EPA/600/A-94/088
91-20.4
Evaluation of Nutritional and Operational
Requirements for Biodegradation of
Chlorinated Phenols by the White Rot
Basidiomycete, Phanerochaete
Chrysosporium in RBC Reactors
Henry H. Tabak and John A. Glaser
U.S. Environmental Protection Agency
Cincinnati, Ohio
Susan Strohofer, Margaret J. Kupferle,
Pasquale Scarpino, M. Wilson Tabor
University of Cincinnati
Cincinnati, Ohio
AIR & WASTE MANAGEMENT
ASSOCIATION
•
SINCI 1907
For Presentation at the
84th Annual Meeting & Exhibition
Vancouver, British Columbia
June 16 - 21,1991
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TECHNICAL REPORT DATA
Please read /nstrjci:aif u/i :•.. re; crsc otiore curnrvc
REPORT NO.
FPA/6QQ/A-94/Q88,
TITLE A\D SUBTITLE Evaluation of Nutritional i Operation
Requirements for Biodegradation of Chlorinated Phenols
by the White Rot Basidiomycete, Phanerochaete
Chrysgsporium in RBC Reactors
5. REPORT DATE
'6. PERFORMING ORGANIZATION CC^E
AUTHOR^) Henry H. Tabak and John A. Glaser (1)
Susan Strohofer, Margaret J. Kupferle, Pasquale
Scarpino, M. Wilson Tabor (2) .
8, PERFORMING ORGANIZATION
PERFORMING ORGANIZATION NAME AND ADDRESS
(1) U.S. Environmental Protection Agency
Cincinnati, OH
(2) University of Cincinnati
Cincinnati, OH
10. PROGRAM ELEMENT
1 1. CONTRACT/GRANT NO
2. SPONSORING AGENCY NAME AND ADDRESS
Risk Reduction Engineering Laboratory —Cincinnati, OH
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268 " '
13. TYPE OF REPORT AND PERIOD COVERED
Piihl i
14. SPONSORING AGENCY CODE
EPA/600/14
5. SUPPLEMENTARY NOTES
John Glaser (513)569-7568 -,
Presented at the 84th Annual Meeting & Exhibition - Vancouver, British Columbia
e. ABSTRACT rp^g akj_iity to degrade and detoxify organic & inorganic constituents requires
2 complementary features of microbial competence; the biochem. means (enzymes) to de-
toxify wastes and the capability of a single organism or a multiplicity of compatible
organisms of complementary competence to effect this required metabolism. An ex. of
a single, highly competent organism is the wood-degrading fungus. Phanerochaete chry-
sosporium, which has the potential to degrade the aromatic components of toxic and
hazardous waste. This capacity is based on its ability to degrade lignin, a persistent
biogenic polymer, via the nonspecific extracellular enzymes, liginases.- This fungus has
been utilized to treat liquid-phase wastes in a rotating biological contractor(RBC),
the MyCoR process.
This research program explores the use of the MyCoR process as a hazardous
waste site clean-up technology, addressing issues of application and practicality to
emphasize cost effectiveness and efficiency. In these studies, biochemical and mechan-
ical parameters were optimized for biodegradation by the use of Pshrysosporium biofilms
in bench-scale and pilot-scale RBCs.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
c. COSATi Field/Group
biodegradation,
chlorinated phenols,
white rot basidiomycete,
Phanerochaete chrysos-
porium, RBC Reactors
18. DISTRIBUTION STATEMENT
Release To Public
19, SECURITY CLASS (i
Unclassified
32
20. SECURITY CLASS (Tilts pagei
-Unclassified
22. PRICE
EPA Form 2220-1 (R»v. 4-77) PREVIOUS EDITION is osspLETE
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91-20.4
INTRODUCTION
The ability to degrade and detoxify organic and inorganic watte constituent* requires two
complementary features of microbiai competence: the biochemical mean! (enzymes) to detoxify wastes and the
capability of a tingle organism or * multiplicity of compatible organismf of complementary competence t0 efTecE
tint required metabolism. An example of a tingle, highly competent organism is the wood-degrading fungui,
Phane,rochaejf chrvaot|»QriiHa. which baa the potential to degrade the aromatic component! of toxic and
hazardous waste. Thii capacity la-based OB it* ability to degrade ligniii, • persistent biogeeic polymer, via the
nonspecific extracellular enzymes, Iigninases. This fungus bit been utilized to treat liquid-phase wastes in a
rotating biological contactor (RBC), (he MyCoR process.
This research program exploraa the use of the MyCoR prooees as * hazardous wa&le site clean-up
technology, addressing issues uf application and practicality lo emphasize cost effectiveness and efficiency. In
theae studies, biochemical and mechanical parameters were optimiied for biodegrmdalion by the use of L
chrvsogporium biofUms in bench-scale and pilot-scale RfiCl. The specific objectives were: (I) to optimize the
carboo source mil other nutrient requirements in the primary growth phase and IB th* secondary enzyme
synthesis/metabolic phase; (2) lo optimize ihe operational parameters, (e.g., head space oxygen, temperature.
rotational speed of RBC discs) in support of pilot-scale testing; and (3) to investigate degradation kinetics and
identify Hilcnnediele and end products of the metabolism of several representative chlorinated phenols. The
variable* were evaluated using s wood-processing pulp and paper mill (Kraft liquor) effluent. Translation of
bench-scale operation was demonstrated in parallel bench- aad pilot-scale 2,4-dlchlorophenol (DCP) treatment
studies
BACKGROUND
Lignolytic fungi, found throughout the northern hemisphere, appear to he unique among
microorganisms in ihsl they can rapidly depolymenze and ttuaeralile ligiun. a complex, biorecslcursnl,
biogeruc, irregular, nonhydrolyiable, and environmentally persistanl wood polymer of phenol propane units
(Kiric and Farrell1; Cbeo and Chang1). Lignm degradation by most of these basidiomyceles is part of their
secondary metabolism, occurring at high rates only under conditions of nutrient limitation, and is comeiabohc,
i.e.. a primary growth substrate such as cellulose or glucose is required. Lignin contains numerous
substructure* also found ie common'organic pollutants, phenolict, anisoles, sryi-o-elhers, and bipbenyls, and iu
exceptional recalcitrance lo attack by moat microbes has led to a belief thai any organism capable of
mineralizing it must have highly nonspecific oxidizing systems that could be applicable to aromatic pollutants as
well (Kirk aad Chang1; Bumpus ef oj.*).
Phanerpchaeie corvsQt|»ri.yra. a white rot Baaidiomycet*, is a species that has been shown to have s
superior ability lo degrade lignin*13. Ligninaset are peroxidaaea thai utilize hydrogen peroxide to oxidize
orgatuc compounds. Although these enzymes resemble convantioaal peroiioaaes in some respects, their H:Q]-
oxidized suua an evidently electropositive (Hamroel m at."). They catalyze one-electron oxidations of a wide
variety of lignin-relsted, polyallcoxylated model compounds to give carbon-centered, substrate-free radicals si
jjutiaj transient products, end post enzymatic reactions of Ilieae radicals account for the many end products
found1'-11, la the caae of lignina, carbcmI«* «•"• «»ad at the feed source u the initial optimization
eipenmenla. All factors ware held constant lor *» opraiim of • gives reactor throughout the growth and
secondary metabolic phasea of each sxpsnmeot with das Meaplion of oiygen tupply. In telected experimenu,
growth was initiated under an air atmosphere Iktu wta raplaoad with a pure oxygen supply afler 41 hourt of
operaiion. .-.• .'.:', '• ,
Materials
Chemicals All chemicals used in eipenmenuHioo were reagent grade or better. Gates (eg. 99 5 percent
oiygen) wire purchased from Wnghl Bros. Internal standards for gaa cbromstogrtphy and gat
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91-20.4
chromalograpby/mass spectrometry analyses were provided by ifee EEE laboratory al U.S. EPA, Cincinnati,
OH.
Waslawsler Souret. Kraft liquor samples of 10 to SO gallons were obtained from Mead Paper Mill,
Chjllieothe, OH, as needed, to maimaiii * steady source for reactor treatment studies. These effluentj contained
approximately 80 percent of the total mill color effluent (Tyler & Fiugerald"), The sample Ion were from boih
hardwood and aoftwood processing; color value* were determined to range from 1900 10 6200 platinum color
unit intensity (PCU).
