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WfraJJ-.J WATER POLLUTION CONTROL RESEARCH SERIES •
^wflMiiigy
16050DXN07/70
DEVELOPMENT OF
IMMOBILIZED ENZYME SYSTEMS
FOR ENHANCEMENT OF BIOLOGICAL
WASTE TREATMENT PROCESSES
ENVIRONMENTAL PROTECTION AGENCY • WATER QUALITY OFFICE
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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 , develop-
ment, and demonstration activities in the Water Quality
Office, Environmental Protection Agency, through inhouse
research and grants and contracts with Federal, State,
and local agencies, research institutions, and industrial
organizations.
Inquiries pertaining to Water Pollution Control Research
Reports should be directed to the Head, Project Reports
System, Office of Research and Development, Water Quality
Office, Environmental Protection Agency, Room 1108,
Washington, D. C. 20242.
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DEVELOPMENT OF IMMOBILIZED ENZYME SYSTEMS FOR
ENHANCEMENT OF BIOLOGICAL WASTE TREATMENT PROCESSES
by
Grumman Aerospace Corporation
Bethpage, New York 11714
For the
WATER QUALITY OFIICE
ENVIRONMENTAL PROTECTION AGENCY
Program #16050 DXN
Contract #14-12-562
July, 1970
For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402 - Price 70 cents
Stock Number 5501-0113
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EPA Review Notice
This report has been reviewed by the Water
Quality Office, EPA, 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 recommendation for
use.
11
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ABSTRACT
In existing biological wastewater treatment systems, the degradation and uti-
lization of wastewater nutrients are controlled by complex enzyme systems.
The objectives of this study were the biochemical fractionation, concentration,
and evaluation of a lyophilized immobilized enzyme preparation for its effec-
tiveness to enhance the biological degradation of domestic sewage.
A method was developed to biochemically fractionate the microbial enzymes
from activated sludge, to concentrate and characterize their activity, and to
immobilize this activity by entrapment in a polyacrylamide gel. The enzyme-
gel preparation was tested for its effect on the biological degradation of a bench-
scale batch activated sludge process.
The conclusions were: (1) the soluble enzymatic components of activated sludge
can be readily separated from the particulate components of the cell; (2) the
soluble system thereby obtained can be fractionated in such a manner as to
maintain the activity of the catabolic enzyme systems of interest while remov-
ing nonessential components; (3) the enzymatically active preparation can
then be immobilized within the matrix of a polyacrylamide gel; (4) the gel can
maintain the activity during storage, repeated washings, and repeated exposure
to substrate; and (5) the limited bench-scale activated sludge experiments failed
to produce meaningful results due to possible incomplete polymerization of the
polyacrylamide gel and an improper activated sludge culture.
The recommendations were: (1) utilize inorganic carriers such as porous
glass or nickel oxide on nickel screening to insolubilize the enzymes from
activated sludge organisms; (2) perform further work to substantiate whether
or not insolubilized enzymes extracted from activated sludge organisms will
enhance the biological degradation process; and (3) conduct further studies on
a synthetic substrate representative of the constituents found in the waste flows
being investigated.
This report was submitted in fulfillment of Program # 16050 DXN and Contract
# 14-12-562 under the sponsorship of the Federal Water Quality Administration,
Department of the Interior.
iii
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CONTENTS
Section Page
I CONCLUSIONS 1
II RECOMMENDATIONS 3
III INTRODUCTION 5
Need for Study 5
Objectives of Study 5
Approach Rationale 6
IV LITERATURE REVIEW 7
Introduction 7
Enzyme Additives . 8
V EXPERIMENTAL PHASE 17
Biochemical Investigations 17
Materials and Methods 17
Experimental Laboratory Studies 18
Preliminary Bench-Scale Activated Sludge Investigations ... 34
Construction and Operation of Apparatus 34
Substrate 34
Activated Sludge 34
Laboratory Procedure and Analyses 34
Results 36
VI ACKNOWLEDGMENTS 45
VII REFERENCES 47
VIE APPENDIX 49
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FIGURES
Page
1 Leominster Plant 20
2 Determination of Optimal Source of Bacterial Sludge 21
3 Preliminary Fractionation: Membrane Disruption Techniques . . 22
4 Preliminary Fractionation: Storage Techniques 22
5 The Effect of Correcting for Non-Specific Interference Upon
Detection of Esterase Activity Immobilized in Polyacrylamide
Gels 32
6 Series 2 Experimental Set-Up 35
7 Series 1 Experimental Results,
Volatile Suspended Solids vs Aeration Time 39
8 Series 1 Experimental Results,
Soluble COD vs Aeration Time 40
9 Series 2 Experimental Results,
Soluble COD vs Aeration Time 42
10 Series 2 Experimental Results,
Mixed Liquor COD vs Aeration Time 43
11 Series 2 Experimental Results,
Volatile Suspended Solids vs Aeration Time 44
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TABLES
No. Page
I Fractionation Techniques ....... 24
II Values Obtained with Spot Monitoring During
Fractionation of Sonicate 25
III Comparative Biochemistry of the Sonicate and the
Mixed Supernatant Fraction. . . . 26
IV Variations in Co-polymer Concentration in
Polyacrylamide Gel Preparations 28
V Immobilization of Resuspended Lyophilized Sonicate 28
VI Determination of Concentration of Mixed Supernatant
Derivative Yielding Optimal Retention of Immobilized
Enzyme Activity . . 30
VII Comparative Summary of Enzyme Activity Retained After
Immobilization in a Polyacrylamide Gel 33
Vin Series 1 Experimental Results 38
IX Series 2 Experimental Results 41
IX
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SECTION I
CONCLUSIONS
The overall objective of the research described in this report was to prepare,
characterize, and evaluate an immobilized enzyme preparation from the ma-
terial known as the microbial floe or zoogleal film, demonstrated to be the
material responsible for the biological removal and degradation of sewage in
the activated sludge process.
Specifically, the research concluded that:
• The soluble enzymatic components of activated sludge can be readily
separated from the particulate components of the cell.
• The soluble system thereby obtained can be fractionated to maintain
the activity of the catabolic enzyme systems of interest while remov-
ing nonessential components.
• The enzymatically active preparation can then be immobilized within
the matrix of a polyacrylamide gel.
• The gel will maintain activity during storage, repeated washings, and
repeated exposure to substrate.
• The limited bench-scale activated sludge experiments failed to pro-
duce meaningful results due to possible incomplete polymerization
of the polyacrylamide gel and an improper activated sludge culture.
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SECTION II
EE COMMENDATIONS
Inert inorganic carriers such as porous glass or nickel oxide on nickel screen-
ing should be utilized to insolubilize the enzymes extracted from activated
sludge organisms.
In view of the tentative nature of the conclusion of the limited benchscale
activated sludge experiments, it can only be recommended that further work
be carried out to substantiate whether or not insolubilized enzymes extracted
from activated sludge organisms will enhance the biological degradation pro-
cess.
The studies should be carried out on a synthetic substrate representative of
the constituents typically found in the waste flows being studied. It should be
reproducible, and there should be a relatively easy method of separating the
substrate from the cell material.
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SECTION III
INTRODUCTION
NEED FOR STUDY
The proper treatment and disposal of sewage for small communities having up
to 500 people and for individual isolated houses has been for many years one
of the most difficult problems in the sanitary engineering field. For these
isolated houses, septic tanks have long been the standard answer, but the main-
tenance required and the results produced have left much to be desired.
For the small communities, oxidation ponds — either alone or in combina-
tion with an Imhoff tank or septic tank — have failed to provide either the
necessary protection from a public health standpoint or the aesthetic require-
ments now demanded by society. Maintenance costs and poor treatment pro-
cess results, resulting in odor and pollution problems, have left the small
community in a poor position to improve its sanitation standards.
The small "package plant" sewage disposal treatment has provided a partial
solution to these problems. These treatment devices, such as activated sludge
units and trickling filters, essentially are modifications of naturally occurring
processes. The same biological phenomenon of microbial metabolism under-
lies the removal of waste materials. The major difference in the "package
plant" and the naturally occurring purification process is that the man-made
devices are designed for relatively short detention times in order to make
them economically feasible. However, increasing the degree of treatment as
measured by the percentage removal of waste matter requires longer detention
times and increases the cost markedly. It follows that the increasing need for
more complete treatment of wastewaters, which is projected for the future,
will stimulate a search for cost/effectiveness in the design of treatment systems.
Design of conventional treatment facilities is based on an impressive accumula-
tion of many years of practical experience. Within the context of these corre-
lations many process design improvements have been developed and cost reduc-
tions achieved; ft seems unlikely, however, that any major design improvement
or cost reduction for conventional treatment facilities will be forthcoming from
this approach in the future.
An alternate approach is to develop a more fundamental understanding of the
underlying principles of biological treatment in the hope of arriving at new
system design concepts or uncovering new ideas.
OBJECTIVES OF STUDY
The objectives of the research can be stated as follows:
• To prepare, characterize, and evaluate an immobilized enzyme pre-
paration from the material known as the microbial floe or zoogleal
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film, demonstrated to be the material responsible for the biological
removal and degradation of wastewater effluents in the activated
sludge process.
• To conduct limited studies of a bench-scale activated sludge system
treating domestic sewage in order to determine the effects of the
immobilized enzymes.
APPROACH RATIONALE
A review of the literature revealed no pertinent reference to the isolation,
characterization, purification, and immobilization of enzymes from micro-
organisms in the microbial floe of activated sludge. Many references were
found describing the addition of crude enzyme preparations, usually from
yeast, to activated sludge.
These studies concluded that enzymes added in this manner were not effective
in enhancing the efficiency of the activated sludge process. Two possible ex-
planations for these failures are apparent: (1) the enzymes were crude pre-
parations and were not isolated from the microorganisms involved in the ac-
tivated sludge process, and (2) no attempt was made to protect the enzymes
from instant destruction by the viable microorganisms in the activated sludge
to which they were added.
Using this rationale, the investigation first developed a method to biochemically
fractionate the microbial enzymes from activated sludge, to concentrate and
characterize their activity and to immobilize this activity by entrapment in
polyacrylamide gel. The enzyme-gel preparations were then tested for their
enhancement effects on a bench-scale activated sludge process.
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SECTION IV
LITERATURE REVIEW
INTRODUCTION
Domestic wastewater is composed of suspended solids, colloidal matter, and
soluble organic substances. The settleable portions of the wastes are frequent-
ly removed prior to the aeration reactor in the activated sludge process. Other
impurities are to be reduced or eliminated through various mechanisms during
the detention of the wastewater in the aeration tank. To more fully understand
these mechanisms, the ecology of activated sludge will be described.
The principal and most numerous biological "workmen" in the aeration tank
are saprophytic microorganisms, including autotrophic bacteria and gelatinous
masses constructed by bacteria with filamentous organisms such as Z ooglea
ramigera. Bacteria are responsible for the stabilization of the organic matter
through biochemical processes. The suspended solids are removed by floccu-
lation and enmeshment in the biological floe. The colloidal matter is absorbed
on the biological surfaces. The protozoa present in the wastewater assist in
removal of dispersed microorganisms to produce a clarified effluent in the
final sedimentation tank.
The hypothetic biosorption mechanism to remove soluble organic constituents
present in the wastes has been recently disproved by Krishnan and Gaudy (Refl),
Siddiqi (Ref 2), and many other investigators. It was found that soluble organic
substances would be removed from biological systems primarily under enzym-
atic reactions. The study of organic transport into cell interiors has been re-
ported by Siddiqi, et al (Ref 2). It was concluded that intracellular enzymes,
permeases, and extracellular enzymes were the three systems to operate the
whole biochemical reactions between organic substances and enzymes.
The intracellular enzymes comprise (a) hydrolases and (b) synthesis and re-
spiration enzymes. Permeases facilitate the transport of exogenous substance
into the cells. The extracellular hydrolases are secreted by the cells to hy-
drolyze long polymeric substrates into smaller units.
In the conventional activated sludge process, the sludge organisms are in con-
tact with the waste for a period of three to six hours. This period is sufficient-
ly long to permit the organisms to synthesize inducible enzymes which may be
required for the utilization of particular waste, but which are not present ini-
tially in sufficient quantity. On the other hand, in the contact stabilization pro-
cess, the sludge is in contact with the waste in the contact unit for only a short
period of time, usually 0.5 to 1.0 hour. The sludge is then separated from the
waste and returned to the stabilization unit in which the assimilated organic
compounds are used for biosynthesis and maintenance of microbial life under
the reaction of intracellular enzymes with the assimilated substance. There-
fore, satisfactory operation of the contact-stabilization activated sludge process
can be achieved when the sludge organisms process a complete set of preformed
enzyme systems at the time they are introduced to the wastewater.
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In the extended aeration process, rate of substrate removal per unit weight of
activated sludge decreases as the aeration time prolongs. The loss of activity
is due to inactivation of synthesis and respiration enzyme systems. The achieve-
ment of a high degree of purification is attributed to high concentrations of the
mixed liquor suspended solids.
The transport of substrate into cells is the limiting step. Subsequent endogen-
ous utilization of the substrate by hydrolysis, synthesis and respiration enzymes
permits additional permeation of substrate into cells.
