable tubes were used  so that high dilution tubes could be
maintained for  subsequent anaerobic counts.  At  the termination of the ex-
periment, high  dilution tubes became  available and were used for the final
methanogen estimates.

          Tables 27 and 28 show the resultant  total anaerobic  counts for
N2/C02 and H2/C02  atmospheres, respectively.  With the exception of the
samples taken from the  Olathe digester, there would appear to  be no signifi-
cant difference in total anaerobic  counts.  In this case, a significant dif-
ference is apparent from the  initial  to the final count.  These counts were
initially the highest  (significantly  so),  but  showed very little increase
in total count  over the 35-day counting period.  This is partially clarified
by the aerobic  plate counts  (Table  29).  The facultative anaerobic count
(aerobic plates) from the Olathe sludge was much higher than the other three
digesters; hence,  the initially high  total anaerobic count.

          Tables 30 and 31 show the numbers of methanogenic colonies recorded
at each counting interval.  These results  are  surprising in that high num-
bers of methanogenic bacteria are indicated for  the digesters  known to pro-
duce very little methane (Kansas City and  Olathe).  This, obviously, is not
an expression of actual digester condition.  It must be emphasized that the
AELS used to supplement all media1 came from sludge taken from  a digester
(Indian Creek sewage disposal plant)  which was normal with respect to methane
production.  It is then quite possible that either growth of methanogens and
methanogenesis or methanogenesis, per  se, is inhibited in the unbalanced or
upset digesters.   Methanogenesis is obviously  restored when the organisms
from the upset  digesters are  placed in a more  favorable environment, such
as that present in the  culture medium supplemented with AELS from a digester
functioning  normally.   It appears that the methanogens are present in equal
or greater numbers in the upset digesters  as in  "normal" digesters but that
methanogenesis  is  being inhibited.  As will be subsequently explained, we
feel that the lack of methanogenesis  in the upset digester is  due to the
lack of a sludge factor; required for methanogenesis, rather than due to
the inhibition  of  growth of the methanogenic bacteria.  The presence of this
factor in the AELS used to prepare  all media would thus allow  methanogenesis
to occur in  the roll tube but not in  the digester.

          Further  studies need to be  performed in which media  containing
AELS are compared  to media supplemented with clarified sludge  supernatant
taken from the  digester in question at the time  of sampling and compared
for total anaerobic counts, aerobic and/or facultative counts  and methano-
genesis.  The experiments which could now  be performed with the information
and techniques  provided in the current study would yield new information
pertaining to digester function, and  could quite possibly lead to better
means for digester control.
                                    63

-------
                                                TABLE 27

                       COMPARISON OF TOTAL COLONY COUNTS OF SLUDGE  SAMPLES  FRCM
                          FOUR DIFFERENT DIGESTERS WITH AN N2/C02 ATMOSPHERE

                      	Days Incubation at  37°C	
                      4               1_               14              21                2_8              35

Indian Creek      3.1 x 107  x-7  8.8 x 107  x    2.0 x 108  x    8.6 x 108  x     1.0 x 109  x    1.2 x 109   x
  basal + 0.1%   (1.7 x 107)  12-' (1.5 x 107) 12   (0.4 x 108) 12   (2.8 x 108)  12   (0.2 x 109) 12  (0.3 x 109)  12
  acetate

4800 Nail         2.0 x 107  x    3.9 x 107  z    2.2 x 108  x    7.9 x 108  xy    1.0 x 109  x    1.1 x 109   x
  basal + 0.1%   (1.8 x 107)  12  (2.6 x 107) 12   (1.7 x 108) 12   (2.7 x 108)  12   (0.2 x 109) 12  (0.3 x 109)  12
  acetate

Kansas City,      1.8 x 107  x    4.3 x 107  z    1.2 x 108  x    9.3 x 108  x     1.1 x 109  x    1.2 x 109   x
  Kansas, basal   (1.6 x 107)  12  (2.2 x 107) 12   (0.4 x 109) 12   (1.7 x 108)  12   (0.2 x 109) 12  (0.3 x 109)  12
  + 0. 1%
  acetate

Olathe, Kansas,   3.3 x 108  y    3.7 x 108  y    4.8 x 108  y    5.7 x 108  y     6.4 x 108  y    6.6 x 108   y
  basal + 0.1%   (1.8 x 108)  12  (2.1 x 108) 12   (2.7 x 108) 12   (3.5 x 108)  12   (3.3 x 108) 12  (3.2 x 108)  12
  acetate
a.b/  See Table 1.