Method!
RBC Blorcacton, Bench-scale routing biological coQiactort (RBC) (Figure I) were constructed from
plexiglaa, baaed upon the design description to the patent of Chang #f fl/.***3" and by Huynh a al.n. Alteration*
to ilia design were made as follows: tbe reaction chamber, a half cylinder conaiiting of four baffled iectioai
thai can be plugged of yaod for flowihitsugh, had valve fittings OB either end of the outaide chamber to permit
flow daring continuous operation or to remain sealed for batch operations, y*^ section contained two pleiigiu
disks, O.S m in diameter by 8 mm thick, roughed by a lathe to facilitate fungal attachment. The eight disks per
RBC provided • nirface area of 0.2« m'/RBC. Forty percent of the iota) disk area wai iubmerged in 2 L
mixtures of growth and/or treatment solutions for experimentation. A water jacket nirrounded the RBC
chamber 13 allow temperature control, and a half cylinder shaped lid enclosed the lop of the disks to prevent
contamination and evaporation, ud to Eft***?**;** increased oxygen environments. Two round apertures on
opposite sides of (lie lid were used for the eir/oxygen inie) and gas sampling ports, respectively. Oxygen was
supplied by cylinder and distributed to four individual RBC units through manifolds, containing valves to
distribute the flow of oxygen to each reactor individually. The flow to each unit was adjusted via Cilmont (F-
1100) ileel ball flowmelen. Two RBC units were driven by a Daylon Gearmolors (3M096) dual drive shaft
motor mounted at the ends of respective RBCs. Motor speed was adjusted via a rheostat controller (Dart 125-
SOC). A schematic of the while rol fungus pilot-scale RBC unjl is shown m Figure 1. This unil ia operated at
the Teal and Evaluation Facility, U.S. Environmental Protection Agency, Cincinnati, OH.
While Rol Fungus. Culture of £» cnrvtosnonum iiraui BKM F-l W was obtained from Or. Thomas w.
Joyce of the North Carolina State University School of Forestry. Stationary cultures of fungus were maintained
on basic media consisting of 3 percent agar, 1 percent malt, 1 percent glucose, (Kirk el of.0) and minimal salts,
at pH 4,5, and grown at both room temperature and 34 C. Cultures were grown in Roux flasks and transferred
to the RBC reactors.. A concentration of aporea equivalent to approximately 400 per mL at A^n^O-S was used
per each RBC (Chang «f ai,D).
Reactor Operation The composition of growth and treatment solutions added to the RBC media mixtures was
baaed on tin work of Campbell*1. Synthetic media were prepared asepUcally with sterile deionixad water. Each
sample lot of Kraft liquor waa refrigerated at a temperature of 4 C until use. Buffer was not added to any
mixture containing Kraft liquor. The RBC unila for Kraft liquor decoloriiation eiperimenta were operated in
both growth a&d treatment phases is a constant temperature room maintained at 33 to 33 C, The pH of the
reactor media wia maintained at 4.23 to 47 via daily adjuatments to 4.4 and to 4.5 with IN HCI or NsOH as
required.
DichJoropbenol (DCP) treatment medium was prepared by adding the required volume of slock
solutions (2,000 or 200.000 ppm DCP) into the reactor media. Treatment batches of DCP ai final
concenfratioaa of 200 sad 100 ppm were prepared. Biodegradation of the DCP wax assessed during
experimental rani in the laboratory hood by duplicate sampling of each RBC unit; this was accomplished by
dividing each of 2 RBC unila into two individual I L reactors by plugging the central baffles of the umis
according to the method of Yin". To maintain a temperature of 30 to 33 C in the RBC unila, water was
circulated constantly by a pump from a temperature-controlled waicrbath through the water jackets of the RBCs.
91-20,4
RBC inoculation and development of fungal biofilms were usually conducted in accordance with
liierarur* reports (Chang n al."). The initial inoculation media containing sport* and synthetic growth media
was grown for 2 days, following which the media was replaced with frcah growth media without spores. After
2 additional days of growth, nitrogen had been depleted ud tigninolylic conditions were eaubliahed (Kirk*).
Biofilms grown in the presence of Kraft liquor remained under operation with the initial inoculation media until
decolorixation of thai Kraft liquor occurred, generally after 5 days. This acclimated the biofiln 10 the Kraft
liquor substraie, but ii in contrast to work performed by Campbell**. Experiments lo aaaeas the loss of DCP
continued for one week or until leas than detectable limit (1 ppm) amounia wen measured by CC analysis.
During experimental RBC runs, sample collections and pH adjuatmenu were conducted daily. Sample
removal and pH adjustments were conducted via a camiula and a syringe. In Kraft liquor decolorizauon
experimenia, approximately M mL reactor mixture aamplea wen taken for measurBBesu of absorbsnce, PCU,
COD, and DOC. This sampling did not change the reaction mixture volume by more than 10 percent during
the course of a given experiment. In DCP degradation eiperimenta, 15 mL samples were withdrawn daily and
analyzed for DCP only. To maintain sufficient volume in the RBC, reactor liquid matrix samples were filtered
from the withdrawing syringe through a filler bolder coniaining a 2.4 cm glass fiber filter (Whatman GF/C)
before use of the sample in analytical procedures, eicept for selected aamplea for phenol and volatile organic
analysts (VOA). Atmospheres wen established by allowing free gas exchange with the ambient atmosphere for
air mediated experiments, whereas elevated oxygen concentrations were established by sealing the lid of the
RBC with tape and supplying oxygen to the reactor. When oxygen was used as the atmosphere, the headspace
of the reactors was flushed aggressively with oxygen for 5 to IS minutes before the oxygen flowrate was
edjusltd'to a constant rale of 10 mL/min. Wet weight! of disk biofilms wire measured upon completion of the
reactor batch runa after draining the liquid phase from each RBC unil.
Final sample* from the RBC units (at the end»of baich runs) of 30 lo 100 ml were filtered through
glass fiber fillers aad analyzed for DOC, COD, TICK, ammonia and nitrite. Abeornance and PCU
measurements were taken for the final samples from Kraft liquor decolorizaiion eiperimenla. Once an
experiment waa completed the biofilm was scraped off the disks, the RBC waa washed with soap and warm
water, and then sterilized by ethylene oxide gaa.
Analytical Techniques. Dissolved oiygen waa measured by a YSI Model 54A meter with a stirring BOD
probe YSI Model 5720A. Ambient atmospheric oxygen concentration was measured with a Fisher Model 1200
Csa Penilionei. Speelroscopic meanireineiiu of UV/visible wen taken with a Beckman Model 25
speclrophouuneiet. Measurements were made on filtered liquid samples, Wavelengths were chosen to monitor
the reduction of auhatrau concentration throughout the batch treatment periods, which ranged from 2 days to
one week. Became of the heterogeneity of (he Kraft liquor, a single wavelength was considered inappropriate
to measure me* of conversion; wavelengths °' 5*° nm (measuring the yellow-orange color region), 450 nra
(for bumic substance detection), 2SO sin (for aromatic structure and ligrun fragment detection) were used
(lanshedar a al.*1; Alen A Hattus"). In Knft liquors, a strong abjorhance was observed at 220 tun, and a
wide flattened peak was observed at 280 nro. Coloi was measured as platinum color units (PCU), according it>
the ilaodard meihod developed by (he paper and pulp mill industry (Oellman").
Continuous liquid-liquid extraction (EPA Method 1250) waa used to extract phenolic compounds,
including DCP. for GC/MS analyais (EPA Method .1270). Quality assurance and quality control (QA/QC)
measures for extractions included a daily blank plus a duplicate, a matrix spike and a spike duplicate for every
10 samples. A 5-poini calibration curve for UwGC/MS was established and rechecked daily. Additionally,
separatory funnel extraction (EPA Meihod 3510) were performed on aamplea from DCP expenmenii for quick
turnaround of results. Extracts were analyzed via gaa chromatography with flame ionitaiioa detection (EPA
Method 1040). For these deternunalioni, QA/QC measures before and after every series of injections included
ihe use of a 5-poini calibration curve of the DCP and 2 acid surrogates, 2-fluorophenol and phenol d-i. VOA
analysis used EPA Meihod 1624. An internal standard of bromofluorobenzene was used for semiquantitanon of
identified compounds for select samples,
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91-20.