Recirculation of sludge to the aeration tank, of course, is intended to maintain
microbial populations and to decrease the organic loading on a unit weight of
sludge. In addition to the above, the assimilated organic matter within the
cell cytoplasm are stabilized during detention in the final sedimentation tank
prior to introduction into the inlet of the aerator to mix with incoming wastes.
Such recirculated sludge possesses high ability for further permeation of exo-
genous soluble organic substance.
ENZYME ADDITIVES
A survey of the general field of enzyme additives to the biological waste de-
gradation process revealed the following material.
Chamberlin (Ref. 3) presented a review of organic catalysts ( enzymes) as well
as laboratory work conducted to determine "in what quantities they appeared
during the course of digestion and their effect on gasification and liquefaction
of sewage solids."
The enzymes used in the initial study to determine the effect of enzymes on
digestion when added to properly seeded mixtures, consisted of additions of
trypsin, rennet, pepsin, lipase, and diastase with and without pH and tem-
perature adjustment to 1 liter laboratory digesters. Results on digesters
(with and without adjustment) shows in general that the addition of enzymes
causes only a "slight" or no increase in gasification and no increase in solids
or volatile matter. More specifically, of the enzymes used, only trypsin "has
shown „.. increase in reduction over the control (20%). " The author explains,
"trypsin converts all of the solid proteins, proteoses and peptones to lower
nitrogenous compounds. An enzyme like this demonstrates digestion much
better by solid reduction than by gasification. " With regard to lipase and pepsin
which also reduce solids in a "similar manner . „. both of the materials which
they attack are not only solids but also heavy liquids." Lipase, he considers,
"may not have been the type (of enzyme) which would attack the heavy oils, fats
and grease." Trypsin, he considers to be the only enzyme that increased
digestion "from the standpoint of liquefaction."
From the standpoint of gasification," lipase and diastase were the only enzymes
that increased digestion unter optimal conditions, while lipase and rennet, "in
the absence of fresh solidssupernate" behaves similarly. For the first experi-
ments the author concludes that the rate of digestion is not increased.
The second set of experiments to determine the "effect of liquefaction of fresh
solids" by enzymes used basically the same experimental approach. The author
concluded that liquefaction of fresh solids resulting from the action of added
enzymes, "although slight in most instances, is appreciable in the case of lipase."
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He notes that lipase gave a 12. 6 per cent reduction in solids (per 100 cc fresh
solids) over the control, whereas rennet gave 7. 3 per cent and diastase 6.2
per cent. Liquefaction was determined by filtration, with lerric chloride added.
An evaluation of the effect of enzymes on swelling of fresh solids indicated
that the great swelling is caused by lipase, pepsin and a mixture of the five
enzymes used. The lowest swelling was caused by trypsin. Lipase, it is
noted "hastens swelling immensely" at the beginning of the test and then "falls
below the usual degree of swelling after a few hours." The author, neverthe-
less, concludes that with the exception of lipase and pepsin, the addition of
enzymes does not affect the swelling of fresh solids.
The effect of enzymes on dewatering of fresh solids indicated that in the ab-
sence of ferric chloride there was no effect, but that in general ferric chloride
together with the enzymes appeared slightly better than the ferric chloride
alone. Trypsin was the only enzyme "in the presence of ferric chloride" which
cause appreciable decrease in final moisture.
Heukelekian and Berger (Ref. 4) present a general discussion of the biochemical
activities of bacteria in sludge digestion including their role in hydrolysis and
liquefaction as well as a brief consideration and review of the "claims made
for the addition of bacterial cultures or enzymes with regard to promoting di-
gestion.
The authors consider that "if bacterial cultures or their enzymes are to prove
of any value they might be used in two different ways. First, they might be
used as initial cultures to shorten the "working-in" period required to est^b-
lish digestion. Second, they might be added to a tank in which digestion has
been established in order to accelerate the digestion. Heukelekian and Berger
consider the first method as showing "greater promise,"
The purpose of the investigation is to "produce experimental evidence to deter-
mine the value of some biological preparations consisting of enzymes and/or
bacterial cultures on the digestion of sewage solids. " The work, however, also
includes an experiment to determine the effect of yeast additions on the diges-
tion process and the addition of a preparation added to sewage "to determine
whether an influent with a lower BOD could be obtained after sedimentation in
settling or septic tanks..."
Parameters ("yardsticks") used to evaluate these experiments included for the
raw sludge experiments, the BOD of the supernatant,, For experiments using
ripe and raw sludge mixtures gas production and solids reduction was used.
In all the experiments, the concentration of additives were according to the
instructions of the manufacturer, or "in many instances several times higher."
Preparations used included:
1. Pure preparations
(a) Special Diastase HT concentrate - "obtained from a strain of
Bacillus subtilis with considerable proteolytic activity in
addition to diastatic activity converting starches and glycogen
to maltose
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(b) Clarase 900 - "A very concentrated diastatic enzyme obtained
from Aspergillus oryzae and similar in its action to Special
Diastase."
(c) Cellulolytic enzyme - from Rhizopus. "capable of hydrolyzing
cellulose and pectin."
(d) Pectinase - "Stronger in its action on pectin than the cellulolytic
enzyme, but free from cellulolytic activity."
(e) Pancreatin, "with a hydrolyzing action on fats, proteins, and
starches."
2. Preparation A - "claimed to consist of more than 70 enzymes belong-
ing to... esterases, sulfatases, carbohydrases, amylases, amidases,
and proteolytic enzymes, for use in home septic tanks. "
3. Preparation B - living bacteria and enzymes
4. Bakers yeast
Results from the addition of pure enzymes (Group 1, Pure preparations), to
sterilized (autoclaved) fresh solids for two runs, shows in general a "definite
increase in the BOD of the supernatant liquor after 12 hour incubation with
each of the enzymes used separately and with the combination of the five
enzymes. The minimum increase was obtained with pancreatin and the max-
imum with the combination of all five enzymes. Greater increases in BOD
were obtained after 5 days of contact than after 12 hours with each of the
enzymes, although in the case there is a question about the validity of the BOD
value of the control." The results of the addition of enzymes to unsterilized
fresh solids for the two runs indicates" no significant increase in the BOD of
the supernatant in the first run with any of the enzymes or the combination of
all five enzyme additions. " In the second run, "only diastase and clarase gave
appreciable increases in the BOD after 18 hours, but none of the other enzymes
or the combination of enzymes gave an increase.
The addition of pure enzymes on the digestion of a "properly seeded fresh solids
ripe sludge mixture" showed after 39 days that the "addition of enzymes did not
increase the gas yield or affect the volatile matter reduction and pH values. "
The results of the addition of preparation "A" to sterilized and unsterilized
fresh solids indicates that for both sterile and unsterile fresh solids the ad-
dition of this enzyme preparation "did not give a significant change in BOD of
the supernatant over the control." A similar study with "B" gave essentially
the same results.
Yeast additions to fresh solids (56 day runs) indicated that a "somewhat greater
volume of gas is obtained from the fresh solids to which yeast was added than
from the fresh solids alone. The yeast alone produced a considerable volume
of gas. When the volume of gas produced from the yeast itself is subtracted
from the fresh solids yeast mixture, the gas yield from the fresh solids por-
tion of this mixture is not appreciably higher than the fresh solids digesting
without yeast addition. " They further note that pH values of the fresh solids-
yeast mixture was "somewhat higher after 53 days," and indicate that the solids
content of the yeast alone "decreased greatly, (67. 5% reduction in volatile solids),
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which probably accounts for the higher volatile solids reduction of the fresh
solids-yeast mixture than the control." In summary, Heukelekian and Berger
consider that "it does seem likely that the addition of yeast resulted yi a great-
er reduction of volatile matter in fresh solids. "
The addition of Preparation "A" to septic tanks which were then allowed to re-
main quiescent for six days (20°C) gave "no indication that the addition of
enzyme preparation resulted in any improvement in BOD of the sewage."
The authors consider that the addition of biological preparations such as en-
zymes or bacterial and yeast cultures to promote the digestion of sludge in
septic tanks or sludge digestion tanks "would be of value in initiating new tanks
or in accelerating digestion of overloaded tanks if there were proof of their
beneficial effect. " The authors believe, however, that in general, laboratory
investigations are "in disagreement with this line of reasoning. "
On an a-priori theoretical basis Heukelekian and Berger consider that the use
of enzymes "does not offer a promising outlook, and point out that while" it
is generally true that of the two types of reactions - bacterial and enzymatic -
enzymatic is less efficient than bacterial." The authors note that enzymes "as
true catalysts" should theoretically not be consumed by the processes they are
bringing about and "should be regenerated so that starting with a given quantity
of an enzyme, the process should go to completion without their utilization or
destruction. In bacteria-free preparations this may be true to a certain extent,
although even under this condition the rate of reaction is not as high as with
bacterial cultures because:
(a) Certain enzymes such as trypsin inactivate themselves;
(b) Enzymatic reactions are reversible and the products formed,
which in bacteria-free enzyme reactions would naturally accu-
mulate, bring this reaction to a stop by virtue of the equilibrium
established; and
(c) Each enzyme has its own optimum pH value.
The authors therefore consider that in the presence of mixed bacterial cultures
the inactivation, utilization, and destruction of the added enzymes would be
greater than in pure enzyme reactions. "
Heukelekian and Berger recognize that the above discussion is "from the stand-
point of pure enzyme reactions in substrates containing relatively simple
soluble, organic compounds. " In sewage sludge, "sludge compounds do not
exist in a similar state of subdivision. There are no soluble sugars, starches
and proteins but rather complex polysacharides, cellulose, proteins and fats
associated with large suspended particles. " "Since hydrolyticenzyme reactions
are apparently surface contact phenomena, it can readily be seen why an en-
zyme preparation cannot bring about as efficient a reaction when added to the
substrate containing coarse solids. They further note that "in the case of hydrolysis,
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brought about by a bacterial culture with coarse suspended particles, bacteria
can attach themselves to these particles and secrete enzymes in-situ to hydro-
lyze and solubilize these particles in preparation for diffusion into and assimi-
lation by cells. " The authors further consider that in many of the hydrolytic
reactions with complex organic materials more than one enzyme is involved,
"which the bacteria produce readily in proper sequence for the specific reac-
tion, whereas in the arbitarily selected enzyme preparations some of the im-
portant enzymes required may be overlooked. "
The authors note that the hydrolytic phase of complex organic materials in
the presence of bacteria proceeds rapidly and does not constitute the rate
controlling factor. They note that "diffusion into and the breakdown and
assimilation of the products of hydrolysis within the cell is the slower and
rate controlling reaction." It is therefore reasoned that the addition of extra-
cellular enzymes into a substrate "by no means can accelerate this phase nor
can this phase be brought about by extra-cellular enzymes in the absence of
living cells."
Other arguments advanced against the addition of bacterial culture preparations
for materially improving digestion includes:
(1) "All bacteria necessary for digestion are present in the raw
solids."
(2) "These bacteria are normally few in number in the raw solids, "
but soon multiply and establish themselves under a favorable
environment.
(3) "Even during the initial period prior to the establishment of flora
responsible for digestion, the addition of cultures of bacteria
normally does not result in shortening the period of maturation
and adjustment unless proper environmental conditions are
established, and if these conditions are an optimum the rapid
rate of multiplication of bacteria normally present in the raw
sludge could establish the necessary flora without the aid of
artifical additions of cultures. In other word, one can add a great
number of bacteria artifically, but unless conditions are conducive
for their multiplication, the benefit derived will be negligible and
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if the environmental conditions are an optimum the organisms
present in the raw sludge multiply and establish the necessary
flora. The important consideration is the multiplication of the
bacteria, with the resultant biochemical activity, rather than
the existence of a large number of bacteria. Furthermore, it is
unlikely that the diverse bacteria necessary for the digestion are
included in the artificial culture because so far the exact types
of organisms involved in the digestion have not been isolated
or studied."
Heukelekian and Berger further consider "practical considerations, " with regard
to the additions of enzymes or bacteria to septic tanks. Generally these
additions are made to the sewage before entering the tank. They consider that
since these preparations are in a "highly dispersed state" it is " very probably
that the major portion will pass out of the tank and will not settle to the sludge
zone where the digestion takes place. " They state that in order to bring these
preparations in contact with the sludge, either the entire tank contents have to
be disturbed by mixing, which is contradicted from the standpoint of septic tank
operation, or some means have to be found to introduce and distribute these
preparations in the sludge.
Heukelekian and Berger conclude that evidence available in the published
literature seems rather " inconclusive and is not based on rigidly controlled
experimental work." They consider that the results present in this paper on
the basis of "controlled comparisons does not show any evidence of improve-
ment due to the addition of any enzymes, preparation, of enzymes, bacterial
cultures, and yeast to sterile and non-sterile fresh solids and to sewage,
with a single exception. The exception is the indicated higher BOD values of
supernatant liquor when pure and strong enzyme preparations were added to
sterile fresh solids." They further note than "on the other hand when these
same enzymes were added to the unsterilized fresh solids there was no increase
in the BOD of the supernatant, .. . (indicating) that when the bacteria and their
enzymes are destroyed it is possible to get liquefaction of the complex organic
materials by use of artifically added strong enzymes, "but in the presence of
bacteria and their enzymes as found in fresh solids, artificial addition of
enzyme does not increase liquefaction. "
Ingols (ReL 5) presents a general review of the literature of sewage treatment
and more specifically the activated sludge process with regard to enzymes.