-------
                                                     TABLE 28
                             COMPARISON OF TOTAL COLONY COUNTS  OF  SLUDGE  SAMPLES -FROM
                                FOUR DIFFERENT DIGESTERS WITH AN H2/C02 ATMOSPHERE
Ul
                                                         Days  Incubation at 37°C
                                                             14
                                                                       21
Indian Creek     3.8  x  107   *-   7.3 x 10?  x    9.1 x 107  x     2.2  x  108  x
  basal + 0.17c   (1.2  x  107)  12-'(1.9 x 107) 12  (1.8 x 107) 12   (1.2  x  108)  12
  formate

4800 Nail        2.0  x  107   x    3.1 x 107  x    4.1 x 107  x     1.4  x  108  x
  basal + 0.1%   (1.7  x  107)  12  (1.8 x 107) 12  (2.3 x 107) 12   (0.4  x  108)  12
  formate

Kansas City,     1.7  x  107   x    3.7 x 107  x    1.0 x 108  x     4.0  x  108  y
  Kansas,basal   (1.5  x  107)  12  (2.5 x 107) 12  (0.2 x 108) 11   (0.9  x  108)  12
  + 0.1%
  formate

Olathe, Kansas,  1.7  x  108   y    2.0 x 108  y    2.4 x 108  y     3.5  x  108  y
  basal + 0.1%   (0.2  x  10s)  12  (0.3 x 108) 12  (0.2 x 108) 12   (0.8  x  108)  12
  formate
28
35
                                                                                          5.0 x 108  x    7.5 x  108   x
                                                                                         (1.5 x 108) 12   (0.9 x  108)  12
                                                                                          5.6 x 108  x    7.2 x  10b   x
                                                                                         (1.7 x 108) 12   (2.7 x  108)  12
                                                                                          1.0 x 109  y    1.1 x  109   y
                                                                                         (0.1 x 109) 12   (0.2 x  109)  12
                                                                                          3.8 x 108
                                                                                                  8^
                                                                                                     x
                                                                                         (0.9 x 10°) 12
            4.0 x  108   z
           (1.0 x  108)  11
      a,b/   See  Table 1.

-------
                                TABLE 29

              RESULTS OF AEROBIC PLATE COUNTS FOR ANAEROBIC
                   SLUDGE FROM FOUR DIFFERENT DIGESTERS

                             	Days Incubation  at  37°C
                             I            1             1             4

Indian Creek             1.5 x 106*/  3.3 x 106         -         6.8  x 106

4800 Nail                7.5 x 104    9.1 x 105                   1.2  x 106

Kansas City, Kansas      2.2 x 105    3.3 x 105     4.4 x  105     4.8  x 105

Olathe, Kansas           6.2 x 106    1.3 x 107     2.6 x  107     4.1  x 107
al  Total count per milliliter.


                                TABLE 30

      TOTAL ESTIMATED METHANOGENS BASED UPON METHANE DETERMINATIONS
                ON ROLL TUBES WITH AN N2/C02 ATMOSPHERE

                           	Days Incubation at 37°C	
                           4       1_        ]A       21        28_        35

Indian Creek             > lO2^/ > 102    > 103    > 103    >  104    >  105

4800 Nail                > 102   > 102    > 103    > 103    >  104    >  105

Kansas City, Kansas      > 102   > 103    > 104    > 10^    >  105    >  10$

Olathe, Kansas           < 102   > 102    > 103    > 103    >  104    >  104
a/  Methanogens per milliliter, see d/, Table 23.
                                    66

-------
                                TABLE  31

      TOTAL ESTIMATED METHANOGENS BASED UPON METHANE DETERMINATIONS
                ON ROLL TUBES WITH AN  H2/C02 ATMOSPHERE

                           	Days  Incubation at  37°C	
                           4       ]_        14        2A        28        15

Indian Creek             > 102a/ > 1Q3    > 104    > 1Q5    > 1Q5    > 106

4800 Nail                > 102   > 1Q3    > 104    > 104    > 104    > 105

Kansas City, Kansas      > 102   > 103    > 104    > 10$    > 106    > 106

Olathe, Kansas           > 102   > 103    > 104    > 10^    > 105
aj  Methanogens per milliliter,  see  d./,  Table 23.
                                      67