RESULTS
Growl h. Ho discernible difference in growth or activity was observed in ihe biofflms inoculated wsih spores
culliviied at room temperature compared to those at 34 C. Sufficient number) of sporei for the inoculation of 2
lo 6 re*£tori were grown within 10 days at 34 C whereas 1 weeks were required for room temperature growth
of spores- No significaai differences were observed in the consecutive treatment performance of bio films
developed ut we pre*esce of pulp mill effluent compared with those developed oo synthetic caedia, En both
cases. an initial growth tod ace I i nation penod of one week wai required after start -up. Under all comJinons of
growth and treatment, but aoi at ill times, black and red pervaiiva coloration of the biofilm developed and
occasionally ibe coloration waj observed in the form of localized speckled areas, Such coloration is believed to
result from one or taore of the manganese-dependent hgninase enzymei When environments of purified
oxygen were eatablished with the initial inoculation of • reactor, some of the rung •! rsas* was observe*! lo group
into spherical clumpi on ihe discs. This was not observed when biofilm growth was begun under air.
Decolorization. lo these bench-scale studies with the pulp and paper null effluent, visible color removal from
Kraft liquor occurred in experiments in which oxygen, glucose, and roumomil speed of the RBC discs were
viried. Effects of tir verms oxygen head space were compared; the best decolonuiion kinetics were found
using oxygen rather than air (Glaser ei o^,50'1' JJ). The biamaw developed under these two oxygen
coaeenlraliona differed cim»iderably. The nitrogen-rich biomus retained! greater •mounu of water lhan (he
nitrogen -deficient biomaii The color bodies in the Kraft bleach effluent were belter sorbed by the nitrogen-nth
bictma&i than by ihe niiragen-defieienl Biomiis. The btonmss grows under oxygen was more stable
mechanically and had greater decolonxing activity,
Nitrogen-Rich and Ligninolylic Conditions. Figure 3 i! lust rites thai Itgmnolyiic conditions (nitrogen-deficient
media) produce effective treatment of Kraft liquor waste Experiments using nitrogen-rich media (conditions
thii do oat induce the hgnjn-degriding secondary metabolic state is sonmuunt fungi! species) throughout ihe
treatment resulted in a "nonactive* condition, Phyucal attachment of color bodies was noted on th« biofilm for
a period of one fa 2 days, fallowed by their release back into the liquid matrix. Substrates monitored at the ISO
nm wavelength also displayed this uptake/ release phenomenon This is lo be contrasted with conditions of
observed "inactivity* m which no initiit uptake by the fungus 11 observed and no treatment occurs- In slower
acting systems (i.e., under air), residual attached color from a previous bitch is decolorized upon the addition of
a new batch of effluent containing • freah supply of glucose (i e. . H,0,|.
Physical attachment or adsorpiion of substrate to Ihe fungal bio film is primary to inuiiiing subsequent
degradation by the fungus. The treatment removal was bused on measurement of ihe liquid concentrations of
color and individual organic substrates For example, tnchlorophenol concentrations of IB ^g/L in the pulp mil!
effluent were sol- defected after treatment eves in the case where the desired enzymsuc conditions for treatment
had DO! been established. This loss of phenol was ntnbuted to the great relentive characteristics of the
developed BonligBiaolyMc hiolilm.
Oxygen Concentration, The effect of 100 percent oxygen OB concentration of Kraft liquor effluent is
illustrated in Figure 4, which compares treatubtlity data for concentrated and diluted waste iff the presence of
100 pereenl oxygen and air. No difference in treatment was noted when cultures were placed initially under an
increased oxygen atmosphere despite the smoother appearance of the bkjfilm. The easi of oxygen supports the
use of air for early growth in pilot- and field -scale studies. Invariably with all other parameters held constant,
more biamas* developed under air than under increased oxygen. When treatment failed in a set of reactors
under increased oxygen, the fungus visually appeared to grow to « weight comparable to thai grown m the
presence of aif ,
Initial acclimation to the pulp mill effluent wu found lo be slower in the presence of air than in the
presence of higher oxygen concentrations (Figure 5). These reactors decolorised dilute effluents comparably,
The oxygen-assisted reactor systems showed compirable treatment activity for both dilute ( *; 3,000 PCU) and
concentrated (2:4,,QGO PCU} effluents. However, reactors with air ttOTOspheres showed greatly diminished
91-20 4
decolonzaiion raws for concentrated pulp null effluent compared with the rate observed for ihe more- dilute
effluents (Figure 4). la similar studies, Yin « a/.*1* demonstrated thai the kinetics of color removal by L
chrvsoyporium are concentration dependent with effluents containing fi8,000 PCU- The effluent concentration
used in our ifudie* was 2,000 to 5,000 PCU; hence, oxygen concentration and its availibility are deierrnimng
factor* for the biological tfeaiiaea! of laeae effluent* in RBCi- These remits miggcst strong correlation between
oxygen requirement and pulp mill effluent color concentration. Such relationships are integral to the
commercial operation of a Ireatrnent tysiem of this design.
Glucom. Bench-K»le RBC studies evaluating the effect of glucose concentration on treatment efficiency
generated dtia suggesting that glucose concentration at the levels studied did no! affect the degree of treatment,
bui m conjunction with oxygen concentration, did affect the rate of treatment. Scudiea showed thai following
acclimation, a penod of one day was considered significant time far effective treateoest (decoloritatjon) of the
Kraft mill liquor waste. Accordingly, evaluation of data at one day indicate* thai treatment time can be
considered a more central determinant of the treatment.
The data in Table 2 indicate that at 100 percent oxygen, the amount of glucose (1 versus 0,5 percent)
and the disk shaft rotational speed (I versus 6.5 rpm) had Uttle effect on treatment efficiency. Figure 6
illusiraiea the effect of glucose concentration under air in RBC* thai are treating dilute Kraft liquor effluent.
Glucoee is used fit nevo to produce hydrogen peroxide from molecular oxygen and is used by the ligmnases in
the oxiditivo depolymenulion of lignin (Bar-Lev & Kirk'1). Tha dual role for glucose in both growth and
secondary melaboliim is supported by the described refulta,
The amount of fctofilra growth ts not directly proportional to glucose concentration. Under increased
oxygen concentrations with O.S percent glucose, treatment performance waa as efficient aa with 1.0 percent
glucose (see Table 2). Under air, with only dilute effluent, the activities of the initial acclimauon/treaimeni
batch and the second consecutive batch were comparable al the two glucose conceal rations. The treatment of
tiler batches were accompanied with • decline in the rale of decolorizing activity n the 0.5 percent glucose
level. However, the extent of decolonzittoa tiltirraftly waa maintained by employing longer treatment periods.
The decolorization rate was maintained at I percent glucose for dilute effluent feedstock*. An additional factor
of carbon source is indicated here to terms of concentration, oxygen, and kinetics1. A critical concentration
berween 0.1 and 0.2 percent glucose has been identified as necessary for decolonution with supplied oxygen
(Yin a ai*•**), Our result* ahow UIBI activity at 0-1 percent glucose proceeded at • decreased rate Oxygen
concentration in conjunction with Kraft pulp null effluent feed strength end glucose were shown to have ihe
greatest impact on treat ment-
BufTen. No difference in growth or treatment extent was observed when potassium Urtrmie (KTar) WM used
ui the place of 2,2-diraeiliyl succtaaie (DMS) buffer for biofilre development at 1 and O.S percent glucose
concenlrujonj with the reactor speed at one rpm. The DMS buffer hai been previously used for the
optimization of lignisaie production (Fenn & Kirk 1979). Buffer choice n of critical economic importance for
economic considerations for larger scale operation!, In bench-scale studiea. 2,1 DMS was shown to stabilize
pH for «n effective treatment and GO aubiliia pH more effectively than K-Tar, The effect of buffers on
treatment of Kraft liquor, supplemented with I and 0,3 percent glucoae, it shown in Figure ?.