The laboratory studies presented include methods of analysis for hydrolytic
enzymes. The principal work concerns studies in activated sludge using the
hydrolytic enzymes diastase, pepsin, trypsin and lipase. The presence of
other hydrolytic enzymes such as cellulase, cellobiase, maltase and chitinase,
although noted, require methods for study "so cumbersome as to render their
inclusion (in this study) „.. impossible. "
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The study includes enzymatic activities during the development of activated
sludge, enzymatic activities of sludge mixtures during aeration, environ-
mental effects (substrate, temperature, reactants, salts), effects of poisons
(arsenate, copper and mercury at 1. 5 and 6. 0 mg/L), and a correlation
between activity and clarification.
From these studies it is concluded that there is a "marked" increase in pepsin
during the development of activated sludge from sewage. Lipase, pepsin and
diastase are found only on the surface of the sludge floe whereas trypsin is
also found in the liquor surrounding the floe.
The enzymatic activities of activated sludge and sewage are considered to
"change little" during a 6-hour aeration period. "Slight" fluctuations in pH
have little effect on the enzyme studies.
When activated sludge is aerated continuously for several weeks the "importance
of enzyme is demonstrated because the enzyme is increased during the first
three days and only after that decreases gradually."
Diastase may be increased 500 per cent by feeding the sludge organisms starch
at 5°C. The rate of hydrolysis of a given substrate will be influenced by the
rate of assimilation of its hydrolysate by the sludge organisms. The author
considers that diastase requires a certain salt concentration for action.
Ingols (Ref. 6) also presented a review of the oxidation-reduction enzymes
present in activated sludge, but also included references to studies using
hydrolytic enzymes. The author considers that these enzymes are important
in providing the energy "necessary for the other functions of the organism. "
An increase in number of cells brings a concurrent increase in activity in the
oxidation-reduction enzymes "but that after a period of time the food supply
is used up and the rate of cell metabolism decreases and the cells are said to
be resting. As the cells approach the "resting" stage there is a decrease
in the activity of the oxidation-reduction enzymes, but no decline in the
number of organisms."
It is concluded that oxidation-reduction enzymes are necessary in the activated
sludge process for purification of sewage and that for this process the activity
of activated sludge is dependent upon the concentration of these enzymes.
McKinney and Sawyer (Ref. 7) present a general review of biocatalysts including
a presentation of the "Fundamental Facts" of waste treatment describing the
roles of enzymes in the metabolism of waste treatment. The authors point out
that one "important" difference between catalysts of inorganic nature and
biocatalyst is that the inorganic catalysts have a long life and need rejuvenation
or replacement "only at infrequent intervals." Biocatalysts, on the other
hand, "have a relatively short life owing to denaturation which occurs during
use." The authors also point out that loss of enzymes released by bacteria
are lost to the effluent making it "imperative" that regenerative capacity of
the organism be kept at a satisfactory level." For this reason "emergency"
addition of biocatalysts "has to be on a continuous or frequent basis to obtain
results" and can hence "become an expensive measure."
14
-------
The availability and contents of biocatalysts are discussed. The contents of
"the commercial biocatalyst are basically either concentrated cultures of
bacteria or concentrated enzymes and are used in specific treatment units
such as digesters, trickling filters, Imhoff plants, activated sludge plants and
septic tanks. Various claims for commercial biocatalysts are examined
("there is little specific information,") and evaluated in light of "what they
have done." Although the authors consider that little information is available
in this respect, "little by little information is becoming available directly
from operators and from independent research studies. "
The cost of commercial biocatalysts is discussed in light of claims made by
manufacturers in terms of savings. " The authors note that "where the prod-
ucts have been used with limited success there is some question as to the eco-
nomics of use.
The authors consider that "at present, there is considerable doubt as to the
value of these commercial products," and that "results have shown that these
commercial biocatalysts can bring about improvement in certain operating
problems, but that for the most part, it is not dramatic nor do they appear to
afford an economical solution. "
Rudolfs (Ref. 8) reports on a group of experiments in which pure enzymes
were added to fresh solids, fine screenings and activated sludge. The enzymes
used included trypsin, pepsin and lipase at various pH's and temperatures.
Results are reported for both laboratory and plant-scale operation.
The author considers that the experiments conducted do not show that digestion
time is decreased "even with the addition of comparatively large quantities
of enzymes." However, using lipase "under optimum conditions" gave some-
what more gas per gram of volatile matter destroyed, but with trypsin the
reverse occurred." The author noted that trypsin also "seemed to stimulate
liquefaction, " and notes that "with our present knowledge it would seem that
the bacterial groups produce sufficient different enzymes during the digestion
process that any further addition is unnecessary. "
Evaluation of the results in terms of digestion time, gas production, material
handled, drainability and odors indicates "very little practical difference, if
any," as compared to controls.
Wooldridge and Standfast (Ref, 9) present their laboratory findings supporting
previous work which considered that "the most important factor in sewage
purification is a series of catalyzed reactions present in either living or dead
bacterial cells or liberated by them into the fluid of the reaction system."
For oxygen absorption by sewage or sludge, a Barcroft microrespirometer
was used in the studies, "simulating, on a small scale, those applying to the
activated sludge process of sewage purification." Oxidation of the constituents
of sewage and of sludge depends upon the presence of certain oxidative enzymes
(dehydrogenases and oxidases) of microorganisms. These enzymes may be
effective whether the organisms are alive or dead, "provided the method of
15
-------
killing has not destroyed the enzymes. " Although protozoa possess enzymes
that can oxidize the constituents of sewage, "the greater proportion of the oxi-
dation is brought about by bacterial enzymes, "the bacteria being both far more
active. "
Sewage oxidation is mainly dependent on the presence of bacterial enzymes
which may be associated with either living or dead cells. It is "probable" that
the activities of the living proliferating cells for certain oxidations is greater
than that for dead cells. Dead cells may be considered to be important in
oxidation in activated sludge since the sludge contains a high proportion of
dead bacterial cells "many of which are probably enzymatically active. "
The most active microorganisms as a source of enzymes in sewage oxidations
are Bacillus alkaligenes, Proteus vulgaris, Pseudomonas pyocyanea, and
Pseudomonas fluorescens. Bacillus coli appeared to be 'less active. "
Of the protozoa examined Polytomella uvella and Euglena gracilis had an oxi-
dizing power less than B. coli, and "in general less active than bacteria. "
16
-------
SECTION V
EXPERIMENTAL PHASE
BIOCHEMICAL INVESTIGATIONS
Materials and Methods
The materials for this investigation are readily divided into two groups. The
first group includes those compounds used as standards within the various
assay protocols:
1. Crystallized Bovine Plasma Albumin, Armour Pharmaceutical
Company, Chicago, Illinois.
2. Proteinase, Nutritional Biochemicals Corporation, Cleveland, Ohio.
3. Lipase Steapsin, Nutritional Biochemicals Corporation, Cleveland,
Ohio, Lipase value 3. 5 x USP.
4. Alpha Amylase, JB. subtilis, Nutritional Biochemicals Corporation,
Cleveland, Ohio, 1800° Lintner or 2500 8KB units/gm0
5. Acetylcholinesterase (horse serum), Nutritional Biochemicals Cor-
poration, Cleveland, Ohio, 1 unit hydrolyzes lyuM acetylcholine/min
at 25°C (4 units/mg).
The second major group consists of those specific reagents employed in the
immobilization protocol:
1. Acrylamide, Eastman Organic Chemicals, Rochester, New York.
2. N, N1 - Methylenebisacrylamide, Eastman Organic Chemicals,
Rochester, New York.
3, Riboflavin, Eastman Organic Chemicals, Rochester, New York.
4. Ammonium Persulfate, A.C.S. crystals, Matheson Coleman and
Bell, Cincinnati, Ohio.
The detailed procedures for the specific assays employed during the investiga-
tion are presented in the Appendix (Section VIE) of this report. A general
description of each method is as follows:
10 Proteolytic Activity; Two different assays were used to quantitate the
proteolytic activity of experimental samples. The first of these entailed the
use of a substrate composed of an insoluble hide protein to which the dye,
Remazolbrilliant Blue, is chemically coupled. During the attack of proteolytic
enzymes on the substrate-dye complex, the dye molecules are released. Sub-
sequently, the reaction is terminated by filtration, and the absorption of the
filtrate, now containing dye molecules, is determined at 595 myu . Initially
enzyme activity was expressed as a function of absorption units, but later ex-
periments converted these value into the equivalent milligrams of a reference
proteinase required to obtain the identical absorption reading.
17
-------
The second proteolytic assay entailed the use of casein as a substrate,, Di-
gestion of the latter compound released amino acids into the media. With
termination of the assay via trichloracetic acid (TCA) precipitation, separation
of the precipitated enzyme and non-digested substrate from the acid soluble
components was accomplished by centrifugation. The increased absorption due
to the release of soluble amino acids was then determined at 280 mM with a
Beckman spectrophotometer.
2. Amylytic Activity; Two different assays were employed. The first en-
tailed the use of an insoluble substrate-dye complex similar to that employed
in the proteolytic assay, but in this case the substrate was starch. The en-
zyme activity is measured as a function of the number of dye molecules
released absorbing at 595 m/z.
The soluble substrate system used was that of the classic KI-I2 interaction
with starch molecules„ The soluble starch substrate is incubated with an en-
zyme source. If amylase activity is present, the starch is broken down. Since
iodine interacts with starch to form a blue complex absorbing at 650 rcifj.,
activity is expressed as a function of absorption of the control (no digestion of
starch) minus the experimental absorption. As with the other enzymes, these
activities were converted into the equivalent mg of a standard alpha amylase
preparation which would yield the same change in absorption.
3. Dehydrogenase Activity; Was measured fluorometrically using resazurin
as the indicator. This assay is based on the conversion of the nonfluorescent
resazurin to the highly fluorescent resorufin. The rate of change in fluores-
cence ( AF/min) is then used as a measure of enzyme activity.
4. Lipase Activity; Was measured by the interaction of an enzyme source
with the nonfluorescent substrate fluorescein dibutyrate. The rate of forma-
tion ( AF/min) of the fluorescent product fluorescein is then converted into the
equivalent mg of lipase steapsin required to yield the same enzyme activity.
5. Esterase Activity: Is measured using the very non-specific substrate
indoxyl acetate. This nonfluorescent compound is hydrolyzed by esterase into
highly fluorescent indoxyl. The rate of indoxyl formed (AF/min) is then con-
verted into the equivalent mg of acetylcholinesterase required to yield the same
fluorometric response.
6. RNA and DNA; Analyses were performed via the now classical colorimetric
tests employing orcinol and diphenylamine as the color reagents.
7. Total Protein: Was determined via colorimetric Biuret test. By compar-
ing the samples absorption at 540 m/u with the absorption by known quantities
of bovine serum albumin, the results were readily converted to equivalent mg
protein per ml sample.
Experimental Laboratory Studies
1. Determination Optimal Source. Authorization to obtain sludge samples
was secured from the Leominster, Massachusetts, Plant. It was found advan-
tageous to limit our initial concern to determining which of five strategically
located sources within the plant would yield preparation of optimal enzymatic
18
-------
activity, A diagramatic representation of the Leominster plant is depicted in
Figure 1. Since biological variation is inherently severe during in-plant
processing, it was deemed useless to define "daily" enzymatic activity. The-
oretically it would be expected that aeration (which activates the sludge -
Tank I) and exposure to sewage (which could potentially induce synthesis of
specific enzymes - Tanks II, IE and IV) mightproduce a source of increased
activity. On this basis, probably only source V would prove less active en-
zymologically. However, preliminary determinations of dehydrogenase,
amylytic and proteolytic activity (Figure 2) demonstrated that none of the five
sources appeared to exhibit significantly less activity when compared to the
other four. On the basis of these results, it was arbitrarily decided that
future experiments would be conducted on the activated sludge obtained from
Tank I. This source represents sludge derived from the final settling tank
and which, through a process of vigorous aeration, is activated prior to re-
cycling through the inlet sequence.
2. Preliminary Fractionation; Membrane Disruption Techniques. Our con-
cern was now focused on determining the method whereby cell disruption was
most efficiently achieved. Using amylytic and proteolytic activity as tracers,
it was found (Figure 3) that sonication yielded a much higher release of en-
zymatic activity into the supernatant fraction than did either freeze-thaw or
homogenization techniques. Dehydrogenase activity was lost by all cell dis-
ruption procedures. Althoughpressurebombdisruption would theoretically
enhance the yield, we were unable at this time to procure the instrument from
commerical sources.
3. Preliminary Fractionation; Storage Techniques. It was important to
determine early in the program adequate means for storing the enzymatically
active preparations derived from the original activated sludge source. Of the
methods examined, freezing represented the simplest and fastest means of
maintaining activity. In Figure 4, the 48 hours frozen sonicate exhibited com-
plete retention of activity. Indeed, subsequent work has demonstrated stability
after over 12 weeks of storage at 0°C. Lyophilization is well documented for
its gentleness toward enzymatically active preparations and has ^he additional
advantage of offering an opportunity to concomitantly concentrate activity.