-------
    PHASE IV - DETERMINATION OF GROWTH  SUBSTANCE(S)  IN SEWAGE SLUDGE
              THAT ENHANCES GROWTH OF SLUDGE MICROORGANISMS
          An unknown factor was found in rumen fluid which is essential to
the growth of Methanobacterium ruminantium and possibly other species of
methanogenic bacteria.  An extraction procedure and a microbiological assay
were developed for the factor, the presence of which was determined in
both rumen fluid and sludge supernatant.  Subsequent studies on the factor,
not extractable with ether, showed it to be of relatively low molecular
weight (Syphadex G25, dialyzable) and stable to autoclaving at acid or at
neutral pH, but roughly 50% of the activity was lost during autoclaving in
alkali.22/

          The factor has not been identified nor has a metabolic role been
assigned to it.  The requirement has never been satisfactorily replaced by
known vitamins, amino acids, purines, pyrimidines, etc.

          Since the factor is present in sludge supernatant and has been
shown to be necessary to the growth of some methanogenic species, it is
quite probable that the factor plays an important role in sludge digestion.

          Experiments were carried out during this phase of work to deter-
mine the levels of factor which occur in sewage sludge, and to determine
whether factor concentration might vary as a function of digester perfor-
mance.  A description of the assay procedure and the method of factor
extraction follows.
Methods and Media for Sludge Factor Assay

          Stock cultures of M. ruminantium were maintained on slants in a
H2/C02 (50-50) gas atmosphere.  Cultures were transferred with a platinum-
indium loop by stab into the base of the slant.
                                     69

-------
          Slant Medium

          RF (rumen fluid)                         30.0 %
          Mineral No.  li/                          3.75%
          Mineral No.  2i/                          3.75%
          Resazurin solution                       0.1 %
          No formate                               0.2 %
          Trypticase                               0.2 %
          Agar                                     1.5 %
          Distilled H20 to volume
          Cysteine-HCl                             0.025%
\J Mineral No. 1:
2_l Mineral No . 2 :




K2HP04
KH2P04
(NH4)2S04
NaCl
MgS04-7H20
CaCl-2H20
0.6%
0.6%
0.6%
1.2%
0.245%
0.159%
6.0 g; i
6 g "
6 g
12 g
2.45 g
1.59 g.
                                                         1 liter.
                                                   1 liter
          All ingredients were placed in a 500-ml round-bottom flask and
boiled under C02 until the resazurin was reduced (colorless) .   The medium
was cooled in a water bath to 47 to 48°C and NaHC03 to yield  a concentra-
tion of 0.5% was added.  Na2S-9H20 reducing solution (0.025%  final concen-
tration) was added to the flask just prior to tubing.   The medium was tubed
in 8-ml amounts as described in Phase II.

          The tubes were then autoclaved as previously described (15 Ib for
15 min) and cooled in a slant position.

          Salts were dissolved in order given in 700 to 800 ml distilled
water and made to 1 liter.  The solution was distributed (100-120 ml) into
labeled, screwcap dilution bottles (Pyrex) and autoclaved 15  psi for 15
min.

          Contamination was checked microscopically and by inoculating car-
bohydrate agar slants which were maintained under a C02 (100%) gas atmos-
phere.  Growth on the carbohydrate agar slants indicated contamination.
Ingredients are listed below.
                                    70

-------
          Carbohydrate Agar Slants

          CC-2 gas phase used to check sterility of assay organism.

          Resazurin (0.1% solution)                0.1%
          Whole rumen fluid                       30.0%
          Mineral No. 1                            3.75%
          Mineral No. 2                            3.75%
          Glucose                                  0.05%
          Cellobiose                               0.05%
          Soluble starch                           0.005%
          Agar                                     1.5%
          Trypticase                               0.5%
          Distilled H20 to volume
          Cysteine-HCl                             0.025%
          NaHCOs                                   0.9% added after medium
                                                     cooled to 47-48°C
          Na2S solution (0.25% final volume) added just prior to tubing
            (0.04 ml/5 ml medium)

          The same basal medium was used for assay medium and for medium
in which cells were grown for inoculum.