Two inorganic buffers were tented a* alternative* to DM1. One buffer waa a Na1HPQjl/KH,PO«
mixture adjusted to pH 4.5 with HCL and the other was 0.05 M KJH,FO4. The reactors were operated ai 0 5
percent glucose and 4-week-old Kraft liquor al one rprn, TJbe funguj w*j grown under air for the first 48 h
(GR1) and then under oxygen for the second 40 h (OR2). The pH was measured al 0, 24, and 49 h for each
batch of growth solution. The pH was adjusted wiia NaOH as necessary. The pH data ire lummartzed in
Tihle 1. Both buffers performed acceptably tltlioii|h both reactors required a small addition of base to bring
the pH to 4.5 after the first 24 h following introduction of fresh growth media.
Disk Rotation Speed. Development of biofilm at one rpm invariably resulted in bridging berween ai least two
of the discs, whereas thif never occurred ai 6.5 rpm. In addition, it appeared thai the films at 6.S rpm were
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91-20.4
more evenly distributed with la overall smoother surface No difference IB meatmen! activity was observed,
•tthough dissolved oxygen coacetjrrmitoBS at 6.5 rem were geaermlly double those it one rpm (Table 2). The
influence of did! rotation nie may be more pronounced for larger scale spent tons.
Temperature, Bencli-acale RSC trudjes indicated an improvement in growib and treatment at higher
temperatures. A temperature range of 33 to 3$ C was found to be optimum for growth and treatment, whereas
temperature at 23 C provided a delay of growih cycle for 24 h.
Trefitment af Organic Components of Wood-Processing Wait*. Is addition to monitoring the decolonzalion
of Kraft liquor waste during treatment, aarnplea were analyzed by GC/MS for residual organic compounds and
chlorinated! phenols. The GC/MS icajai of wajte maple before aod after bench-scale fungal treatment showed *
reduction of organic! including chlorinated pbeaoti (2,4-dicWorophenot and 2,4,6-inchloropbeneI) (Figure 8).
Extraction surrogate, internal standard, and Kraft liquor effluent indigenous compounds and their concentration
levels in Kraft liquor before and ifter treatment with Pj, chrygipQUjm are listed in Table 4.
Kraft Pulp Mill Effluent Variability, Difference* ta tunpluig lot* of the Kraft pulp mill effluent and changes
m the lot during the time of storage may explain the variability of treatment results, Among the five ton of
effluent treated by our lyuem. ireaiabiUiy varied diiimetly. The sample lau can differ iigrHficaitily. Their
composition depends on daily mill protease* and feedstock;, pulp mill effluent for softwood u typically dark in
color, where** the hardwood wastewater is lets concentrated. No tread can he observed to identify the time
interval for greatest ireatmeBl activity; maximum treatment effects were observed for both early and late periods
IP ihe lifetime of the biofilma. An apparent loxiciiy developed within the effluent lots over time, normally
encountered within two months after collection of tbe effluent supply, Ai this stage, hingtl ireitmenl became
less effective and ultimately tbe fungus would not grow in the presence of that effluent lot. BiofUm became
fragile and peeled off tbe diik. Toxins showing activity u>ward fungi have been previously noted m these
effluents. Bacterial contamination is also suspected.
One batch of Kraft liquor was completely characterised, and the effect of holding time and aging prior
to treaimenl waa studied at four reactor operating conditions. Fresh biomass was grown up on the RBCs for
each experiment. One-week-old, 4-week-oid, and fl-week-old Kraft liquors were tested. Color units as well u
tbaorptius at 580 am, 450 BID. and 180 em were measured at 0, 24, and 48 h, and the removals are compared
on • percentage basis m Tible 5. Treatment was not ifjfocted ie any of the reactors at one and 4 weeks, but at
S weeks iymptom* of toxiciiy to the fungal bsomais developed: ihe fungus began iloughing from the disks after
introduction of th< 8-week-old Kraft liquor.
Tremtibiliiy of Kraft liquor, held for 5,5 rnonthi, wai UKWB& pcnodicallj; during the balding time
uaifig the bencb-icaJe RSC units. For each Instability run, the liquor was supplemented with 0.5 percent
glucose and wit maintained under an atmosphere of oxygen. Following 24 h of fungal treatment, Kraft liquor
concentration wai quintitsted uiing the •bsorbance it 580 urn, and the platinum color units were derived using
regretaion data. The results (data not shown) indicated thai treat men t efficiency decreased more than 70 perceni
during the lime period. These results suggeil that either imcrobial growih in the Kraft liquor or production of
toxins from these organisms are inhibiting the action of me f^ chrvBOfPorium biofilm,
2,4-Diehloroph*nol T realm en i in Bench-Scale RBC Reactors. Two concentrations of DCP were treated in
replicate. These melon were operated with 0.5 percent glucose and 6,5 rprn The fungus was grown under
sir for the first 48 h and then under oxygen for the remainder of the study The fungal biomass wu grown on
two consecutive 48>h growth-solution batches prior io introduction of DCP and limitation of nitrogen for the
treatment pbsje. DCP concentration ID the reactor was measured with lime, and these dau are summarized m
Table 6, The concentration of DCP waa reduced from P.I mg/L to < i oijt'L wiihin 48 h. uid from 84 8
mg/L to < I mg/L within 120 to 144 h.
Additional beach-&cale studies with 2,4-DCP provided dita oo removalt of the compound it three
different initial concentrations (20, 50, and 100 mg/L target concentrations). No nitrogen source was lupptied
91-20.4
during treatment for tbeae hatches. Tbe removal of 2,4-DCP at the three cooceotraiioa levels veraus lime is
shown is Figure 9, Tbe final conceotratioa of 2,4-DCP wu leaa lh*n one mg/L for all three initial
concent mtionj. Al tbe two lower concectntioDj, thja level of nsductioa waa obtained in 4g b or le&s. In the
100 rog/L reactor, 120 b were required, Tbe biofilm was adversely affected at tbe 100 mg/L coBCCDlration;
partial sloughing of the eaublished growth waa observed. A kinetics study wma performed in which 2,4-DCP
removal at 50 mg/L initial concentration wu nuauured *t 0, 2, 4, S, 12, 24, 36, 48, mud ?2 b of incubation.
Tbe maximum removal look place bcrweno 0 and 24 a. with Uie sbjupefi drop between 12 and 24 b, Dai*
bezwoen 0 and 24 h ate plotted is Figure 10.
Studiej 'were alto performed with consecutive batch treatmatits of 2,4-DCP ai 20 and 50 mg/L initial
concentration*, with and without a nitrogen source (NH,Cf) in the synthetic modi*. Tbe effect of the addition of
33 mg/L nitrogen to the treatment batch reactors oa the biodegradation rate of 2,4-DCP was measured ID these
consecutive batch treatment experimenta. Tbe cooftecuuve beocb -scale RBC baicb (rrjtment d*u for 2,4-DCF
at SO mg/L imtial ooocetitratiaa in the reactor with *ad without addod nitrDgea showed oompvable removtli of
the target compound after 24 h. DCP removal for up to three caaMeuUve batch treatmaiu ai 20 and SO mg/L
initial coocezitimttofi with and without added nitrogen showed reduction to leveli of leta than ooe mg/L within 48
h. For treatment of DCP at 100 mg/L initial concent rations, reductions 10 less than one mg/L were achieved
Control bench-scale studies of 2,4 DCP trcatability were cooductud in duplicata as follows: (I)
Positive Control — fungus plus glucose/buffer/salu media plus 50 mg/L DCP: (2) Negative Media Control —
bufTer/aalti awdi« phit SO mg/L DCP; and (3) Negative Compleu Media Control — glucose/buffer /ults medis
plus 50 mg/L DCP. The results showed DCP ircsiabiliiy to a tovel of less than one mg/L in the positivo
control {experiment I) wiiaia 24 h, and laiigrajficwM loss of DCP to the reactor or atmosphere m both negative
controls (experiment* 1 and 3), A
2,4-DCP Treatment in Pilot-Scale RBC Reactors. Whems the bench scale tyttein as being used to study
decolontsiiea of ICraft-felemch-piaat efHueat. aurrogate waste streams conUuing 2,4-DCP are being used to
evaluate the pilot-scale unit. Adherence of the fungus io the *ugh-4enjiiv polyethylene (HDPE) discs of the
pilot -Kmie usji has bees execlleat1^11.