As might be predicted, on this basis, lyophilization did indeed prove to be an
effective storage technique. Acetone powder preparation was found to have a
detrimental effect upon amylytic activity and was, therefore, discarded from
use in future protocols.
4. Preliminary Fractionation - Partial Enrichment Studies. The three frac-
tionation steps proposed represent the simplest and most basic employed by
investigators of microbial enzyme purification. Protamine sulfate precipita-
tion is specifically used to remove nucleic acid contaminants from the remain-
ing soluble components. The latter have been shown to interfere with many
enzyme assay systems and, in any event, would not contribute to total bio-
chemical activity during future immobilization attempts. Dilute acetic acid
precipitation will fractionate on the basis of enzyme isoelectric points via
variations in pH. Ammonium sulfate precipitation is perhaps the most widely
applied of the fractionation techniques investigated, but entails a lengthly time
lag to permit extensive dialysis of the preparation prior to enzymatic analysis.
Ammonium sulfate fractionates proteins on the basis of their isoelectric point
as determined by variations in ionic strength.
-------
Aeration Tank
iiiiiliijililijijliiiljiilli Sludge
'Xy&&&. Soluble Sewage
Soluble
Wastes
Solid
Wastes ~*r
Incoming Sewage
ng Sewage
Figure 1. Leominster Plant
20
-------
Original Samples (1, 2, 3, 4 and 5)
Centrifuge 1,500 rpm, 15 min., refrig,
International
Wash 3x 0.05M Tris buffer, pH 7.8
Determine packed cell volume wet weight
Remove aliquot for dry weight determination
Make up remainder to 25% suspension in buffer
w
SL
hole Cell
spension
— Proteolytic
— Amylytic
— Dehydrogenase
Description
Whole Cells
(1 ml 25% suspension)
Tank I Derivative
Tank n Derivative
Tank HI Derivative
Tank EZ Derivative
Tank 3T Derivative
Dehyrogenase
Activity**
0.323
0.309
0.352
0.298
0.317
Amylase
Activity***
0.119
0.125
*
*
0.110
Freeze Remainder
Proteolytic
Activity***
0.161
0.153
0.135
0.157
0.127
*Not Assayed
**Absorption values at 430 mju
***Absorption values at 535 m;u
Figure 2. Determination of Optimal Source of Bacterial Sludge
21
-------
Tank I
Centrifuge, freeze supernatant
Determine packed cell volume wet weight
remove aliquot for dry weight determination
Wash 2x buffer
Protease
abs 0.595
Amylase
abs 0.595 m/U
I I I \
Whole Cell Sonicate 10X Homogenize
(25% Suspension) , Freeze-Thaw
I *
Supernatant Supernatant Supernatant
0.377
0.234
0.398
0.209
0.109
0.043
0.117
0.123
FigureS Preliminary Fractionation: Membrane Disruption Techniques
Sonication
Protease*
I
Orig. Sonicate
3.25
9.75
I
Frozen
Sonicate
3.09
9.68
I
Acetone
Powder
3.16
1.99
I
Lyophilizf
4.12
9.21
*abs 595 rryu/mg protein/hr
Figure 4. Preliminary Fractionation: Storage Techniques
22
-------
Preliminary experiments indicated that the enzymatic activity of the acetic acid
supernatant fraction exhibited maintenance of proteolytic and amylytic activity.
Protamine sulfate precipitation encountered some problems due to the difficulty
in determining the end point for nucleic acid removal. The addition of excess
protamine sulfate to the suspension subsequently led to apparent loss in enzyme
activity in the supernatant fraction. Ammonium sulfate precipitation similarly
appeared to have a negative effect on amylytic activity although the precipitate
demonstrated a 2-3x enrichment of proteolytic activity.
These preliminary results had several important ramifications upon the gen-
eral investigational protocol. First of all, the already apparent efficiency of
the fractionation systems selected obviously required the monitoring of addition-
al enzymes to assure optimal maintenance of the hydrolytic bacterial enzymes
of interest. Thus, both a general esterase and lipase assay were incorporated
into the enzyme monitoring system. Secondly, it became apparent that further
attempts at enrichment would entail the inevitable loss of component enzymes
among the widely diverse catabolic enzymes derived from activated sludge. As
a result of these conclusions, a series of experiments were performed to verify
and improve upon the applicability of the aforementioned fractionation tech-
niques. In Table I, a summary of the data obtained from these experiments is
presented.
From these results, the decision was made to use a protamine sulfate precipi-
tation followed by a dilute acid precipitation for the preparation of the sonicate
derivatives to be used in immobilization. However, it was found that incomplete
nucleic acid precipitation was obtained in fractions #1 and #2, and therefore,
a second protamine sulfate precipitation was done on these two derivatives of
the acetic acid fractionation,, Subsequently, the preparation of large scale quan-
tities of the soluble enzymatic derivatives was completed according to these
procedures and a chart detailing the data obtained via the enzyme assay mon-
itoring system is shown in Table n. The three final fractions (Protamine
Sulfate supernatant Ib, Protamine Sulfate supernatant 2b, and Acetic Acid
supernatant #3) were than combined (Mixed Supernatant Fraction) and tested
to determine the biochemical characteristics of the final product. In Table
III, a summary of these biochemical parameters for both the original sonicate
and the final combined sludge derivatives is presented.
5. Immbolization in Polyacrylamide Gels. Initial attempts at immobilization
of enzymatically active preparations according to the procedure of Updike and
Hicks (Ref 10 and 11) yielded a very unsatisfactory product. Thus, experiments
were designed to determine the co-polymer concentrations which would yield
both maximal retention of enzymatic activity and optimal gel consistency for
our particular application. The basic components of the polymerization system
are as follows: 40% solution of acrylamide, 2.3% solution of N, N-Methylene-
bisacrylamide (MBA) and solutions of riboflavin and potassium persulfate such
that a 0.1 ml contained 0. 03 mg. In all these solutions 0.1 M phosphate buffer,
pH 7.4 was used as the solvent. The preparative protocol entailed the bubbling
of nitrogen gas separately through the previously measured copolymer compo-
nent for a minimum of fifteen minutes to remove any traces of dissolved oxy-
gen. The latter solutions are then combined and both riboflavin and persulfate
are added in the stipulated catalytic amounts. After thorough mixing, the en-
zyme preparation is added to the mixture, stirred and placed in an ice bath.
23
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TABLE I. FEACTIONATION TECHNIQUES
(25% WHOLE CELL SUSPENSION - 11/10/69)
ACTIVITY
Proteolytic
Total Enzyme Units
Sp Activity
Amylase
Total Enzyme Units
Sp Activity (xlO~2)
E sterase
Total Enzyme Units
Sp Activity (xl(T2)
Lipase
Total Enzyme Units
Sp Activity (xlO~2)
Total Protein
Sonicate
Sonicate
5,340
3.12
112.0
6.6
46.8
2.7
22.6
1.32
1,710
Protamine Sulfate
Precipitation
super,,
5,400
3.26
111.0
6.7
38.6
2.3
19.1
1.16
1,650
ppt
*
*
*
*
1.5
1.5
none
none
100
Acetic Acid
Precipitation
super.
5,850
4.31
102.0
7.5
41.6
3.1
21.9
1.61
1,360
ppt
330
0.62
8.0
1.5
1.4
0.3
201
0.40
530
Ammonium
Sulfate
Precipitation
super. ppt
840 3,260
1.43 9.30
12.0 15.0
2.0 4.3
32.6 14.1
5.5 4.1
none 7. 20
none 2. 06
590 350
1. Enzyme Units:
Proteolytic
Amylase
Esterase
Lipase
2. Specific Activity:
*Not Assayed
— equivalent to mg proteinase/ml/30 min
— Equivalent to mg alpha amylase/ml/30 min
— equivalent to mg acetylcholinesterase/ml/min
— equivalent to mg lipase (steapsin)/ml/min
equivalent enzyme units/mg protein
24
-------
TABLE H. VALUES OBTAINED WITH SPOT MONITORING
DURING FRACTIONATION OF 1/19/70 SONICATE
Sonicate proteolytic - 7. 00 mg proteinase
(1-19-70) amylytic 0.300 mg alpha amylase
esterase 00376 mg acetylcholinesterase
lipase 0.298 mg lipase (steapsin)
Protamine Sulfate Fractionation
Supernatant Precipitate
#1 Prot-8.81 (8.63) #1 Ester -0.127(0.151)
Amyl- 0.201 (0.185) Lip -0.069(0.084)
Ester 0.278 (0.366)
Lip - 0.250 (0.292)
#2 Prot 8.48 (8,63) #2 Ester -0.122(0.151)
Amyl 0.160(0.185) Lip -0.091(0.084)
Ester 0.439 (0.366)
Lip - 0.335 (0.292)
#3 Prot- 8.60 (8.63) #3 Ester -0.203(0.151)
Amyl - 0.195 (0.185) Lip - 0,122 (0. 084)
Ester - 0,374 (.366)
Lip -0.247(0.292)
Acetic Acid Precipitation
~~2nd Protamine Sulfate PPT.
Precipitate Supernatant (Super)
#1 Ester -* : #1 Prot -5.88(7.09) #lb Prot - 6.52 (7.00)
Lip -* Amyl - 0.195 (0.153) Amyl - 0.175 (0.144)
Ester -0.229(0.230) Ester-0.238
Lip -0.242(0.241) Lip-0.235
Protamine Sulfate #2 (Super)
#2 Ester - 0 087 (0.105) #2 Prot -8.22(7.09) #2b Prot -7. 28 (7. 00)
Lip -0.257(0.350) Amyl - 0. 155 (0. 153) Amyl -0.136 (0.144)
Ester - 0.271 (0.271) Ester -0.220
Lip -0.242(0.242) Lip -0.232
#3 Prot - 7.19 (7.00)
Amyl - 0.120 (Oo 144)
Ester - 0.189 (0.230)
Lip - 0.239 (0.241)
* not assayed
Note: The numbers in parenthesis represent the average values obtained by the
three different samples within the same fractionation step.
25
-------
TABLE IE. COMPARATIVE BIOCHEMISTRY OF THE
SONICATE AND THE MIXED SUPERNATANT FRACTION
Enzyme Activity*
Proteolytic Activity
1. Equivalent m.g Proteinase/hr
2. Specific Activity
Amylase Activity
1. Equivalent mg Amylase/hr
2. Specific Activity
Lip as e
1. Equivalent mg Lipase (Steapsin)
2. Specific Activity
Total Protein/ml
Total Nucleic Acid/ml
Sonicate
6.46
1.46
0.331
7.5 x 10~2
0.343
7.8 x 10 ^
4.41 mg
38.3 mg
Mixed Fraction
5.76
2.11
0.295
10.8 x 10~2
0.268
9.9x 10 2
2. 73 mg
14. 5 mg
*Numerical values represent the equivalent milligrams of a reference standard
proteinase, and amylase, acetylcholinesterase and steapsin lipase required to
obtain the enzymatic activity exhibited by each of the various fractions.
The area directly in front of the preparative flask is cleared of ice and the
vessel moved as close as possible to the glass wall of the ice bath. A number
2 photo flood lamp is placed directly opposite the preparative flask and func-
tions as the light source for the photocatalytic initiation of polymerization.
During the immobilization process nitrogen is gently blown down on the experi-
mental mixtures in order to both exclude oxygen from the system and to provide
for slight agitation of the polymerization components. The latter requirement,
in direct contrast to the procedure of Bernfeld (Ref 12 and 13) was found essen-
tial to obtaining a homogeneous consistency within the individual polyacrylamide
gel preparations. During the procedure the temperature of the bath was also
carefully maintained at 0-4°C in order to prevent possible thermal denaturation
of the constitutent enzymes (Ref 14). Initially the resulting block of polymer-
ized enzyme-gel was mechanically dispersed into particles by extrusion through
a syringe. However, this procedure soon proved feasible only for the less firm
gel produced in the presence of high concentrations of either sludge derivatives
or MBA0 The optimal gel consistency eventually selected required the use of
26
-------
sieving devices (mesh screen or modified garlic press) to achieve the force
necessary to obtain satisfactory particles. The particles prepared by the
latter procedures were then thoroughly washed with liter quantities of fresh
buffer and stored at 0-4°C prior to enzymatic analysis.
A summary of the results observed during experimental variation in co-polymer
concentrations is shown in Table IV. No significant change in enzymatic
activity was apparent once a homogeneous consistency was obtained (Sample
2-5). However, both the time required for the reaction to go to completion
and the characteristic integrity of the gel (as measured by its ability to resist
externally applied force) changed drastically as a function of co-polymer
concentration (Ref 15). In addition, it was found in subsequent experiments that
increasing the total reaction volume resulted in disproportionate increase in
the required reaction time. Although the time lag had no visible effect on the
resulting gel product characteristics, it was found to have drastically reduced
recoverable enzyme activity (for example, 6.2 ml total volume-6 min. and
20% recoverable enzyme activity; 62. 0 ml total volume-2. 5 hours and less
than 3% recoverable enzyme activity.)
It must be noted, however, that a more detailed investigation of possible mod-
ifications in batch preparation would probably permit large-scale gel synthesis
feasibility. However, on the basis of the available data a proportion of 4 ml
acrylamide and 1 ml MBA was selected as providing maximal firmness while
still permitting division of the gel into small bead-like particles. Similarly, a
maximum volume of 30 ml/preparation was selected as providing the optimal
balance between needs for large scale preparation vs minimizing polymerization
time.