          Basal Medium (for 200 ml; 5x concentration)

          (NH4)2S04                                0.5 g
          Na formate                               2.0 g
          Yeast extract                            2.0 g
          Trypticase                               2.0 g
          VFA solution                             3.1 g
          FeS04-7H20                               0.002 g
          Resazurin                                1.0 ml
          Mineral No. 3                            5.0 ml
          Hemin (1 mg/ml)                           1.5 ml
          Distilled water                         97.0 ml

          Ingredients were placed in a 400-ml beaker,  on a magnetic stirrer
and the pH was adjusted to 6.5 with 3N NaOH.  The medium was then brought
up to 200 ml with distilled H20.  One hundred milliliter amounts were trans-
ferred to Pyrex screwcap bottles; autoclaved 15 psi/15 min, and stored in
the refrigerator.  For use, 10 ml was placed in a 100-ml round-bottom flask
and the indicated volume of material to be assayed was added.  Cysteine-HCl
(0.025%) was added, the solution was reduced, and the total volume brought
to 50 ml.
                                     71

-------
          NaHCC>3 (0-5/0 was added and the medium was tubed in 5-ml amounts
anaerobically and autoclaved.  Na2S (0.04 ml/5 ml) reducing solution was
added 2 to 24 hr prior to inoculation.  Twenty percent rumen fluid was
used in the medium in which the inoculum was grown and maintained.

          Mineral No. 3

                                     1 Liter           Final Min Med

          KH2P04                      18.0 g            6.62 x 1CT3
          NaCl                        18.0 g            1.54 x 10~2
          CaCl2 (CaCl2-2H20)           0.4 g            1.80 x 10~4
          MgCl2-CH20                   0.4 g            9.85 x 10~5
          MnCl2-4H20                   0.2 g            5.05 x 10~5
          CaCl2-CH20                   0.02 g           4.20 x 10~5

          Salts were dissolved in 700-800 ml distilled water and made up to
a liter.  Mineral solution was distributed in Pyrex bottles and autoclaved
15 psi/15 min (0.0066 M PC>4 final) (5% v/v added to media).

          VFA (Volatile Fatty Acid) Mixture

                                                       Molar Concentration*

          Acetic acid (glacial)      17 ml               2.9 x 10~2 M
          Propionic acid              6 ml               8.0 x 10~3
          Butyric acid                4 ml               4.3 x 10~3
          Isobutyric acid             1 ml               1.1 x 10~3
          v-Valeric acid              1 ml                 9 x 10~4
          Isovaleric acid             1 ml                 9 x 10~4
          DL-a-methylbutyric acid     1 ml                 9 x 10~^
   Final molarity when added to assay basal medium at level of 3.1 ml/200 ml
     medium and subsequently diluted 5x for final volume.
Assay Procedure

          Cells for inoculum were grown in basal medium plus 20% rumen
fluid.  Cells from an actively-growing culture (OD 0.3 to 0.4) were centri-
fuged (4,000 rpm, 2,000 g's for 20 min) and the pellet was washed in ADS
under  aseptic and anaerobic conditions.  The cells were resuspended in
ADS for inoculation of assay media.
                                    72

-------
          ADS (Anaerobic Dilution Solution)

          Mineral No. 3                           5.000%
          Resazurin                               0.1  %
          Distilled water
          NaHC03                                  5.000%
          Cysteine-HCl                            0.025%
               reducing solution added before use (0.04 ml/5 ml medium)
          The O.D. of the cell suspension was measured at 600 my.   Dupli-
cate or triplicate tubes were inoculated for each level of each fraction
to be assayed.  Cultures were incubated at 39°C on a shaker.  After the
first day, the tubes were flushed twice daily with H2/C02 (50-50)  gas.
Growth was estimated as O.D. at 600 my.  Readings were taken prior to
flushing the tubes .  Maximum growth was reached in approximately 5 days .

          A unit of activity of the factor is defined as the amount in
50 ml of medium required to allow growth equal to O.D. of 0.3.  Net O.D.
was determined by subtracting the amount of growth (O.D.) in the control
medium with no factor addition from the O.D. of the experimental medium
(containing various concentrations of factor) .
Extraction of Factor

          For experiments in which crude concentrates of the factor were
assayed, the following procedure was carried out as outlined in Figure 5.

          Fresh gauze-filtered sewage sludge was autoclaved, cooled, and
centrifuged or lyophilized and extracted with hot water.  The supernatant
or extract was treated, batchwise, with Dowex 50, hydrogen form, to lower
the pH to about 2.5.  The resin and acid precipitate was separated by
vacuum filtration and the supernatant was passed through a column of Dowex
50 to remove any remaining positively-charged ions remaining.  The eluate
was extracted with ethylacetate to remove lipids, organic acids, and other
relatively nonpolar materials.  The aqueous phase, still quite acid, was
adsorbed on Norit.  The Norit was recovered by filtration through a pad of
hyflosupercel and the cake was washed twice with hot water.  The factor
was eluted from Norit with 0.1 M ethanolic NH40H and immediately concen-
trated in a vacuum evaporator to remove NH3 and  concentrate the factor to
a small volume.  Aliquots of this concentrate were diluted for assay.