In iniiial pilm-scaJe atudras, 2,4-DCP wai redocad from 45 mg/L to
-------
91-20.4
REFERENCES
I. T. K. Kirk and R. L. Funll. Enzymatic 'Combiution': The Microbiil Dcgndition of Ugnin. Ann,
Review Microbiol. 14:450(1987).
2. C. L. Chen and H. M. Chug. Chemitlry of Lignin Biodegredalion. In Bioiynlheiii ud
Biodemdalion of Wood Comnonenla: T. Higuchi, ed. Academic Preai, Inc.. New York. 1985. pp
535-556.
). T. K. Kirk ud H. M. Chug. Potential Appliuioni of Bio-Ligninolytic Syelemi. Enzyme Microb.
TechnolQg. 3: 189(1981).
4. I. A. Bumpui, M. Tien, D. Wrighl. ud S. D. Aim. Onidation of Pertinent Enviroamenul Polluunu
by • White Rot Fungui. Science. 228: 1434 (1985).
5. 1. A. Buiwell ud E. Odier. Lignin Biodegndalion. Cril. Revi. Biolechnol. 6: 1 (1987).
6. K. E. Hunmel. M. Tien. B. Kalyanareman, ud T. K. Kirk. Mechaniim of Olidalive a-S Cleavige
of • Lignin Model Dimer by Phucrochaete chrvaoBporium LJgninue: Stoichiometry ud Involvement
of Free Radicali. J. Biol. Chem. 260, 15: 8348 (1985).
7. T. W. Jeffrie*. S. Choi, ud T. K. Kirk. 1981. Nutritional Regulation of Lignin Degradation by
Phuerochmete chrvioroorium. Appl. Environ. Microbiol. 42:290(1981).
8. L. J. Forney, C. A. Reddy, M. Tien, ud S. D. Ami. The Involvemul of Hydrolyl Radical Derived
from Hydrogen Peroxide in Lignin Degradation by the While Rol Fungui Phuerochaele
chrvaonxmum. J. Biol. Chem. 237, (19): 11455 (1982).
9. H. E. Schoemiker and M.S.A. Leiioli. Degradation of Ljymn by Phuerochaele chrvaosoorium. L
Biolechnol. 13: 101 (1990).
10. R. L. Kdley and C. A. Reddy. Glucnae Oiidaae nf Phanerorhaele chrnoiporium. In Melhodt in
EnrvmolotY: W. A. Wood and S. T. Kellogg, edl. Academic Preaa, Inc., New York, Vol. 161,
B1OMASS J-lrt B. Lignin. Peclin. and Chilin, 1988, pp 307-315.
11. P. J. Kenten ud T. K. Kirk. Involvement of a New Enzyme. Clyoial Oiidaae, in Eitncellular H,O,
Production by Phanerochaele chryaoiporium. J. Bacleriol. 169:2195(1987).
12. T. K. Kirk. S. Crou. M. Tien. K. E. Murtaugh. and R. L. Farrell. Production of Multiple
I igniniffi by Phuerochaele chrnoiporium: Effect of Selected Growth Condition! and Uae of Muunl
Slrain. EM. Mitrffh. Ttth. 8: 27 (1986).
13. J. Vole and K. E. Erikaeoa. Pvranoae-2-Oxidaaa from Phuerochaele chryaoiporium. In Melhodi in
Enzyme-logy. W. A. Wood ud S. T. Kellogg, eda. Academic Preaa, Inc., New York. Vol. 161,
BIOMASS Part B. Lignin, Pectin, and Chilio, 1988, pp 322-326.
14. B. D. Paiaon and T. K. Kirk. Faciora Involved in the Regulation of a Ugninaie Activity in
PlliniTnrlllfH chrnomorium. Appl. Environ. Microhiol. 49: 299 (1985).
15. M. Tien and T. K. Kirk. LJgnin-Degrading Enzyme from the Hymenomycete Phuerochaete
chrvaoanmium Burda. Science 221:661(1983).
10
91-20.4
16.
17.
18.
19.
20.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
K. E. Hammel, B. Kalyanaraman. and T. K. Kirk. Subatrale Free Radicali Are Intermediates in
Ligninaae Calalyiii. Proc. Nail. Acad. Sci. U.S.A. 83:3708(1986).
P. J. Kenlen. M. Tien, B. Kalyanaramu. ud T. K. Kirk. The Ligninaae of Phmerochaele
chrvioiponum Generalel Canon Radicali From Melhoivbenzenei. J. Biol. Chem. 260:2609(1985).
H. E. Schoemeker. P. J. Harvey. R. M. Bowman, and J. M. Palmer. On the Mechanism of
Enzymatic Lignin Breakdown. FEBS Lett. 183:7-12(1985).
P. Keyaer, T. K. Kirk, ud J. G. Zeikui. Ligmnolylic Enzyme System of Phanerochaeie
chrvaotporium: Synlheaited in the Abeence of Lignin in Response to Nitrogen Starvation. J. Baclenol.
135: 790(1978).
P. Fenn and T. K. Kirk. Relaiionatup of Nitrogen to the Onset and Suppression of Ligmnolylic
Activity and Secondary Mecaboliim in Phanerochaele chrvaoiporium. Arch. Microbiol. 130: 59
(1981).
T. Fukuzumi, A. Niihida. K. Aoahima. and K. Mmami. Decolourilalion of Kraft Wale Liquor wuh
White-Rot Fungi. Mok. Oak. 23: 290 (1977).
V. B. Huynh. H M. Chug, and T. K. Joyce. DechJorinalion of Chloroprganics by a While Roi
Fungui. Tappi J. 68:98(1985).
H. M. Chug, T. W. Joyce, T. K. Kirk, ud V. B. Huynh. Proceai of Degrading Chloroorganict by
ibe While Rol Fungi. US Patent No. 4.554.0V (1985).
H. M. Chug. T. W. Joyce, ud T. K. Kirk Proceai of Treating Effluent From Pulp or Paper-
Making Operation. US Patent No. 4.655.926(1987).
G. A. Lewandowiki. P. M. Armenante, and D. Pak. Reactor Design for Hazardoui Waste Treatment
Uiing a While Rol Fungi. Walei Rea. 24: 75 (1990).
A. L. Prouly. Bench-Scale Development and Evaluation of a Fungal Bioreaclor for Color Removal
form Bleach Effluenta. Anpl. Microbiol. Biolechnol. 32:490(1990).
D. C. Eaton. H. M. Chug. T. W. Joyce. T. W. Jeffrie*, and T. K. Kirk. Method Obtains Fungal
Reduction of the Color of Extraction Stage Kraft Bleach Effluents. JjnnjJ, o5, (6): 89 (1982).
C. F. Yin. C. W. Joyce. H. M. Chug. Role of Gli
Bleaching Effluents. J. Biolechnol. 8: 67 (I989a).
in Fungal Decolorization of Wood Pulp
C. Yin. T. W. Joyce, ud H. M. Chug. Kineiica of Bleach Plul Effluent Decolorization by
Phuerochaele ehrnunponum J. Biolechnol. 10: (7 (I989b).
•.iJi^j - - IS '
J. A. Glaaer. H. H. Tabak. S. Slrohofer. M. Kupferle, P. Scarpino. and M. W. Tabor. 'Bench-icale
biodegradation atudiea with organic polhllantj iiaing • while rol fiinguj,' in Remedial Action.
Treatment, ud DUPOM! of Hazatdoua Waiinf- Proceeding! of 16lh Annual EPA Research Symposium.
U.S.E.P.A.. Cincuinau. OH: April 3-5. I990» (in preu).
J. A. Glaaer, H. H. Tabak, ud E. J. Opalkea. •Treelroenl of Aqueoui Slreami of Hazardoui Waile
by a Wood-Degrading Fungui.' in Chemical ud Biochemical Deloiificalion of Haiardom Wasie 11. J.