Immobilizations of commercially obtained proteinase, acetylcholinesterase
and lipase were initially done to affirm that these enzymes would indeed with-
stand the polymerization reaction. Subsequently, an attempt was made to
immobilize an aliquot of sonicate, but no detectable enzyme activity was ob-
served. However, upon concentrating the sonicate via resuspension of a
lyophilized preparation, all four enzymatic parameters exhibited partial re-
tention of activity. In Table V is found a summary of these initial results.
Caution must be observed in interpreting these results since they represent only
preliminary testing in which the emphasis was on the presence vs absence of
enzyme activity rather than precise quantitation of the retention. However,
it was established that: (1) immobilization of the activated sludge derivatives
was indeed feasible; (2) that the gels were capable of retaining activity for at
least three weeks at 4°C; and (3) that repetitive use of the same gel fraction
exhibited continued enzymatic activity.
It was at this point that the two major phases of the preparative program met.
The preparation of the soluble fraction derived from activated sludge and pos-
sessing optimal catabolic enzyme activity (Table III) was essentially completed,
and it remained to be determined the degree of concentration necessary to ob-
tain optimal recovery of enzymatic activity in the immobilized form. In essence,
this consisted of lyophilizing a sample of the fractionated sonicate, resuspending
at a concentration of about 50 mg protein/ml and making several dilutions
27
-------
TABLE IV. VARIATION IN CO-POLYMER CONCENTRATION IN POLY-
ACRYLAMIDE GEL PREPARATIONS
1_
Sample
No.*
1
2
3
4
5
6
ml Acryla-
mide
1
2
3
4
4.8
5 ml tris
buffer
ml
MBA
4
3
2
1
0.2
Approx
Reaction
Time
35 min
25 min
15 min
10 min
5 min
Gel
Description
opaque, curds &
whey consistency
opaque, junket-
like consistency
transparent, orange
jello consistency
transparent, orange
firmer
transparent, orange
rubber -like
firmness
Proteolytic Acti-
vity Absorption
595 myu
0.197
0.284
0.264
0.273
0.258
1.056
* All samples also contained 0.1 ml riboflavin and persulfate catalysts and 1
ml proteinase (20 mg/ml).
TABLE V. IMMOBILIZATION OF RESUSPENDED LYOPHILIZED SONICATE
11/9/69
Enzyme Units /ml
Protease
Amylase
Esterase
Lipase
Sonicate
1.062
2.55
0.25
1.04
Immob Gel
0.186
0.320
0.01
0.07
Est % Recovery
17.5
12.6
4.0
6.8
28
-------
through 5 nag/ml and immobilizing each concentration„ The gels were then
compared on the basis of total activity in terms of the lipase and esterase as-
says, required time, and the final characteristic gel consistency.
It was predicted that, the rate of increase in enzymatic activity corresponding
to increasing higher concentration of total enzyme immobilized would eventually
reach a point of diminishing returns. Although this prediction might yet be
valid, it was found that the physical characteristics of the gel itself proved to be
the limiting factor. In Table VI the results of this experimental series are pre-
sented. At concentrations greater than approximately 12 mg of protein/ml (3x
concentration of the mixed supernatant fractions) the consistency of the gel
became increasingly viscous. At maximum concentrations, the gel had become
so amorphous as to be resistant to breakdown into the desired particles and
exhibited a high degree of adhesion for almost any surface. Also in Table VI
the results of this experiment are summarized. It will be noted that with in-
creasing enzyme concentration used for immobilization a corresponding increase
in enzyme activity was indeed observed.
The experimental protocol also included an analysis of the protein content of
the initial 10 ml wash of the gel particles containing immobilized enzymes. It
was of interest to note that none of these five concentrations tested exhibited
any release of protein into the wash water. In one of Bernfelds most recent
papers (Ref 12), he has used a radioactive enzyme sample to verify that in this system
all activity also remained associated with the polymerized gel. Bernfeld (Ref
16 and 17) has also reported in his original article that no leakage was detected
into the buffer during storage of his immobilized gel. However, Kalchalski's
review article (Ref 18) cites Bernfeld as reporting a continual low level of leak-
age. In any event, several spot checks of the buffers in which the immobilized
sludge derivatives were stored revealed no leakage of protein within the sen-
sitivity of the Biuret test. Immobilization of protein components within the gel
matrix appears to be complete within the concentration ranges examined.
Consequently, any loss of enzymatic activity is directly attributable to some aspect
of the polymerization process rather than to a non-specific removal of unbound
protein by multiple washings,,
In addition, a series of experiments was done in an attempt to further delineate
the causative factors in the formation of the heterogenous viscous gel occuring
when high concentrations of the mixed supernatant fraction were used. The
first of these entailed varying the co-polymer concentrations in a similar pro-
tocol as that described for Table IV. However, it was found that the char-
acteristics of the various proportions described in Table IV were again re-
produced but this time in combination with the constant viscous-gel type elicited
by the use of the concentrated enzyme preparation. A highly concentrated sample
of bovine serum albumin (75 mg/ml) was also subjected to immobilization and
yielded a very firm clear gel. On the basis of these results it can be assumed
that the difficulty experienced was not simply due to a need for different co-
polymer proportions or too high a protein concentration. It appears, therefore,
that the unknown causative agents were present in the fractionated derivatives
of activated sludge.
29
-------
TABLE VI. DETERMINATION OF CONCENTRATION OF MIXED SUPERNATANT
DERIVATIVE YIELDING OPTIMAL RETENTION OF IMMOBILIZED
ENZYME ACTIVITY
Description
Lyophilized
Concentration
Dilution A
Dilution B
Dilution C
Original Mixed
Supernatants
Gel Derivative
of Concentrate
Gel Derivative
of A
Gel Derivative
of B
Gel Derivative
of C
Gel Derivative of
Original Mixed
Supernatant
mg protein per
ml of
enzyme source
61.2
36.2
27.6
9.72
2.85
Esterase
Activity
330/ml
197/ml
113/ml
58. 8/ml
41. 2/ml
6.62/gm
3. 01/gm
1. 59/gm
1.33/gm
0.90/gm
Lipase
Activity
27 8/ml
180/ml
406/ml
52. 0/ml
42. 0/ml
6.00/gm
2. 56/gm
1. 71/gm
2. 82/gm
1. 62/gm
Physical
Characteristics
of Gel
Viscous, sticky
Heterogeneous
Viscous, sticky
Slightly less
Viscous
Homogeneous
Gels semi-firm
Characteristics
Firm, Clear Gel
On the basis of these results, the mixed supernatant fraction was lyophilized
and adjusted to final concentration of 12 mg protein/ml. The latter prepara-
tion was then used for immobilization according to the proportions 5ml en-
zyme preparation, 5 ml MBA, 20 ml acrylamide, 0.5 ml persulfate and 1. 0
ml riboflavin. As previously stated, attempts to increase the reaction volume
beyond 30 ml had entailed a disproportional increase in the time required to
complete polymerization and an observed loss in recoverable enzyme activity.
30
-------
Using the above proportions for polymerization a total volume of 1 1/2 liters
of immobilized enzyme gel was prepared over a period of 9 days. In addition,
two different control polyacrylamide gels were prepared. The first of these
consisted of the standard polymerization components to which distilled water
was added instead of the enzyme source. The second control preparation en-
tailed the substitution of bovine serum albumin (BSA, 12 mg/ml) for the en-
zymatic component. It is important to note that an absolutely valid control
for the enzymatically active immobilized gel is extremely difficult to obtain.
The boiling of a gel to destroy enzymatic activity presents two potential dis-
advantages. First of all the structural integrity of the gel itself may be
altered and secondly, a structural alteration of the enzymes (denaturization)
definitely occurs. The potential ramifications include loss of small (or even
large) molecular weight components into the system, or conversely a possible
increased absorption of soluble components from the assay system. The
acrylamide gel alone can show possible non-enzymatic effects that the poly-
acrylamide has on the system. However, since the polymerization system
lacks the "structuring effect" mediated by the sludge derivative, the gel type
is not necessarily identical to that which entrapped enzymes. Similarly, the
entrapped BSA can potentially serve to mimic an immobilized protein without
enzymatic activity. On the other hand it also lacks the ability of the enzyme
derivative to vastly effect gel structure.
At the completion of these preparations, the gels were stored at 4 C for an
additional two weeks. During this time various biochemical assays were
performed on the immobilized derivatives of activated sludge. The assays used
were the standard esterase and liapse fluorometric assays, the casein test for
proteolytic activity, and the starch-iodine test for amylase activity. In both
the fluorometric and the amylytic assays the control used was a heat-inactivated
sample of the immobilized enzyme gel. The latter was prepared by subjecting
the gel to a boiling water bath for 20 minutes after which the gel was thoroughly
washed to remove any components which might have initially been solubilized
by this procedure. The caseinolytic assay provides for the control to consist
of incubation of substrate and enzyme source separately and then combination
of the two components in the presence of 10% trichloracetic acid (TCA). All
assays also include a calibration curve for a commercially obtained enzyme
source, and the gel activity was therefore computed in terms of activity re-
lative to that of the standard enzyme. And finally, a preliminary "interference-
curve" was also obtained to determine the effect (i.e., quenching of fluores-
cence, non-specific absorption of substrate or product, etc.) that the gel it-
self might have upon the particular assay system. The effect of the gel was
determined by combining a constant concentration of the commercial enzyme
with an assay system to which varying amounts (gms) of gel were also added.
The fluorescence of absorption between the system to which no gel was added
and those with increasing quantities of gel were then compared. The data which
was obtained enabled the investigator to correct upwards the experimental
values obtained for the enzymatic activity of the immobilized sludge derivative.
An example of the effect of correcting for interferences is found in Figure 5.
31
-------
18r
16
14
12
10
c
'E
X - Corrected Values
0 - Uncorrected Values
0.5 1.0
Immobilized Gel (gms)
0
2.0
Figure 5. The Effect of Correcting for Non-Specific Interference Upon Detection of
Esterase Activity Immobilized in Polyacrylamide Gels
32
-------
However, it must be emphasized that even with this correction for interference
one cannot directly equate the activity of a soluble system with that exhibited
by the immobilized polyacrylamide gel. The inherent problem this system
presents is one of access. The gel matrix presents an obstacle across which
potential substrate molecules must pass. The difficulty with which passage is
accomplished is primarily a function of the degree of cross-linking between
the two copolymers and as previously stated is also tremendously influenced by
the components of the preparation immobilized within the polyacrylamide gel.
However, the further delineation of the effects of these factors and of their
combinations upon accessibility represents a major investigational problem in
itself, and is, therefore, far beyond the scope of this study.
With these reservations in mind, Table VH presents a summary of the results
of experiments performed to determine the enzymatic activity retained by the
gel. The activity of the gel was corrected for interference and then compared
to the soluble system in order to determine an estimated percentage of activity
retained. It is of interest that at least in Bernfelds (Ref 16) work only a 3-6%
retention of enzyme activity among various enzymes was observed. The single
disturbing factor is the low retention of proteolytic activity. In previous work
this class of enzymes had proven resistant to any and all inactivational forces
mediated by either fractionation or immobilization techniques. The investigation
to determine rationale behind the observed loss in activity is beyond the immed-
iate scope of this contract, and it is felt that it might prove to be an exception
within the probable range of proteolytic enzyme recovery.
The final experiments conducted to complete this phase of the contract include
analysis of a sample of the gel to determine the enzymatic response to repetitive
use. In summary initial experiments using the lipase and esterase systems
indicated that when the gel source was removed from a depleted assay system,
thoroughly washed, and recombined with additional substrate, the maximal rate
of enzymatic activity was once again observed. The immobilized gel samples
were tested a total of 7 times within a two day period for these preliminary
findings. In terms of storage, the enzymatic activity was retained for up to 5
weeks if the gel was maintained in a liquid (distilled water or buffer) environment
and kept refrigerated. No attempts were made at this time to determine the ef-
fects of either lyophilization or freezing upon the enzyme activity or the physical
integrity of the immobilized gel.
TABLE VII. COMPARATIVE SUMMARY OF ENZYME ACTIVITY RETAINED
AFTER IMMOBILIZATION OF A POLYACRYLAMIDE GEL
Enzyme
Activity
Esterase
Lipase
Amylase
Proteolytic
Commercial
Enzyme
Standard
Acetylcholinesterase
Lipase Steapsin
Alpha Amylase
Protein ase
mg Std
Enzyme Per ml
Lyophilized Prep*
0.0718
0.114
0.173
0.912
mg Std
Enzyme Per gm
Immob Gel
0.006
0.019
0.010
0.008
Esti-
mated
Retention
8.41%
18.50%
5.78%
0.88%
*The concentrated lyophilizate is diluted with buffer to duplicate the dilution to
which it is subjected during the immobilization procedure.
33
-------
PRELIMINARY BENCH-SCALE ACTIVATED SLUDGE INVESTIGATIONS
Construction and Operation of Apparatus
Completely mixed reactors, consisting of two ten gallon aquarium tanks for
the Series 1 experiment and three ten gallon aquarium tanks for the Series 2
experiment; in which the liquid volume in each tank was maintained at 18. 925
liters for Series 1 and 35 liters for Series 2. The reactor contents were
mixed with 2-inch X 1-inch stirring paddles with a propeller on the end of the
mixer shaft, driven at 150 rpm. Laboratory compressed air was introduced
to the reactors at a rate of 1400 cc/min for Series 1 and 500 cc/min for Series
2 through porous stone diffusers after passing through an oil trap, an adsorber
containing Purafil to remove any organics, a microbial filter, water scrubbers
and rotameters.