          The effect of sludge treatment on factor recovery was studied
using the following samples:  aqueous extract of lyophilized sludge; raw
sludge, filtered and sterilized; raw sludge preincubated with 0.4% yeast
extract, sterilized and filtered; factor extracted from hot 80% alcohol
extract of sterile lyophilized sludge; factor extracted from AELS.

                                     73

-------
                RAW OR PREINCUBATED SLUDGE
                        AUTOCLAVE
                      LYOPHILIZATION
                   HOT WATER EXTRACTION
                            V
                        DOWEX 50-HH
RESLN + ABSORBED
MATERIAL (DISCARD)
 AQUEOUS ELUATE
                                     ETHYLACETATE EXTRACTION
             v
     ETHYLACETATE PHASE
         (DISCARD)
ADSORB ON NORITE
                                         HOT WATER WASH
                                   ELUTE WITH 0.1 M ETHANOLIC
                                             NH4OH


                                                  V
                                        VACUUM EVAPORATE
   Figure 5 - Scheme for Purification and Concentration
                       of Sewage Sludge Factor
                             74

-------
          The results shown in Table 32 indicate that the factor levels
found in sludge are comparable to those found in rumen fluid  (0.8-1.0
unit/ml by Bryant).  The factor concentration increased twofold when the
sludge was preincubated at 37°C for 40 hr under nitrogen.  This increase
is much less than  the increase found in rumen fluid  (up to eightfold).  The
0.3 unit/ml recovery from lyophilized sludge, which had been refluxed with
80% EtOH for 2 hr  prior to extraction, was rather disappointing.  It had
been anticipated that preextraction would not only result in releasing more
factor, but also the preparation would contain less debris and be more
readily extracted.  The 80% EtOH did not increase factor yield signifi-
cantly and, in addition, contained much more ethylacetate extractable
material which interfered with the extraction procedure.

          The factor recovery from AELS (0.2 unit/ml) appeared to be low.
To determine at which point the factor was lost and whether the yield could
be increased by treatment of the activated charcoal, the following experi-
ment was performed.  A 2-liter batch of freshly prepared AELS was taken
through the extraction procedure.  At the Norit stage, the extract was
split into five samples and equal amounts were treated with Norit and Darco
which had been treated as follows:  untreated Norit; acid washed Norit;
NH^-EtOH, HC1 washed Norit; acid washed Darco; and NH^-EtOH,  HC1 washed
Darco.  Samples from all phases of the extraction procedure (except the
wash water) were assayed using the M-l strain of Methanobacterium rumi-
nantium.  The results shown in Table 33 indicate the losses at each step
in the extraction  procedure.  Treatment of either Norit or Darco had little
effect on total recovery; however, significantly more factor was recovered
with Darco than with Norit.  The final recoveries are higher than those
shown in Table 32, even with Norit.  This is probably due to substitution
of a medium-porosity, sintered-glass funnel for the pad of hyflosupercel
formerly used to recover the charcoal adsorbed factor.  It is quite pos-
sible that some factor was adsorbed to the hyflosupercel pad and lost when
it was discarded.

          Although time would not permit, the status of the factor extrac-
tion procedure is  such that large batches could be extracted and used to
determine the actual significance of the factor in sludge digestion.  With
further purification and mass spectrometry, gas chromatography methodology
 which exists at MRI, we are also in a position to further characterize
 and identify the  factor.

          As the final experiment carried out in this work, factor levels
were determined in the sludge supernatants prepared from the digester
samples discussed  in Phase III.  The results of these comparisons, shown
in Table 34, appear to be highly significant in terms of digester per-
formance.  As will be noted, the sludge supernatants from "normal" digesters
(Indian Creek and  4800 Nail) are much higher than those from unbalanced or
upset digesters (Kansas City Pollution Control and Olathe, Kansas).