A. Glaaer, Ed.; Lewii Publiahen; Cheliea. Ml. 1991.
-------
91-20.4
32. I. A. Gluor, H. H. Tabak, B. J. Opalken. T. W. Joyce, H. M. Chug, S. Strobofef, md C. Hummel,
"Uie of while-rot fungus in a RBC," in Abstracla of F-PA SYrflffffffiuni fffl Bioremediition of Hazardous
Wanes. EPA'i Biosyslem Technology Development Progrmm, Arlington, VA, 1990b, pp 5-8.
33. I A. Bumpuj ud S. D. AIM. Biodegradation of Environmental Pollutants by ibe While Rot Fungui
PhanerDchaeie chrysosporium: Involvement of Ibe Lignin Degrading Syilem. BioEmvi 6: 166
(1917).
34. K. E- Hammel- Orguopolluuni Degradation by Lignioolytic Fungi. Enrvme Microbiol. Technol. 1:
776 (1989).
35. K. E. Hunmel ud P. Tardone. The Oxidalive 4-DechlorinaIion of Polychlorinaud Phenols ii
Catalyzed by Extracellular Fungal Lignin Peroxidaaea. Biochemistry 27: 6563 (1988).
36. O. I. Miloski. 1. A. Bumpua. M. A. Junk, and S. D. Auat. Biodegra-danon of Pealachlorophenol by
the While Rol Fungui Phanerochaele chrv«o«Dorium. Appl. Environ. Microbiol. 54: 2885 (1988).
37. J. A. Bumpua. Biodegradalion of Penlachlorophenol by the While Rol Fungua Phuerochaele
chrnofporiuin. Appl. Environ. Microbiol. 54: 2185 (1988).
38. J. B. Un, H. Y. Wang, and R. F. Hickey. Degradation Kinelici of Penlachlorophenol by
Phanerocbaelc chrvsosporium. Biolechnol. Bioeng. 35: 1125(1990).
39. K- E. Hammel, B. Kalyanaramu. and T. K. Kirfc. Oxidation of Polycyclic Aromalic Hydrocarbons
and Dibemofp>dioxing by Phanerochaete chrvsoroorium Ligninaae. J. Biol. Chem. 261: 16948
(1986).
40. M. S. Sanglard. D. Sanglard, and A. Fiechler. The Role of Extracellular Ligninue in Biodegradalion
of Beuo(i)pyrene by Prunerochaele chrmnporium. Enzyme Microbiol. Technol. 8: 209 (1986).
41. J. A. Bumpui. Biodegradalion of Polycyclic Aromatic Hydrocarbon! by Phanerochaete chrvgosporium.
Aool. Environ. Microbiol. 55: 154 (1989).
42. S. D. Haemmerii, M.S.A. Leiaola, D. Sanglard. and A. Fiechier. Oiidaikmof Benzo(a)pyrene by
Extracellular Lipiinases from Phanerochaete chrvsoroonum. J. Biol. Chem. 261:6900(1986).
43. O. C. Eaton. Mineralization o Polychlorinaled Bipbeoyla by Phanerochaele chrvsoaporium: a
Ugninolylic Fungui Enrvme Microb. Technol. 7: 184 (1985).
44 J. A. Bumpua and B ). Brock. Biodegradalion of Crystal Violet by lite While Rol Fungua
Phanerochaele chrvsoroorium. Acol Environ. Microbiol. 54: 1143(1988).
45. J. K. Glenn and M. H. Cold. Decolorization of levenl Dyes by Ihe Lignin Degrading Buidiomyccle
Phanerochaele chrvsoaporium. Aopl. Environ. Microbiol. 45: 1741 (19B3).
*6. C. Crippi, J. A. Bumpua, and S. D. Alia!. Biodegradation of AID and Heterocyclic Dyea by
Phanerochaete chrvsosporium. Appl. Environ. Microbiol. 56: 1114(1990).
47. J. A. Bumpua and S. D. AUM. Biodegradation of DDT[ 1.1.1 -trichloro-2,2-bis(4-chlorophenyl)ethane}
by the While Rol Fungua Phanerochaete chrysotporium. Appl. Environ. Microbiol. 53: 2001 (1987).
12
91-20.4
48. A. Kohler, A. Jager, H. Willerhausen, and tL Graf. Extracellular LigDinaae of Phanerochaele
chrvsosporium Burdsall Haa No Role in Ihe Degradation of DDT. Apol. Microbiol. Biotechnol. 29:
618(1988).
49.
50
51.
52.
53.
54
55.
56.
57.
58.
59.
60.
T. P. Ryan and J. A. Bumpua. Biodegradalion of 2,4,5-Trichlorophenoiy-acelic Acid in Liquid
Culture and Soil by Ihe While Rol Fungua Phapemfhrtflfi cfu-yaosporium. Appl. Microbiol. Biolcchnol.
31: 302 (1989).
D. W. Kennedy, S. D. Auat, and J. A. Bumpua. Comparative Biodegradalion of Alkyl Halide
Inaecticidea by the While Rol Fungua. Phanerochaele chrvaoEnorium (BKM-F-1767). April. Environ.
Microbiol. 56: 2347 (1990).
T. Fernando. J. A. Bumpus, and S. D. Auat. Biodegradation of TNT (2.4,6-Trinitroioluene) by
Phanerochaele chrvsoiporium. Appl. Environ. Microbiol. 56: 1666 (1990).
T. Fernando and S. D. Auat, 'Biological decontamination of w»ltr contaminated with explosives by
Phuerochaete chrvsogporium." in Proceedings of 1GT Symposium. International Gas Technology, New
Orleans, LA, Dec. 3-5, 1990 (in press).
H.W.H. Schmidt, S. D. Haemmerli, H. E. Scboemaker. and M.S. A. Lnaola. Ondative Degradalion
of 3.4-Dimethoxybenzyl Alcohol ud its methyl ether by the Lignin Peroiidaae on Phuerochsele
chryaosnonum. Biochemistry 28: 1770 (1989).
M. Arjmand and H. Sandennann. Mineralization of ChJoroaniline/Lignin Conjugates ud of Free
Chloroanilmes by Ihe White Rot Funtua Phanerochaelt chrvsoroorium. J. Atnc. Food Chem. 33:
1055 (1985).
J. L. Popp and T. K. Kirk, 'Studies on ihe oxidation of melhoxybenzenes by manganese (111) and the
manganese-dependent peroxidase of Phanerochaete chrysosporium." in Abstracts of Fourth International
Conference: Biotechnology in Ihe Pulp and Paper Industry. Raleigh, NC, May 16-19, 1989.
H. Slroo, M. A. Jurek, J. A. Bumpus, M. F. Torpey. and S. D. Ausl. Bioremedialion of Wood
Preserving Wastes Using ihe While Rol Fungus Phanerochaete chrvsosporium. Remediation
Technologies, Inc., 1989
R. T. Lamar, T. K. Kirk, and J. A. Glaser, "Use of while-rot fungi to remediate soils contaminated
with wood-preserving waste,' in Abstracts of EPA Symposium on Bioremedialion of Hazardous
Wastes. EPA's Biosyslem Technology Development Program, Arlington. VA, 1990, pp 31-32.
K. M. Haider ud J. P. Martin. Mineralization of "C-Labeled Humic Acids ud Humic-Acid Bound
"C-Xenobiotics by Phuerochaele chrvsosporium. Soil Biol. Biochem. 20: 425 (1988).
R. Blondeau. Biodegradalion of Synthetic Humic Acids by the While-Rol Fungus Ptianerochseie
chrYsoiporium. Appl. Environ. Microbiol. 5: 1282 (1989).
H. H. Tabak ud W. B. Cooke. Effects of Gaseous Environments on ihe Growth and Metabolism of
Fungi. Botanical Review. 34: 126(1968). _. _..!•/ '-
S. S. Bar-Lev ud T. K. Kirk. Effects of Molecular Oxygen on Lignin Degradalion by Phuerochaele
chrysosporium. Biochem. Biophvi. Res. Comm. 99: 126(1981).
-------
91-20.4
61. T. K. Kirk, E. Schulu, W J Coraor*, 1, F. Loroni. andJ, O. Zeikui. Influence of Culture
Parameter* on Lignin Metabolism by Phmerochaeta cJnyaoBporium. Areh. Microbiol. 117: 277
(197B).