A pictorial diagram of a typical reactor system is shown in Figure 6 (Series 2),
Substrate
The substrate for the laboratory experiments was raw sewage obtained from the
diverter feeding Grumman's extended aeration sewage treatment plant,, Raw
sewage samples were obtained on different days for Series 1 and Series 2 exper-
iments.
Activated Sludge
Activated sludge cultures were obtained directly from the return sludge line
to the aeration tank in Grumman's extended aeration waste treatment plant.
Activated sludge samples were obtained on different days for Series 1 and
Series 2 experiments.
LABORATORY PROCEDURES AND ANALYSES
In the Series 1 experiment, 3. 785 liters of sludge and 15. 14 liters of raw
sewage were added to each of the two tanks. A nylon stocking containing
400 ml of polyacrylamide gel was suspended in Tank I and a similar nylon stock-
ing containing the immobilized enzymes in the polyacrylamide gel was suspended
in Tank II.
The sludge and raw sewage were sampled both prior to and after being mixed.
After suspending the nylon stockings in each tank, samples were taken every
hour for 8 hours and at the end of 24, 28 and 32 hours. These samples were
analyzed for chemical oxygen demand (COD) and suspended solids according to
"Standard Methods". Soluble COD was used as the substrate parameter in these
experiments. The soluble constituents were separated from cell material by
centrifuging the mixed liquor sample at 15,000 rpm for 5 minutes and then analy-
zing the centrate for COD. Cell material in this study was determined as
volatile suspended solids (VSS)0
34
-------
Mixer
Adsorber
Water Scrubber \ / Tank!
Aeration Stones
(Poly)
Microbial Filter
lank HI
Figure 6. Series 2 Experimental Set-Up
35
-------
A second laboratory scale study (Series 2) was conducted using three 10 gallon
tanks equipped as in Series 1. The same polyacrylamides (with and without
the enzymes) that were used in Series 1 were employed again because of insuf-
ficient time to acquire new supplies.
Seven liters of sludges and 28 liters of raw sewage were added to each of the
three tanks. A nylon stocking containing 200 ml of the polyacrylamide gel with
enzymes was suspended in Tank III, and a similar stocking containing 200 ml of
polyacrylamide gel was suspended in Tank IT. Tank I contained only the sludge
and raw sewage.
Samples of the sludge, raw sewage and mixed liquor were obtained before sus-
pending the nylon stockings. After suspending the nylon stockings containing
polyacrylamide gel with and without enzymes, the three tanks were then sampled
every hour for 8 hours and at the end of 25 hours and 31 hours. These samples
were analyzed according to "Standard Methods" for pH, temperature, COD
(soluble and mixed liquor), suspended solids and alkalinity.
Results
Series 1 experimental results are presented in Table VIII. Plots of COD and
Volatile Suspended Solids (VSS) as a function of aeration time are shown in
Figures 7 and 80 The difference in COD between the two tanks were notice-
able and suspect since the COD of the tank containing polyacrylamide gel was
steadily increasing over that of the tank containing polyacrylamide gel with
enzymes. The polyacrylamide gel was removed from the tank and thoroughly
rinsed with distilled water several times. The final rinse w'ater was checked
for soluble COD and found to exert a COD of approximately 15, 000 ppm. Similar
results were obtained from the polyacrylamide gel with enzymes. The cause of
this could be due to the incomplete polymerization of the polyacrylamide gel
thus allowing acrylamide to go into solution; increasing the COD and Volatile
Suspended Solids. Since the samples were centrifuged at high speeds, it is
unlikely that the high COD readings are due to gel in the sample tested.
It is also possible that the organic constituent, acrylamide may be resistant
to biodegradation even though it is readily oxidized in the dichromate COD
procedure,, This material may not contribute measurably to the growth of the
activated sludge organisms. There is an indication of this since the Volatile
Suspended Solid did not appear to increase as might have been expected.
Series 2 experimental results are presented in Table DC. Plots of soluble COD,
mixed liquor COD and Volatile suspended solids (VSS) are shown in Figure 9,
10 and 11.
The sludge had a low pH indicating that it was acidic. This condition tends to
inhibit the biological activity of the sludge. Studies have shown that a sludge
having a pH of 70 5 to 8. 0 gives the best results in an activated sludge digestion
system.
A comparison of the COD and VSS data from the three tanks is inconclusive as
to the effect of immobilized enzyme on the biodegradation of sewage. The
36
-------
erratic results obtained may be attributed in part to an improper activated sludge
sample, incomplete polymerization of the polyacrylamide gel and possible de-
naturation or inhibition of the enzymes.
37
-------
TABLE VIE. SERIES 1 EXPERIMENTAL RESULTS
OS
00
Date
4/8
4/9
Time
0815
0915
1015
1115
1215
1315
1415
1515
1615
0815
1215
1615
Identification
Sludge
Raw Sewage I
Raw Sewage n
Mixed Liquor I
Mixed Liquor IT
Tank I (Poly) 1
Tank II (Poly + Enz)
Tank I
TankH
Tank I
Tank II
Tank I
Tank II
Tank I
Tank II
Tank I
Tank II
Tank I
TankH
Tank I
TankH
Tank I
TankH
Tank I
Tank II
Tank I
TankH
COD
(ppm)
106
267
288
352
338
354
356
348
327
395
333
426
300
420
343
441
331
462
352
472
320
644
370
602
370
544
814
Total
Suspended
Solids (ppm)
3896
468
428
756
796
792
852
772
756
796
772
764
716
752
756
784
732
744
700
752
700
728
680
700
720
652
704
Volatile
Suspended
Solids (ppm)
Bio-Mass
3504
436
400
676
700
680
736
672
672
728
692
696
684
664
Lost
712
668
668
656
688
668
708
652
748
644
612
672
Fixed
Suspended
Solids (ppm)
392
32
28
80
96
112
116
100
84
68
80
68
32
88
72
64
76
44
64
32
20
28
52
76
40
32
-------
a.
o_
CO
760 r
740 -
720
700
ID 680 -
C
CD
Q.
c/>
CO
CD
O
>
660 -
640 -
620-
600
TankH
— —o Tank
0
12 16 20
Aeration Time, hr
24
28
32
Figure 7. Series 1 Experimental Results, Volatile Suspended Solids vs Aeration Time
39
-------
§:
cT
o
O
C/3
1000.-
900 -
800 -
700
600
500
400
300
200
100
•A Tank IT
o— — -o TankI
8
12 16 20 24 28 32
Aeration Time, hr
Figure 8. Series 1 Experimental Results, Soluble COD vs Aeration Time
40
-------
TABLE IX. SERIES 2 EXPERIMENTAL RESULTS
Identification
Sludge
Raw Sewage I
Raw Sewage II
Raw Sewage III
Mixed Liquor I
Mixed Liquor II
Mixed Liquor III
Tank I (Plain)
II (Poly)
m (Poly + Enz)
I
n
m
I
n
m
i
n
m
i
n
m
i
n
m
i
n
m
i
n
ni
i
n
m
i
n
m
i
n
m
Datfi
4/21
4/21
4/22
4/22
4/22
4/22
4/22
4/22
Time
0730
0800
0800
0815
0915
1015
1115
1215
1315
1415
1515
1615
0930
1530
1530
1530
pH
5.90
Y.75
7.89
7.72
7.21
7.30
7.25
7.21
7.33
7.24
7.21
7.28
7.25
7.23
7.28
7.24
7.24
7.30
7.27
7.27
7.30
7.28
7.23
7.30
7.25
7.27
7.31
7.31
7.28
7.24
7.25
7.24
7.27
7.25
7.24
7.28
7.26
7.21
7.14
7.16
Alka-
Linity
(ppm
Ca OO3)
27.5
212.8
208.9
209.5
167.0
172.0
163.4
169.4
168 1
164.9
172.1
176.5
168.3
168.0
174.4
172.0
171.1
176.3
172.5
177.6
171.5
172.4
174.6
177.8
174.5
180.0
180.0
176.6
176.6
180.5
176.9
176.5
182.5
177.5
176.0
183.2
178.0
182.9
181.2
176.5
Temp.
(°C)
23.4
23.2
23.4
23.4
23.3
23.5
23.5
23.5
23.6
23.7
23.6
23.8
23.9
23.8
24.0
24.0
23.9
24.1
24.0
23.9
24.1
23.9
23.8
24.0
23.8
23.6
24.0
COD
Super -
N at ant
(ppm)
3760
168
160
176
908
880
992
872
900
980
840
832
960
840
852
960
852
860
960
840
852
956
840
860
960
840
860
920
812
836
928
820
820
940
780
840
820
748
800
800
COD
Mixed
Liquor
(ppm)
8600
500
688
500
2280
2372
2440
2292
2432
2420
2352
2320
2392
2352
2268
2328
2300
2288
2368
2328
2400
2328
2300
2408
2408
2308
2388
2388
2308
2280
2448
2212
2112
2304
2212
2192
2212
2112
2168
2300
Suspended Solids (ppm)
Total
2952
280
396
256
1100
908
1300
1128
1008
1004
1068
964
1012
1052
1084
1040
1260
1044
1012
1104
1176
1094
960
1016
944
976
1084
1012
1028
1048
992
984
1260
988
996
1008
900
1188
1016
1024
Volatile
2712
244
372
224
1024
824
1220
1060
960
968
1036
908
936
1032
1028
984
1180
940
916
988
1144
948
892
916
856
900
984
908
968
956
940
908
1172
892
908
892
840
1100
920
952
Fixed
240
36
24
32
76
84
80
68
48
36
32
56
76
20
56
56
80
104
96
116
32
96
68
100
88
76
100
104
60
92
52
76
88
96
88
116
60
88
96
72
-------
Q-
D_
Q"
o
o
.Q
J3
O
C/D
800 -
700
o
O— -—OTankUI
Tankn
o- —o TankI
I
I
12 16 20
Aeration Time, hr
24
28
32
Figure 9. Series 2 Experimental Results, Soluble COD vs Aeration Time
-------
2500 r
Q-
Q.
Q"
o
o
1_
o
3
cr
T3
0}
X
2400
2300
2200 -
2100
0
O—— —
• Tankin
Tankll
Tank I
I
12 16 20
Aeration Time, hr
24
28
32
Figure 10. Series 2 Experimental Results, Mixed Liquor COD vs Aeration Time
43
-------
1300r
1200
1100
Q.
Q_
72 1000
o
CO
•a
OJ
T3
c
OJ
d
CO
JE
o
>
900
^ 8001-
700
600
500
0 2
O—-—O TankEI
£* «^ TankE
O- —O Tank I
8 10 12 14 16 18 20 22 24 26 28 30 32
Aeration Time, hr
Figure 11. Series 2 Experimental Results, Volatile Suspended Solids vs Aeration Time
44
-------
SECTION VI
ACKNO WLE DGME NT S
The preparation and evaluation of the immobilized enzyme system was carried
out by Mason Research Institute under subcontract to Grumman Aerospace
Corporation. Dr. Robert G. Sanders was the principal investigator for Mason
Research Institute.
The construction of the bench-scale activated sludge units and the testing of the
systems was done by Mr. Henry Lowman in the Biotechnology Laboratory at
Grumman Aerospace Corporation.
The overall project was under the direction of Dr. Lawrence Slote, Advanced
Civil Systems of Grumman Aerospace Corporation.
The support of the project by the Federal Water Quality Administration and the
help provided by Dr. Robert L. Bunch is acknowledged with sincere thanks.
45
-------
SECTION VII
REFERENCES
1. Krishnan, P. and Gaudy, A.F., "Mechanisms and Kinetics of Substrate
"Utilization at High Biological Solids Concentrations," Proceedings 21st
Industrial Waste Conference, Purdue University Extension Series, 1966
2. Siddiqi, R.H., et. al. , "The Role of Enzymes in the Contact Stabilization
Process," The 3rd International Conference on Water Pollution Research
n-15, Munich, Germany 1966
3. Chamberlin, N.S., "Action of Enzymes on Sewage Solids, " New Jersey
Agriculture Experimental Station Bull. 500, 1930
4. Heukelekian, H. and Berger, M., "Value of Culture and Enzyme Additions
in Promoting Digestion, " Sew. Ind. Wastes. _25(11):1259 1953
5. Ingols, R.S., "A Study of Hydrolytic Enzymes in Activated Sludge, " New
Jersey Agriculture Experimental Station Bull. 66, 1939
6. Ingols, R.S., "Oxidation-Reduction Enzymes in Activated Sludge, " Sew.
Wks. J., 12(5):862 1940
7. McKinney, R0N0 and Sawyer, C.N., "Wonder Drugs - Enzymes - Will
They Cure Waste Treatment His? A Critical Look at the Modern'Panaceas'
for Sewage Treatment," Eng. News Record, 151(14) :34 Oct. 1, 1953
8. Rudolfs, W., "Enzymes and Sludge Digestion," Sew. Wks. J., 4_(5):782-89
1932
9. Wooldridge, W.R. and Standfast, A.B0, "The Role of Enzymes in Activated
Sludge and Sewage Oxidations," Biochem. J., 30:1542-53, 1942
10. Updike, S.J, and Hicks, G.P., "The Enzyme Electrode," Nature, 214:986-88
June 3, 1967
11. Hicks, G.P. and Updike, S.J., "The Preparation and Characterization of
Lyophilized Polyacrylamide Enzyme Gels for Chemical Analysis," Anal.