                                    75

-------
                                 TABLE 32

               EFFECT OF SLUDGE TREATMENT ON FACTOR RECOVERY

          Material                                             Units/ml

Aqueous extract of lyophilized sludge (AELS)                     0.8
Raw sludge sterilized and filtered                               1.0
Raw sludge pre-incubated with 0.4% yeast extract,
  sterilized and filtered                                        2.0
Factor extracted from hot 80% alcohol extract of
  sterile lyophilized sludge                                     0.3
Factor extracted from AELS                                       0.2


                                 TABLE 33

                     FACTOR RECOVERY AT VARIOUS STEPS
                         IN EXTRACTION PROCEDURE

                                                                 Percent
Treatment                                         Units/ml       Recovery

Aqueous extract of lyophilized sludge (AELS)        1.03
Deionized AELS                                      0.86            83
Aqueous layer following ethylacetate
  extraction                                        0.78            76
Untreated Norit                                     0.40            39
Acid washed Norit                                   0.42            41
NtfJ-EtOH, HC1 washed Norit                          0.43            42
Acid washed Darco                                   0.54            52
NH^-EtOH, HC1 washed Darco                          0.54            52
                                    76

-------
                                 TABLE 34

                   COMPARISON OF FACTOR LEVELS IN SLUDGE
                   SUPERNATANTS FROM FOUR SEWAGE PLANTS

                                                         Supernatant Level
Sewage Plant                                                 (units/ml)

Indian Creek                                                     0.81

4800 Nail                                                        0.68

Kansas City Pollution Control                                    0.34

Olathe, Kansas                                                   0.17
Although this work could not be followed up, there are definite indications
that the low factor levels in these digesters could explain why little
methane is formed in the digesters while high levels are formed in cul-
tures grown in media containing AELS from a digester which is functioning
normally (Indian Creek sewage plant).  This work should definitely be pur-
sued further to determine whether factor addition to these sludges would
aid in reversing the unbalanced or stuck condition.
                                    77

-------
                             ACKNOWLEDGEMENTS
          This final report was prepared by Dr. William Spangler-  who also
served as project leader.  Other Midwest Research Institute personnel who
contributed significantly to this work were Joe Rose, John Unrau,  Dee Bjarnson,
Douglas Weatherman, Christine Guenther, Jack Breen, and Dr. William Garner.
Dr. Walter Langston, Head, Bacteriology Section, MRI, acted in a supervisory
capacity throughout the work.

          Drs. M. P. Bryant, Department of Microbiology and Dairy  Science,
University of Illinois, and Dr. P. H. Smith, Department of Microbiology,
University of Florida, served as consultants for the project.

          The support of the project by the Environmental Protection Agency
and project reviews by Mr. C. W. Chambers, the Contract Project Officer,
are gratefully acknowledged.
                                    79

-------
                                REFERENCES
 1.  McKinney, R. E., Langley, H. E., and Tomlinson, H. D., "Survival of
     Salmonella typhosa During Anaerobic Digestion," Sewage and Industrial
     Wastes, 30. pp 1469-1477 (1958).

 2.  Sawyer, C. N., Howard, F. S., and Pershe, E. R., "Scientific Basis for
     Liming of Digesters," Sewage and Industrial Wastes. 26, pp 935-944
     1954).

 3.  Barker, H. A., "Biological Formation of Methane," Industrial and Engi-
     neering Chemistry, 48, No. 9, pp 1438-1443 (1956).

 4.  Smith, P. H., and Hungate, R. E., "Isolation and Characterization of
     Methanobacterium ruminantium N. Sp." Journal of Bacteriology, 75. pp
     713-718 (1958).

 5.  Paynter, M. J. B., and Hungate, R. E., "Characterization of Methanobacterium
     mobilis N. Sp., Isolated from the Bovine Rumen," Journal of Bacteriology,
     95_, pp 1943-1951 (1968).

 6.  Mylroie, R. L., and Hungate, R. E., "Experiments on the Methane Bacteria
     in Sludge," Canadian Journal of Experimental Medicine. 32, pp 295-311
     (1920).

 7.  Barker, H. A., "Studies Upon the Methane Fermentation.  IV.  The Isola-
     tion and Culture of Methanobacterium omelainskii," Antonie van Leeuwenhoek,
     6., pp 201-220 (1940).

 8.  Bryant, M. P., Wolin, E. A., Wolin, M. J., and Wolfe,  R. S.,  "Methano-
     bacterium omelianskii, a Symbiotic Association of Two Species of Bacteria,"
     Archives fur Microbiologie. 59, pp 20-31 (1967).

 9.  Smith, P. H., "Pure Culture Studies of Methanogenic Bacteria," Proceedings
     of the .20th Industrial Wastes Conference, 20, pp 583-588, Purdue University,
     Lafayette, Indiana (May 1965).

10.  Hungate, R. E., "The Anaerobic Mesophilic Cellolytic Bacteria," Bacteri-
     oligical Reviews. 14, pp 1-49 (1950).

11.  Smith, P. H., University of Florida, Gainsville, Personal Communication
     (1969).

12.  Caldwell, D. R., and Bryant, M. P., "Medium Without Rumen Fluid for Non-
     Selective Enumeration and Isolation of Rumen Bacteria," Applied Micro-
     biology, 14. pp 794-801 (1966).