6), M. A. Tylar and A. D, Fitzgerald, *A review of color reduction technology in pulp lad paper mill
effluent, ' in ilih Annual Mceiinf of ihe Piper and Pulp Indiuiiv. Technical Section CPPA, 1972,
64, A.C.Campbell, A Bench-Scale Evaluation of (he MyCoR Proccu for Decolori cation of Bleach. PUni
Effluent U«Bg ihe White-Rot Fusgui, PhaBerpchae^a cfarvioaDoriuiii. Ph.D. Tlieiii, Hoitb Carolina
Sltl£ Umvcnily, Raleigh, NC, 1983
61, C. F. Yin. Kinetiu of Fungal Dccolohution in the MyCoR Proccai and Bioireaunrai of ila Eflluent.
M.S. Thuii, North Carolina Stale Univenily, Raleijb, NC, 1986.
66 T. K Kirk. Smdiei on ihe Phjuologj of Li jmn Meuholiiai by the Whiw-Roi Fungi. In Ijgsua
BiodegntdaiioH: Microbiology. Chemiiiry. aad Pojemial Applica^^. T, K. Kirk, T. HigucbJ, and H.
M. Chang, edi. CRC Pre«. Inc., Boca Raton. FL, Vol. 2. 1980. pp 51-64
67. H. Janihadar, C. Brown, and A. Fiechlcr. Determioition of Lignio From Alkaline Pulping Liquors.
Anal, Chim. Acu. IJO- «l (I9g|),
6B. R. AJen und T. Hartui. UV Spectropholomeinc Detenmnatiog of Ligmirj From Ajjcaline Pulping
Liquora Cell. Chem. Technol. 22:613(1988).
69. 1. Cellmao. An Ifivesligalion of Improved Procedure! for Measurement of Mill Effluent and Recewmg
Water Color NCASI Technical Bulletin No. 253; Ni!ion«l Council of Ihe Paper Indlinry for Air and
Stream Improvement, !nc; Ne^ York, 1971.
91-20.4
TABLE 1. SUMMARY OF DESIGN PARAMETERS FOR BENCH- AND PILOT-
SCALE ROTATING BIOLOGICAL CONTACTOR UNITS. Bench-scale
RBC units were built after the design of Chang et at. (1985. 1987). and piloi-
scale RBC units were 77.SX scaled-up modified versions of this design.
Parameter
Bench
Pilot
Volume
Disk Diameter
Disk Surface Area
Disk Material
Disk Submergence
Shaft RPM
Operation Mode
2L
,015m
0.28m'
Plexiglas
40%
1-10
Batch
155L
0.5m
32.4m'
HOPE '
40%
1-10
Batch with recirculation
-------
91-20.4
TABLE 2, EFFECT OF GLUCOSE CONCENTRATION AND DISC SHAFT
ROTATIONAL SPEED ON TREATAB1LITY OF KRAFT LIQUOR BY L
CHRYSOSPQRIUM. Using bench-scale RBC units, fungal biofilms were
grown and maintained under an atmosphere of 100 percent oxygen and in
Kraft liquor containing 1,0 percent or 05 percent glucose, with RBC unit
discs operated at indicated speeds. Treatabiliry was assessed by triplicate
measurements of Kraft liquor concentration a( 8 and 17 h of treatment using
both absorbance at 280 ntn and platinum color units.
Total Average Removal (%)
Reactor Conditions
Glucose % Disk Speed (rpm)
8hf
17 hr
Absorbance Platinum Absorbance Platinum
280nM Color Units 280nM Color Unils
1.0
1.0
0.5
0.5
1
6,5
6.5
1
10
13
11
5
11
39
41
33
27
39
19
23
19
20
21
57
" 59
55
51
55
91-20.4
TABLE 3. EFFECT OF ALTERNATIVE BUFFERS ON pH MAINTANCE OF RBC
UNIT MEDIA DURING TREATMENT OF KRAFT LIQUOR BY
t CHRYSOSPORIUM. Using bench-scale RBC units, fungal biofilrn were
grown and maintained under an atmosphere of air using a Kraft liquor media
containing 0.5 percent glucose and 0,05 M inorganic buffer, as indicated.
Buffer
Batch
Time
a, pH = 5,0, adjusted with HC1
b. pH = 4.53 with no pH adjustment
c. pH = 4.5, adjusted with NaOH
pH Value
Na,HPO,
+ KHjPO, '
KHfOt b
Growth
1
Growth
2
*
Growth
i
Growth
2
Ohr
24 hr
48 hr
Ohr
24 hr
48 hr
Ohr
24 hr
48 hr
Ohr
24 hr
48 hr
4.52
4.00 c
4.53
4.50
3,70 c
4.48
4.53
4.15 c
4.28
4.53
3.50 c
4.43
-------
91-20.4
TABLE 4. LIST OF EXTRACTION SURROGATES, GC INTERNAL STANDARDS
AND KRAFT LIQUOR EFFLUENT INDIGENOUS COMPOUNDS, AND
THEIR CONCENTRATION LEVELS IN KRAFT LIQUOR BEFORE AND
AFTER TREATMENT WITH £, CHRYSOSPQR1UM.
Compound
/. Extraction Surrogate
2-Fluorophenol
Phenol d-6
2.4 ,6-Tribromophenol
2, CC Internal Standard
d4-l ,4-Dichlorobenzene
d8-Naphihalene
d 10- Acenaphlhene
d!2-Chrysene
dl2-PeryIene
3. Sample Compounds
2,4 Dichlorophcnol
Benzoic Acid
2,4,6 Trichlorophenol
Pentaehlorophenol
Di-n-Butylphlhalate
Bi$(2- Eihylheiyl)Ptuhalate
Di-n-ociyl phthalate
Concentration
Before
68.66
65.95
61.66
40.00
40.00
40.00
40.00
40.00
1.51
9.00
4.10
0,92
1.62
-
(mg/L)
After
49.2
45.46
41.61
40.00
40.00
40.00
40.00
40.00
--
..
--
--
0.78
_
1.89
I. Surrogate Compounds - spiked into Kraft liquor effluent before extraction
2. GC Internal Standards - spiked into Kraft liquor effluent extract before GC/MS analysis
3. Sample Compounds - indigenous to Kraft liquor effluent
91-20.4
TABLE 5, EFFECT OF KRAFT LIQUOR HOLDING TIME ON TREATABIL1TY OF
KRAFT LIQUOR BY £. CHRYSOSPOR1UM. Using bench-scale RBC units,
fungal biofilms were grown and maintained in Kraft liquor (aged 1, 4, and 8
weeks) under the experimental conditions indicated. Treatability was assessed
by triplicate measurements of Kraft liquor eonceniractions at each lime point
using absorbance measurements at 580 nm, 450 nm, and 280 nm, and using
platinum color units.
Reaclor
Age
Reacior 1
1 week
old
4 week
old
8 week
old
Reacior 2
1 week
old
4 week
old
8 week
old
Reacior 3
1 week
old
4 week
old
B week
old
Reactor 4
1 week
old
4 week
old
8 week
old
% Removal % Removal
ai 580 nm at 450 nm
% Removal
at 280 nm
% Removal
of C.U.
- 1 % jlucose/1 rpm/oxveen
24 hr
48 hr
24 hr
48 hr
24 hr
48 hr
23
47
24
40
18
33
18
36
15
35
14
29
4
13
12
20
6
12
21
41
16
35
17
37
- 1% plu^ojS/ 1 ipsnfaii
24 hr
48 hr
24 hr
48 hr
24 hr
48 hr
- 0.5% glucose/6.
24 hr
48 hr
24 hr
48 hr
24 hr
48 hr
- 0.5% glucose/6.
24 hr
48 hr
24 hr
48 hr
24 hr
48 hr
8
21
22
33
15
30
5 rpm/oxv|en
32
60
45
60
3
21
5 rpm/air . •! "
18 -" iv
35
19
41
12
33
« 7
17
12
26
13
22
32
SI
29
47
12
22
"'-, 20
30
13
30
15
25
4.5
12
16
20
7
13
29
32
17
15
10
15
14
17
11
8
11
16
24 .