Chem., 38(6):727-30 May 1966
12. Bernfeld, P. and Bieber, R0E., "Water-insoluble Enzymes:Kinetics of
Rabbit Muscle Enolase Embedded within an Insoluble Carrier, " Arch.
Biochem. Biophysics, 131:587-78 1969
13. Bernfeld, P., et. al., "Kinetics of Water-insoluble Phosphoglycerate
Mutase, " Biochim. Biophysica Acta, 191:570-78 1969
14. Goodfriend, T., et. al., "Antibody in Polyacrylamide Gel, A Solid Reagent
for Radioimmunoassay," Immunochemistry, 6(3):481-4, 1969
47
-------
15. Bauman, E.K., et0al0, " Preparation of Immobilized Cholinesterase for
use in Analytical Chemistry, " Anal. Chem., 37(11) :1378-81, Oct. 1965
16. Bernfeld, P0 and Wan, J0, "Antigens and Enzymes Made Insoluble by
Entrapping Them into Lattices of Synthetic Polymers, " Science, 142:678-
79, Feb. 1963
17. Bernfeld, P., et. al., "Water-insoluble Enzymes rArrangement of Aldolase
with an Insoluble Carrier, " Arch. Biochem. Biophysics, 127:779-86 1968
18. Silman, I.H. and Katchalski, E., "Water-insoluble Derivatives of Enzymes
and Antibodies, " Ann. Rev. Biochem., 35_(2):873-908 1966
19. Rinderknecht, H., et. al., "A New Ultrasensitive Method for the Deter-
mination of Proteolytic Activity," Clin. Chim. Acta, _21:197-203 Aug.
1968
20. Rosenkrantz, H. and Kirdani, E., "Proteolytic Enzymes of Canine Pros-
tatic Fluid," Cancer Chemotherapy Report, No. 15, 9, 1961
21. Rinderknecht, H., et. al., "A New Method for the Determination of a
amylase," Experientia, 23(10):805 1967
22. Banerji, S.K., et. al., "Kinetics of Removal of Starch in Activated Sludge
Systems," Journ. Water Pollution Control Federation, 40(l):16-29, Jan.
1968, 40, (2) :161-73 Feb. 1968
23. McCready, R.M., et.al., "Determination of Starch and Amylase in
Vegetables," Anal. Chem. , 22(9):ll56-58 Sept. 1950
24. Guilbault, G0C. and Kramer, D.N., "Fluorometric Procedure for Mea-
suring the Activity of Dehydrogenases," Anal. Chem. 37(10):1378-81
Oct. 1965
25. Guilbault, G0C0, et.al., "Evaluation of Fluorometric Substrates for
Lipase," Anal. Letters, l(9):551-63, 1968
26. Guilbault, C.G., et.al., "N-Methylindoxyl esters as Substrates for
Cholinesterase," Anal. Letters, 31(6):365-79 1968
27. Ashwell, G., Methods in Enzymology, Colowick and Kaplan, editors,
Academic Press, New York, _3_:87, 1957
28. Dische, Z., "Color Reactions of Nucleic Acid Components," The Nucleic
Acids:Chemistry and Biology, Chargaff, E. and Davidson, J. N., editors,
Academic Press, New York, 1:285-305 1955
29. Gornall, A.G., et. al., "Determination of Serum Proteins by Means of
the Biuret Reaction, " J. Biol. Chem., 17J7:751-66 1948
30. Kingsley, G.R., "The Determination of Serum Total Protein, Albumin
and Globulin by the Biuret Reaction, " J. Biol. Chem., 131:197-200 1939
48
-------
SECTION VIE
APPENDIX
PROTEOLYTIC ACTIVITY #1 (INSOLUBLE SUBSTRATE) (REF. 19)
Reagents; RBB-Hide Protein (C albiochem)
Tris buffer, 0005M, pH 7. 8
Proteinase (2 mg/ml Tris buffer)
Enzyme Source (unknowns)
Standard Curve
1. Weigh out 20 mg RBB-Hide Protein for each of a series of duplicate
25 ml Erlenmeyer flasks .
2. Add Tris-buffer and proteinase solutions to the flasks as stipulated
below:
Final Enzyme Cone
10 mg
8 mg
6 mg
4 mg
2 mg
1 mg
0.2 mg
ml Proteinase
5.0
4.0
3.0
2.0
1.0
0.5
0.1
ml Tris Buffer
-
1.0
2.0
3.0
4.0
4.5
4.9
3. Incubate flasks in a Dubanoff shaker at 37°C for 30 Minutes.
4. The reaction is stopped by filtering the suspension through glass wool.
5. The absorption of the individual samples is then determined using a
Coleman Junior Spectrophotometer - 595 m/u.
Unknowns
1. Set up duplicate flasks for each concentration of the unknown to be tested
and add 20 mg RBB-Hide to each flask.
2. The addition of buffer and the enzyme source are added as previously
described such that the final concentration totals 5 ml.
3. Unknown samples previously boiled for 10 minutes are used as blanks,
in addition to one set of flasks to which no enzyme is added.
49
-------
4. Run standard enzymes (proteinase) at 5, 2 and 1 mg total concentration.
Incubate samples as described and stop reaction by filtration.
5. Absorption is determined at 595 mM
6. Substrate saturation is verified by the observation of increases in enzyme
concentration yielding proportional absorption increases.
50
-------
PROTEOLYTIC ACTIVITY #2 (SOLUBLE SUBSTRATE SYSTEM) (REF. 20)
Reagents
lo Saturated casein solution was prepared by dissolving 1.3 gm
Hammerstein . grade casein in 100 ml 0.05M Tris-buffer, pH 7. 8 and
heating in a boiling water bath for 20 minutes. The suspension is then
centrifuged at 2,000 X G for 15 minutes to remove any excess particu-
late matter remaining. (Can be refrigerated for up to 5 days).
2. Tris buffer - 0.05M, pH 7.8.
3. Enzyme source (unknown and protease at 10 mgm/ml).
4. 10% Trichloracetic Acid (TCA)
Calibration Curve and Unknown Assay
1. Duplicate sets of four test tubes are set up for each enzyme concentra-
tion and to each of three is added 2 ml of the casein solution.
2. Tris buffer is then added to each of three of the test tubes such that,
upon addition of enzyme, the total volume would be 7 ml.
3. A standard enzyme preparation is then added to two of the tubes con-
taining the casein, and the same quantity is added to the as yet empty
tube remaining.
4. The tubes are incubated at 37° for one hour in a Dubanoff shaker.
5. To the three enzyme containing tubes 3 ml of 10% TCA is added to stop
the reaction. Subsequently, the tube containing the buffer and casein
is combined immediately with the one containing enzyme and TCA alone.
6. The suspensions are then centrifuged, the ppt discarded, and the ab-
sorption of the supernatants read at 280 mAi on the Beckman DU
Spectrophotometer to determine the amino acids enzymatically re-
leased.
7. The running of a blank in this manner allowed for an accurate deter-
mination of both the possible non-enzyme partial breakdown of casein
and also the possible non-specific release of entrapped proteins from
the gel during the hour long incubation.
8. The protocol adopted essentially represents that employed in the paper
cited (Ref. 20) with only the above modification for blank determinations
and also an adjustment upward in total assay volume.
51
-------
AMYLYTIC ACTIVITY #1 (INSOLUBLE SUBSTRATE) (REF. 21)
Reagents RBB - Starch (C albiochem)
Tris buffer (0.05M, pH 7.0) - 0. 5M NaCl
Amylase (1 nag/ml)
Enzyme Source (unknowns)
Standard Curve
1. 2 ml of a 2% suspension of RBB-starch (0. 05M Tris buffer, pH 7. 0
0.5M NaCl) is pipetted into a series of duplicate 25 ml Erlenmeyer
flasks.
2. Add Tris buffer and amylase to the flasks as stipulated below:
Final Cone
5
4
3
2
1
0.5
0.1
ml Amylase
5.0
4.0
3.0
2.0
1.0
Oo5
0.1
ml Tris buffer
_
1.0
2.0
3.0
4.0
4.5
4.9
3. The flasks are incubated in a Dubanoff shaker for 30 minutes at 37°C.
4, Initially the reaction was stopped by the addition of acetic acid followed
by filtration but later filtration alone via S. and S, prefolded filters
was used. The absorption of the samples at 595 HIM was determined
with the use of a Coleman Jr. Spectrophotometer,
Unknowns
1. Set up duplicate flasks for each concentration of the unknown to be
tested and add 20 mg RBB-Hide to each flask.
20 The addition of buffer and the enzyme source are added as previously
described such that the final concentration totals 5 ml.
3. Unknown samples previously boiled for 10 minutes are used as blanks,
in addition to one set of flasks to which no enzyme is added.
4. Run standard enzyme (alpha amylase) at 5, 2 and 1 mg total concentra-
tion. Incubate samples as described and stop reaction via filtration.
5. Absorption is determined at 595 m/u .
6. Substrate saturation is verified by the observation of increases in
enzyme concentration yielding proportional absorption increases,,
52
-------
AMYLYTIC ACTIVITY #2 (SOLUBLE SUBSTRATE) (REF. 22 and 23)
Reagents
1. 1 mg/ml starch (soluble starch for lodometry) in 0. 05M Tris - 0. 05M
NaCl pH 7,0,
2. 0.05M Tris buffer - 0.05 NaCl, pH 7.0.
3. KI-I color reagent.
Calibration
1. From 0.1 ml to 7 ml of the starch solution is added to duplicate test
tubes.
2. Using the Tris buffer, the total volume is adjusted to 7 ml.
3. The tubes are incubated for 15 min at 37° and 0.3 ml of KI-L, color
reagent is added.
4. The tubes are incubated an additional 15 min and the absorption at
650 m/i determined with a Coleman Jr. Spectrophotometer.
Assay
!„ 2 ml of the starch solution is added to each tube.
2, The duplicate unknowns are assayed according to the same protocol
as described above at concentration of 0-5 ml.
3. The final volume of all tubes is adjusted to 7 ml with Tris buffer.
4, Blanks are run using enzyme preparations previously heated in a
boiling water bath for 15 minutes.
5. In addition, standard amylase concentrations of 5, 3, 1 and 0.5 mg
are run with all assay systems.
6. Problems inherent in the system include the inability to easily stop
reaction at the end of the 15 min. incubation, and also the occasional
presence of contaminating substances in the sonicate derivatives
which interfered with the reaction.
53
-------
DEHYDBOGENASE ASSAY (Eef. 24)
Reagents
_2
I. Krebs acid intermediate soln (substrate) (10 PO.,pH 8)
_p
2. DPN, + TPN+ soln (confactor) (10 HgO soln)
_3
3. PMS soln (electron acceptor) (light sensitive) (10 , HO)
&
_2
4. Resazurin (fluorescent source) (10 M in cellosolve)
5. PO4 buffer, 0.1M, pH 8.0
6. Isocitric DH. in glycerol
7. Liver Homogenate - 25% in PO4 buffer, 001M, pH 8. 0
Substrate Mixture 1 ml DPN, TPN
1 ml Resazurin
Ues 2 ml/assay
10 ml PMS
KEEP PROTECTED FROM
20 ml Kreb intermed. LIGHT! !
Fluorometer Setting Ex 560 raff}
/ Scalar 10
Em 580 m/yj
Protocol 1. Obtain reading on substrate mixture alone
2. Add enz., invert to mix, replace for reading
3. Obtain reading on enzyme-substrate mixture rate
Series Procedure !„ Substrate + liver homogenate 1 (0.1ml 25%)
2. Substrate + liver homogenate 2 (0.2 ml 25%)
3. Substrate + isocitric DH (0. 5 ml)
4. Substrate + sample cone. 1 (0, 1)
50 Substrate + sample cone. 2 (0»2)
6. Substrate + sample cone. 3 (0.5)
7. Substrate + cone. 1,—» 2,—» 3 boiled WC preparation
0.1, +0.1, +0.3 ml
54
-------
LIPASE ASSAY (Eef. 25)
Reagents
— fi
1. Fluorescein Dibutyrate (1 X 10 M, cellosolve)
2. PO4 buffer 0.1M, pH 6.5
Substrate Mixture 1 ml fluorescein dibutyrate "^
> Use 2 ml/assay
29 ml PO4 buffer, 0.1M, pH 6. 5 J
Fluorometer l"/min chart speed
10 Scalar
Protocol 1. Obtain reagent blank reading c 2 ml substrate mixture
2. Add enz. prep., invert to mix, replace
3. Obtain rate on substrate-enzyme interaction
Series Procedure
1. Substrate mixture + 0.1 ml lipase
2. Substrate mixture + 00 3 ml lipase
3. Substrate mixture + 0.1 ml fraction sample
4. Substrate mixture + 0.3 ml fraction sample
5. Substrate mixture + 0.5 ml fraction sample
6. Substrate mixture + boiled enzyme —»0.3 —>0.5
(0.1ml; + 0. 2 ml; + 0.2 ml)
Note: Both the standard enzyme and the fluorescein dibutyrate are made fresh
daily.