                                     81

-------
13.  Mah, R.  A., and Sussman, C., "Microbiology of Anaerobic Sludge Fermen-
     tation.   I.  Enumeration of the Nonmethanogenic Anaerobic Bacteria,"
     Applied Microbiology. 16. No.  2, pp 358-361 (1968).

14.  Kirsh, E. F.,  "Studies on the Enumeration and Isolation of Obligate
     Anaerobic Bacteria from Digesting Sewage Sludge," Proceedings of the
     25th General Meeting of the Society for Industrial Microbiology, 10,
     pp 170-177, Columbus, Ohio (1969).

15.  Spears,  R. W.,  and Freter, R.,  "Improved Isolation of Anaerobic Bacteria
     from the Mouse  Cecum by Maintaining Continuous Strict Anaerobiosis,"
     Proceedings of  the Society for  Experimental Biology and Medicine, 124,
     pp 903-909 (1967).

16.  Drasar,  B. S.,  "Cultivation of  Anaerobic Intestinal Bacteria, " Journal
     of Pathology and Bacteriology,  94,  pp 417-427 (1967).

17.  Rosebury, T.,  and Reynolds, J.  B.,  "Continuous Anaerobiosis for Culti-
     vation of Spirochetes," Proceedings of the Society for Experimental
     Biology and Medicine, 117, pp 813-815 (1964).

18.  Socransky, S.  S., MacDonald, J. B., and Sawyer, S., "The Cultivation
     of Treponema microdentium as Surface Colonies," Archives of Oral Biology,
     1, pp 171-172 (1959).

19.  Aranki,  A., Syed, S. A., Kennedy, E. B., and Freter,  R. , "Isolation of
     Anaerobic Bacteria from Human Gingiva and Mouse Cecum by Means of a
     Simplified Glove Box Procedure," Applied Microbiology, 17, pp 568-576
     (1969).

20.  Smith, P. H.,  and Mah, R. A.,  "Kinetics of Acetate Metabolism During
     Sludge Digestion," Applied Microbiology, 14. No.  3, 368-371 (1966).

21.  Dirasian, A.,  "Electrode Potentials—Significance in Biological Systems.
     Part 3.   Sludge Digestion," Water and Sewage Works. 115. No. 11, pp
     505-511 (1968).

22.  Bryant,  M. P.,  and Nalbandov,  0., "An Unknown Factor Required for Growth
     by Methanobacterium ruminantium," Bacteriological Proceedings. 90 (1966).
                                   82

-------
                                 GLOSSARY
Anaerobic - The absence of free-molecular oxygen.

Anaerobic Dilution Solution - An oxygen-free, reduced, buffered, salt solu-
tion used to make serial dilutions under anaerobic conditions.

Anaerobic Sludge Digestion - The anaerobic process, generally considered to
be biphasic, during which digestable complex organic substrates are converted
to short-chained fatty acids and subsequently to carbon dioxide and methane.

Aseptic Technique - Generally referred to in bacteriological nomenclature as
manipulations involving the transfer of organisms, preparation of dilutions,
etc., during which no extraneous or unwanted organisms are introduced; i.e.,
sterile technique.

Biomass - The total amount or number of living organisms in a particular
area or volume.

Bunsen Valve - As used in this report, refers to a short piece of butyl
rubber tubing closed to the atmosphere but containing a very small slit which
allows the relief of gas pressure while preventing the entry of air.

Clarified Sludge Supernatant (CSS) - The opalescent aqueous phase of anaer-
obic sewage sludge prepared by filtration to remove large solids followed by
high-speed centrifugation to remove suspended solids.

Inhibitor - An agent which slows down or interferes with the growth of
bacteria.

Methanogenie - Refers to the generation of methane by bacteria.

Microbiological Assay - The use of a microorganism with an obligatory re-
quirement for a material to measure the concentration of that material.
The growth rate or some measurable physiological activity of the test organ-
ism must be directly proportional to the concentration of that material
throughout the limiting range of concentration.

Nonmethanogen - For this report, refers to obligate anaerobic species of
anaerobic sludge which do not form methane.

Obligate Anaerobe - A bacterial species to which free-molecular oxygen is
lethal.
                                     83

-------
Plate Count - A method of enumerating viable bacteria in a sample by count-
ing the total number of colonies produced on an agar surface when a suitable
dilution of material is spread on the surface of the agar medium or mixed
with the medium before solidification and allowed to incubate until visible
colonies appear.