31
14
33
26
38
37
61
24
45
26
33
25
46
7
28
31
37
-------
91-20.4
TABLE 6, Reduction in 2,4-DICHLOROPHENOL CONCENTRATION BY
£» CHRY5OSPQR1UM VERSUS TIME. Using bench-scale RBC units,
fungal biofilms were grown and maintained under an atmosphere of air and in
media containing 0.5 percent glucose and 2,4-dichlorophenol (DCP), as
indicated, Treatability was assessed by triplicate measurements of DCP
concentration at each time poinl by USEPA Method 3250.
Target
Batch
Time
Qhr
24 Kr
48 hr
72 hr
Reactor 1
18,0
0.52
< 0.8
0.52
mg/L PCP,
Reactor 2
20 mg/L
DCP
1 Ohr
24 hr
48 hr
72 hr
96 hr
17.1
2.08
< 1
-
< 0.1
17.1
1.36
< 1
< 0.1
18,0
J.S
100 mg/L
DCP
' 1 Ohr
24 hr
48 hr
72 hr '
96 hr
120 hr
144 hr
84.8
42.1
31,7
24.9
5.55
< 1
™
84.8
56.2
43,3
29.9
17.7
5.22
0.16
NOTE TO EDITORS
Under the new federal copyright law,
publication rights to this paper are
retained by the author(s).
91-20.4
I
X ptf
NU i
a oj.
o
21
-------
BATCH FEED TANK
WITH HEATER
pH MONITOR & CONTROL SVSTEM
RECIRCULATION LINE
FIGURE 2. SCHEMATIC OF THE PILOT-SCALE RBC UNIT FOR STUDIES OF TREATMENT OF WASTEWATLKS BY
Et CHRYSOSPORIUM. Pilot-scale RBC uniu were constructed lo accommodate changes in pH, temperature, and
atmosphere, based on experimental protocol requirements. Additionally, provisions for ^circulation or treatment
media have been incorporated.
0.15*
A
b
8
0
r
b
a
n
c
e
5
8
0
n
m
Nonllg
Liflnolytic
Inactive
P
C
U
t
h
o
u
s
a
n
d
s
24 48
Time (hrs)
FIGURE 3. EFFECT OF CONCENTRATION OF BIOAVAILABLE NITROGEN ON TREATMENT OF KRAFT LIQUOR
EFFLUENT BY E, CHRYSOSPORIUM. Using bench-scale RBC until, ihe fungus was grown in media
containing no added nitrogen (Nonlig); low nitrogen, 2.2 mm NH«CI (Lignotytk); and high nitrogen, 13.2 mm
NH,C1 (Inactive) concentrations, Treatabilily was assessfirl by triplicate measurements of Kraft liquor effluent
concentrations as each time point using boUi absorbance at 580 nm and platinum color uniu.
-------
Dil/Air
Dil/Ox
-*- Undil/Air
Undil/Ox
10
20 30 40 50
Time (hrs)
60
70
p
c
u
t
h
o
u
s
a
n
d
s
FIGURE 4. EFFECT OF KRAFT LIQUOR EFFLUENT CONCENTRATION AND OXYGEN CONCENTRATION ON
TREATABIUTY BY P. CHRYSOSPORIUM. Full strength, 4,000 PUC (Undil). and diluted, Z 3,000 PUC
(Oil) Kraft liquor effluents were treated in bench-scale RBC units by Ihe fungus grown and maintained in the
presence of air (Air) or 100 percent oxygen (Ox). Trcatability was assessed by triplicate measurements of Kraft
liquor effluent concentrations at each time poinl using both absorbancc at 580 nm and platinum color units.
0.45
0.4
A
t> 0.35
s
r
b
a
n
c
e:
8 •
S
m
0.3
0.25
^b;2
0.05
0
20 40
Time (hrs)
60
p
c
u
t
h
o
u
s
a
n
d
8
FIGURE 5. EFFECT OF RBC UNIT OXYGEN CONCENTRATION ON THL ACCLIMATION OF L CHRYSOSPORIUM
Using bench-scale RBC units, fungal biorilms were established and subsequently maintained in atmospheres of air
or 100% oxygen. Trealabilily was assessed by triplicate measurements of Kraft liquor effluent concentrations ai
each lime poinl using best absorbance at 580 nm and plaimum color units.
-------
A
b
s
o
r
b
a
n
C
e
5
8
0
n
m
0.4 -
0.35
0.3 s
0.25-
0.2 -
0.15
0.1
0.05
f\
0.5% Glucoae
1.0% Glucose
20 40 60
Time (hrs)
T~
80
100
p
C
u
t
h
o
u
s
&
n
d
s
FIGURE 6. EFFECT OF RBC UNIT GLUCOSE CONCENTRATION ON TREATABILITY OF KRAFT UQUOR BY
IL CHRYSOSPORIUM. Using bench-scale RBC units, fungal bioftlms were grown and maintained in Kraft liquor
containing 0.5% or 1.0% glucose. Treatability wai assessed by triplicate measurements of Kraft liquor
concentrations at each lime point using both absorbance at 580 nm and platinum color units.
A
b
3
O
r
b
a
n
c
e
5
a
0
n
m
0
0
0
•**•>
0
0
.45
0.4
.35i
0.3
.25
0.2
sl'5-
1
OA
.05
0
KTar 1%
DMS 1%
KTar 0.5%
DMS 0.5%
P
C
U
I
h
o
u
s
a
n
d
s
24 48
Time (hrs)
FIGURE 7. EFFECT OF RBC UNITS BUFFER ON TREATABILITY OF KKAl-T LIQUOR BY £. CHRYSOSpOR|UM.
Using bench-scale RBC units, fungal biofilms were grown and maintained in Kraft liquor containing 0.5 percent or
1.0 percent glucose in the presence of either 20 mm 2,2-dimeihyl succinalc (DMS) buffer (pH = 4.5) or 20mm
potassium lanrate (KTar) buffer (pH = 4.5). Trcalabilily was assessed by triplicate measurements or Kraft liquor
concentrations at each time poinl using both absorbance al 580 nm and platinum color units.
-------
91-20,4
91-20.4
1100 tioo Mid i>->«
IBOGOQ^
i-OQOO-
12000O-
OOOOO-
iQOOO*
0-
1200 l^C-3 S*?0
D5COW
It I
III
• ! 1 I
— i £
I
!' !
s
!
Ill ,
1 1 11
4 1 i-> il 1« i* 1* «i 1^ «•< s- 2j j:- "„£ ?* >. ;«
FIGURE 8. TOTAL ION CHROMATOGRAMS OF THE ANALYSIS OF KRAFT
LIQUOR BEFORE AND AFTER TREATMENT WITH
IL CHRYSOSPOR1UM. Untreated and fungal-treated samples of Kraft liquor
were spiked wiih 2-FIuorophenol, Phenol d-6 and 2,4,6-tribromophcnoI at
concentration levels of 68.66, 65.95, and 61.66 mg/L respectively, and
extracted via EPA Method 3250, following which extracts were analyzed by
GC/MS, Internal standards were detected, as indicated.
28
O
•o
o>
o
IO
o
1/Biu
2
JZ
-------
a.
U
a
60
50
40
30
20
10
12
Time (hrs)
18
24
HGURB IO, KlNl-TICS ()l: DCP REMOVAL BY t gHRYSOSTOKIUM USING NITROGEN-SUPPLEMENTED
GROWTH MHDIA. IIsm| bench-scale RBC units, fungal biofilm was grown and mainuined under an
atmosphere using Kraft liquor media containing 05 percent glucose and 2.2 mM NH,C! Trcatabihty was
assessed al the time points indicated by Inplicate measurements of DCP concentration by USliPA Method 12SO
100
mg/l
8
12
24
Time (hrs)
48
FIGURE II. TIME COURSES FOR THE REMOVAL OF DCP BY £» CHRVSOSPOMUM IN PILOT-SCALE
EXPERIMENTAL TRIALS. The fungus wai grown in the pilot-scale RBC uniu in an air atmosphere and
maintained in an oxygen (100%) atmosphere during DCP treatment. Growth and subsequent DCP treatment
conditions included: disc-shad rotation of 1 rpm, temperature or 39C; pH maintenance at 4.0 to 5.0; buffer of
50mM K-Tar; glucose al 0.5*. Trealability was assessed by triplicate measurements of DCP concentrations at
each time point by USEPA Method 3250.
------- |