55
-------
ESTERASE ASSAY (Eef. 26)
Reagents
1. Indoxyl acetate (0.02M in cellosolve)
2. PO4 buffer, 0. 1M, pH 7. 5
3. ENZ - acetylcholinesterase, alkaline phosphatase, Lipase (all mg/ml)
Substrate Mixture 1 ml indoxyl acetate ^\
> Use 2 ml/assay
29 ml PO4 buffer pH 7. 5 J
Fluorometer Ex 395 m/u. I"/ min chart speed
Em 470 mjj. Scalar 1
Protocol 1. Obtain reading on substrate mixture
2. Remove, add enz., invert to mix, return
3. Obtain reading on enzyme-substrate mixture
Series Procedure
!„ Acetylcholinesterase +2 ml substrate mix (0.1 ml)
2, Acetylcholinesterase +2 ml substrate mix (0. 2 ml)
3, Alkaline phosphatase +2 ml substrate mix (0.5 ml)
4. Lipase + 2 ml substrate mix (0. 5 ml)
5. Fraction sample + 2 ml substrate mix (0.1)
6. Fraction sample + 2 ml substrate mix (0. 2)
7. Fraction sample + 2 ml substrate mix (0.5)
8. Boiled fraction sample + 2 ml substrate mix (0.1, +0.1, +0.3)
56
-------
ENA ANALYSIS (Ref. 27)
Reagents
1. 10% TCA, 5% TCA
2. ETOH: ether (3:1)
3. Abs ETOH, 95% ETOH
4. ETOH: Chloroform (3:1)
5. Orcinol reagent - 6% orcinol in 95% ETOH
6. Ferric chloride reagent (10%) 1 gm Fed . 6H0O in 10 ml H0O
O £1 £i
7. RNA Sources (Std. yeast 10 ,U g/ml and Unknowns)
Preparation of RNA Fraction from Unknowns
1. Precipitate a 5 ml aliquot of sonicate and/or derivative with an equal
volume of 10% cold TCA and allow to stand overnight at 4°C0
2. Centrifuge the suspension for 15 min at 2, 000 rpm - 5°C (IEC) and
discard the supernatant.
3. Wash the pellet 3 times with 2 ml of 95% ETOH.
4. Wash the pellet 2 times with 2 ml ETOH: chloroform (3:1)
5. Wash once with 2 ml of ETOH: ether (3:1) and once with ether alone
6. The solvent is removed after washing by a 10 min centrifugation at
2,000 rpm, -5°C (IEC)
7. The pellet is dried in an open vacuum dessicator for 3 min, the vacuum
valve is then closed, the sample pellets stored overnight in the cold
room (5°C)
8. To each of the dry pellets, 3 ml of hot (50° C) 5% TCA is added and
the tubes maintained at 90°C for 15 minutes. The tubes are centrifug-
ed for 15 min at 2,000 rpm, and the supernatant decanted and saved.
9. To the pellet an additional 1 ml of hot 5% TCA is added and the above
extraction repeated. The supernatant is added to that obtained in the
previous step. The pellet is discarded.
57
-------
Assay (Standards and Unknowns)
10 Duplicate 1. 5 ml samples of the unknowns are placed in 19 x 105
Coleman pH tubes and 0. 5 ml distilled water added.
2. A reagent blank employeng 2. 0 ml distilled water is prepared.
3. Known concentrations of RNA are added to Coleman tubes and the
volume adjusted to a total of 1. 5 ml with distilled water.
4. 0. 2 ml of orcinol reagent and 3 ml ferric chloride solution are added
to all tubes and the samples incubated in a boiling water bath for
10 min.
5. The samples are allowed to cool for 5 min at 0°C and 1.5 ml absolute
ETOH is added to prevent turbidity.
6. The absorption of the samples at 670 HIM is then determined with a
Beckman DU Spectrophotometer.
58
-------
DNA ANALYSIS (Ref. 28)
Reagents
1. 10% TCA, 5% TCA
2. Abs ETOH, 95% ETOH
3. ETOH: ether (3:1)
4. ETOH: chloroform (3:1)
5. DNA sources (Std0 Salmon sperm 100 /u g/ml) in 5% TCA and un-
knowns)
6. Diphenylamine - 1% solution in glacial acetic acid to which 2. 75 ml
H SO is added
Preparation of DNA Fraction from Unknown
(Same protocol as that followed for RNA extraction)
Assay Standards and Unknowns
10 Standard DNA concentrations of from 5-100 /u -g and a maximum volume
of 1 ml are pipetted into Coleman test tubes.
2. Duplicate 1 ml samples of the unknowns are added to the test tubes.
3. 2. 0 ml of diphenylamine reagent is added to each tube and the samples
heated in a boiling water bath for 10 minutes.
4. After cooling the absorption of the samples are determined at 595 m/z
with a Coleman Spectrophotometer.
59
-------
TOTAL PROTEIN - BIURET REACTION (Ref 29 and 30)
Materials:
Volumetric flask - 25 ml
0.1 ml pipettes, graduated to top
15 x 125 mm test tubes
B&L calibrated 4-inch tube type cuvette
Reagents:
Harleco Biuret Reagent
Standard
Albumin Bovine (Armour & Co., Chicago)
Std Soln: 100 mg/ml
Weigh carefully 2. 5 gm albumin and dilute with water
in 25 ml volume flask. Store in refrigerator
Standard Curve:
1.
To prepare a standard curve:
Pipette in duplicate into clean 15 x 125 mm test tubes as follows:
Cone
10 mg
5
2.5
1
Blank
10 mg/ml Std
1.0 ml
0.5
0.25
0. 10
0
Saline HO
^j
0
0.5
0.75
0.90
1.0
2. Continue procedure with the addition of 4 ml Biuret Reagent
3. Follow assay protocol as described below:
60
-------
Procedure for Unknown Samples
1. Set up 2 test tubes in a rack for each sample to be analyzed plus Itube
for a blank.
2. Pipette an appropriate amount of sample into the tubes (See Table
below). Do not use blow out pipettes.
3. To each sample add 0.85% saline to bring volume to a total of 1.0 ml.
Pipette 1.0 ml of saline into blank tube.
4. Using a volumetric pipette add 4.0 ml of Biuret Reagent to all tubes.
5. Mix thoroughly on vortex mixer and allow to stand for 15-20 min. at
room temperature.
6.
7.
Read tubes in Spec. 20 at 540 m>u against blank. Record O.D^and
calculate protein concentration using the formula:
O.D.
2.8
x dilution = % protein
If O.D. is greater than 0.500 repeat test using a higher dilution of
sample.
Dilution
1:10
1:25
*1:50
Sample
(ml)
0.5
0.2
0.1
Saline
(ml)
0.5
0.8
0.9
Biuret Reagent
(ml)
4.0
4.0
4.0
"Dilution used for protein determination of whole serum.
61
-------
BIBLIOGRAPHIC:
Lawrence Slots, Eng. Sc. D., Grumman Aerospace Cor-
poration, Development of Immobilized Enzyme Systems for
Enhancement of Biological Waste Treatment Processes, Final
Report FWQA Contract No. 14-12-562, July 1970
ABSTRACT
A method was developed to biochemically fractionate the
microbial enzymes from activated sludge, to concentrate and
characterize their activity, and to immobilize this activity by
entrapment in a poly aery lamide gel. The enzyme-gel prepara-
tion was tested for its effect on a bench-scale batch activated
sludge process.
The conclusions were: (1) the soluble enzymatic compo-
nents of activated sludge can be readily separated from the
particulate components of the cell; (2) the soluble system
thereby obtained can be fractionated in such a manner as to
maintain the activity of the catabolic enzyme systems of
interest while removing non-essential components; (3) the
enzymatically active preparation can then be immobilized
within the matrix of a polyacrylamide gel; (4) the gel will
maintain activity during storage, repeated washings, and
repeated exposure to substrate; (5) the limited bench-scale
activated sludge experiments failed to produce meaningful
results due to possible incomplete polymerization of the
polyacrylamide gel and an improper activated sludge culture.
ACCESSION NO.
KEY WORDS:
Enzymes
Biological Treatment
Biochemical Oxygen Demand
Sanitary Engineering
Research and Development
Waste Water Treatment
BIBLIOGRAPHIC:
Lawrence Slote, Eng. Sc. D., Grumman Aerospace Cor-
poration, Development of Immobilized Enzyme Systems for
Enhancement of Biological Waste Treatment Processes, Final
Report FWQA Contract No. 14-12-562, July 1970
ABSTRACT
A method was developed to biochemically fractionate the
microbial enzymes from activated sludge, to concentrate and
characterize their activity, and to immobilize this activity by
entrapment in a polyacrylamide gel. The enzyme-gel prepara-
tion was tested for its effect on a bench-scale batch activated
sludge process.
The conclusions were: (1) the soluble enzymatic compo-
nents of activated sludge can be readily separated from the
particulate components of the cell; (2) the soluble system
thereby obtained can be fractionated in such a manner as to
maintain the activity of the catabolic enzyme systems of
interest while removing non-essential components; (3) the
enzymatically active preparation can then be immobilized
within the matrix of a polyacrylamide gel; (4) the gel will
maintain activity during storage, repeated washings, and
repeated exposure to substrate; (5) the limited bench-scale
activated sludge experiments failed to produce meaningful
results due to possible incomplete polymerization of the
polyacrylamide gel and an improper activated sludge culture.
ACCESSION NO.
KEYWORDS:
Enzymes
Biological Treatment
Biochemical Oxygen Demand
Sanitary Engineering
Research and Development
Waste Water Treatment
BIBLIOGRAPHIC:
Lawrence Slote, Eng. Sc. D., Grumman Aerospace Cor-
poration, Development of Immobilized Enzyme Systems for
Enhancement of Biological Waste Treatment Processes, Final
Report FWQA Contract No. 14-12-562, July 1970
ABSTRACT
A method was developed to biochemically fractionate the
microbial enzymes from activated sludge, to concentrate and
characterize their activity, and to immobilize this activity by
entrapment in a polyacrylamide gel. The enzyme-gel prepara-
tion was tested for its effect on a bench-scale batch activated
sludge process.
The conclusions were: (1) the soluble enzymatic compo-
nents of activated sludge can be readily separated from the
particulate components of the cell; (2) the soluble system
thereby obtained can be fractionated in such a manner as to
maintain the activity of the catabolic enzyme systems of
interest while removing non-essential components; (3) the
enzymatically active preparation can then be immobilized
within the matrix of a polyacrylamide gel; (4) the gel will
maintain activity during storage, repeated washings, and
repeated exposure to substrate; (5) the limited bench-scale
activated sludge experiments failed to produce meaningful
results due to possible incomplete polymerization of the
polyacrylamide gel and an improper activated sludge culture.
ACCESSION NO.
KEYWORDS:
Enzymes
Biological Treatment
Biochemical Oxygen Demand
Sanitary Engineering
Research and Development
Waste Water Treatment
-------
Accession Number
r\ Subject Field & Group
05D
SELECTED WATER RESOURCES ABSTRACTS
INPUT TRANSACTION FORM
Organization
Grumman Aerospace Corporation
Bethpage, New York
Title
DEVELOPMENT OF IMMOBILIZED ENZYME SYSTEMS FOR ENHANCEMENT
OF BIOLOGICAL WASTE TREATMENT PROCESSES
i r\ Author(s)
Slote, Lawrence
16
21
Project Designation
FWQA program #16050 DXW
Contract #lU-12-562
Note
22
Citation
23
Descriptors (Starred First)
*Enzymes, Biochemistry, Bacteria, *Biological treatment, Activated sludge,
Biodegradation, Biochemical oxygen demand, *Sanitary engineering, Sewage
treatment, Waste water treatment, *Research and development
25
Identifiers (Starred First)
17
Abstract
method was developed to "biochemically fractionate the microbial enzymes
from activated sludge, to concentrate and characterize their activity, and to
immobilize this activity "by entrapment in a polyacrylamide gel. The enzyme-
gel preparation was tested for its effect on the "biological degradation of a
"bench-scale batch activated sludge process.
The conclusions were: (l) the soluble enzymatic components of activated sludge
can be readily separated from the particulate components of the cell; (2)
the soluble system thereby obtained can be fractionated in such a manner as to
maintain the activity of the catabolic enzyme systems of interest while
removing nonessential components; (3) the enzymatically active preparation can
then be immobilized within the matrix of a polyacrylamide gel; (U) the gel
can maintain the activity during storage, repeated washings, and repeated
exposure to substrate, and (5) the limited bench-scale activated sludge
experiments failed to produce meaningful results due to possible incomplete
polymerization of the polyacrylamide gel and an improper activated sludge
culture .
Abstractor
L. Slote
Institution
Grumman Aerospace Corporation
WR:I02 (REV. JULY 1969)
WRSI C
SEND TO: WATER RESOURCES SCIENTIFIC INFORMATION CENTER
U.S. DEPARTMENT OF THE INTERIOR
WASHINGTON, D. C. 20240
ft GPO: 1969-359-339
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