Reducing Agents - Materials added to the culture medium which reduce the
oxidation-reduction potential to a point suitable for growth of obligate
anaerobes.

Roll Tube - Refers to the process of rolling a tube of melted agar medium
in a horizontal position until the agar solidifies uniformly on the walls
of the tube.

Saprophytic - Living or growing on dead or decaying organic matter.

Sludge Factor - An unknown factor or factors present in sewage sludge and
rumen fluid, necessary for the growth of certain methanogenic species of
bacteria.

"Stuck" Digester - A digester unbalanced to the point that methane produc-
tion ceases.  This condition is generally irreversible and requires shut-
down drainage, and reseeding of the digester.

Symbiosis - A condition occurring when two or  more organisms growing to-
gether, produce a reaction or end-product that none of the individual species
can produce when grown in pure culture.

Total Colony Count - The total number of colonies counted on a plate or in
a roll tube multiplied by the reciprocal of the dilution of that plate or
roll tube.

Viability - The ability to grow and multiply in a given environment.
                                     84

-------
  SELECTED WATER                       i. Report No.
  RESOURCES ABSTRACTS
  INPUT TRANSACTION FORM
               2.        3.  Accession No.
                        w
  4.  Title                                                            5.  Report Date
  DEVELOPMENT OF  TECHNIQUES FOR ESTIMATING THE BACTERIAL
  POPULATION OF SEWAGE SLUDGE                                      .'    .   .  _
 _____	 £.  Performing Organization
  7.  Author(s)                                                           Report No.
  William J. Spangler
   9. Organization                                                       17070DRP
  Midwest Research  Institute, Kansas City,  Missouri
  Biological Sciences Division
                        10.  Project No.
  12. Sponsoring Organization  Environmental  Protection Agency
  15. Supplementary Notes
                        11.  Contract I Grant No.
                         14-12-569
                        13.  Type of Report and
                           Period Covered
  16. -distractThis  research program was  initiated to develop  practical methods for evalua-
 tion of the biomass in anaerobic  sewage sludge and to determine if predictions could  be
 made concerning digester performance.   Sampling and handling methods were improved  and
 standardized  to give maximum anaerobic counts.  A simplified technique for growing
 obligate anaerobes that can be safely  performed by technicians with minimum training  in
 bacteriology  was developed.  Anaerobic media were improved  to yield as high or higher
 counts of methanogenic bacteria than heretofore reported.   A simple freeze-dry technique
 was developed for preparation of  consistent batches of  sludge supernatant used in media
 as a supplement for growth of obligate sludge anaerobes.  The possible relationship
 between concentration of a growth factor required by Methanohacterium ruminantium  (used
 to evaluate potency of growth factor extracted) and digester efficiency could have
 important practical implications.  Limited data obtained  indicated that growth factor
 concentrations  were much higher in "normal" digesters than  in unbalanced or "upset"
 digesters.  Practical applications of  the methods developed can have considerable  impact
 upon future research and development in anaerobic sludge  digestion and could lead  to
 improvements  in our ability to predict impending digester failure and control of
 digester performance.
  17a. Descriptors
 * Anaerobic Digestion, * Methane Bacteria, *  Anaerobic  Bacteria, Sewage Sludge,
 Sludge Digestion, Waste Treatment,  Water Pollution  Control,  Biomass
  17b. Identifiers
 * Anaerobic  Techniques, * Roll-Tube Procedures, * Anaerobic Sludge Extracts,  *  Sludge
 Factor Assay,  Sludge Factors,  Obligate Anaerobes, Anaerobic Culture Media,  Digester
 Failure,  Sludge Supernatant


  17c. CO WRR Field & Group Q5D
  18. Availability             19. Security Class.
                             (Report)

                          20. Security Class.
                             (Page)
21. No. of    Send To:
   Pages

   Prifa     WATER RESOURCES SCIENTIFIC INFORMATION CENTER
   fftCC     , , ™ ,_ C-Q AQTH^tTM-I- *—1C" TLJC" 1MTC"^I*™»D
            U.S. DEPARTMENT OF THE INTERIOR
            WASHINGTON, D. C. 20240
  Abstractor William J. Spangler	| Institution Midwest Research Institute
WRSIC 102 (REV. JUNE 1971)           * u- s- GOVERNMENT PRINTING OFFICE : 1972—484-435/234                      G P o 91 3.26 ^

-------