the ohio journal of sciencethe ohio journal of science vol. 70 may, 1970 no. 3 fungi associated with...

THE OHIO JOURNAL OF SCIENCE

Vol. 70 MAY, 1970 No. 3

FUNGI ASSOCIATED WITH THE ACTIVATED-SLUDGEPROCESS OF SEWAGE TREATMENT AT THE

LEBANON, OHIO, SEWAGE-TREATMENT PLANT1

WM. BRIDGE COOKE

Mycologist, Biological Treatment Activities, Advanced Waste Treatment Research Laboratory,Robert A. Taft Water Research Center, FWPCA, 4676 Columbia Parkway,

Cincinnati, Ohio 1,3226

ABSTRACTSamples of materials in various stages of sewage treatment were obtained at monthly

intervals between October, 1967, and April, 1968, from the Lebanon, Ohio, sewage-treat-ment plant. From both secondary and tertiary processes, a total of 93 species or speciesgroups of fungi, including filamentous and yeast-like types, was recovered. Methods ofsampling and isolation techniques, and data for several chemical and physical measure-ments made on the samples are presented. Comparisons of bacterial and fungal populationstested show that, on the basis of colonies recovered per ml of sample, effluent from theactivated sludge aerator yielded 41 bacteria to one fungus, in contrast to a ratio of 1430bacterial cells to one fungus cell in raw sewage. Many of the more common species offungi present in the Lebanon sewage-treatment plant have also been recovered from similar-process materials in sewage-treatment plants at Dayton, Ohio, a number of sewage-treat-ment plants in the Chicago, Illinois area, a small waste-stabilization pond system in adifferent watershed near Lebanon, and from other sewage-treatment plants elsewhere.Fungi, being reducer organisms, are admirably adapted to a habitat in which their assim-ilative cells are continually bathed in a nutrient medium such as that offered by sewagein the process of treatment by the activated-sludge process.

INTRODUCTIONIn general, throughout the literature dealing with sewage treatment, when

the activated-sludge process is considered, emphasis is placed on the zoogloealbacteria as the organisms responsible for good floe formation in an efficient treat-ment process. Literature of this subject has been reviewed by Cooke and Pipes(1969). That fungi are also present has been recognized for some time, especiallyin reference to one of the several types of bulking for which filamentous organismsare considered responsible, but it is only recently that the presence of fungi hasbeen thought of as beneficial.

The present study was initiated in order to determine the kinds of fungi presentin a small activated-sludge-type sewage-treatment plant, those techniques whichwould develop the fungal populations adequately, and the numbers of bacteriapresent at the same time. At the same time, a search was also made for fungiwhich might be associated with tertiary-treatment processes in an adjacent waste-water-renovation pilot-plant, in which the influent was the final product of thesecondary plant.

METHODS

The data presented here were collected as a result of a search for as manykinds of fungi as possible in materials collected in critical stages of a sewage-

manuscript received May 28, 1969.THE OHIO JOURNAL OF SCIENCE 70(3): 129, May, 1970.

130 WM. BRIDGE COOKE Vol. 70

treatment process. The flow chart in Figure 1 shows from where samples wereobtained: 1. raw sewage, 2. primary settled sewage, 3. aeration-tank influent,4. aeration-tank effluent, 5. secondary effluent (effluent from a secondary settling-tank), 6. return sludge, and 7. primary settled sludge. Samples were collectedonce a month for seven months between October, 1967, and April, 1968. Sampleswere obtained from each of the seven process stages listed above on each date:Oct. 12 and 30, and Dec. 14, 1967, Jan. 25, Feb. 20, March 27, and April 23, 1968.

1 R A W S E W A G E

P R I M A R Y

S E T T L E R

P R I M A R Y E F F L U E N T 2

M I X E D L I Q U O R I N F L U E N T 3

• + 7 P R I M A R Y S L U D G E

6 R E T U R N S L U D G E

A E R A T O R

M I X E D L I Q U O R E F F L U E N T 4

S E C O N D A R Y

S E T T L E R

5 S E C O N D A R Y E F F L U E N T

FIGURE 1. Flow chart of Lebanon, Ohio, secondary sewage-treatment plant (R. V. Villiers).

Samples were obtained in the morning, returned to the Cincinnati laboratory, andprocessed the same day. Samples were also taken from tertiary-treatment unitswhich were in operation in the adjacent pilot plant on the same sampling date.Available tertiary-treatment units are indicated in the flow chart in Figure 2.

At the laboratory, portions of each sample were immediately removed forbacteriological analysis. Total count and the count of Pseudomonas spp. werethe only bacteriological determinations made of these samples. Residues of allsamples were analysed for total organic carbon and for organic nitrogen.

No. 3 FUNGI IN SEWAGE TREATMENT 131

For development of fungus populations, samples were processed as in theLaboratory Guide (Cooke, 1963), using pour plates in replicates of five withneopeptone-dextrose agar, with and without rose bengal, but with Tetracycline,and using shaken-flask culture with YNB-1% glucose and YNB-20% glucose(Cooke, 1965). In order to determine whether or not Candida albicans waspresent, plates were prepared in which ABY and BiGGY agar (Nickerson, 1953),as well as Pagano-Levin Base agar (Difco, 1962), were used.

B I O L O G I C A LO X I D A T I O N

F I G U R E 1

S E C O N D A R Y E F F L U E N T

L I M E

C L A R I F I C A T I O N

D U A L M E D I AF I L T E R S

A N T H R A F I L TOVER S A N D

( R E M O V E S O R G A N I CA N D I N O R G A N I CS U S P E N D E D M A T T E RR E M O V E S P 0 4

A N D M g + + . )

( R E M O V E S S U S P E N D E DM A T T E R E S C A P I N GC L A R I F I E R I

T W O S T A G EC O U N T E R C U R R E N T

P O W D E R E DC A R B O N

P R O C E S S

±D U A L M E D I A

F I L T E RA N T H R A F I L TOVER S A N D

( R E M O V E SS O L U B L E A N DS U S P E N D E DO R G A N I CM A T T E R J

( R E M O V E SP O W D E R E D C A R B O NP A R T I C L E SE S C A P I N GP R O C E S S ]

G R A N U L A RC A R B O NC O L U M N S

8 * 3 0 M E S H

P R O D U C T

E L E C T R O D I A L Y S I SU N I T

( R E M O V E S S O L U B L EO R G A N I C S I

( R E M O V E SD I S S O L V E DS A L T S ]

P R O D U C T

FIGURE 2. Flow chart of Lebanon, Ohio, tertiary sewage-treatment pilot plant (R. V. Villiers).

For relatively even distribution of sample and for partial break-up of floe,1:10 dilution samples were shaken on the rotary shaker for a half hour at 130-150oscillations per minute. Incubation of all preparations was at room conditionsof light and temperature. Agar pour-plates were read after seven days, and otheragar plates were read upon adequate colony development. Shaken-flask cultureswere removed from the shaker after 64-98 hours. Colonies were identified accord-ing to procedures outlined in the Laboratory Guide (Cooke, 1963). It should be


noted that, for counting the fungus colonies, replicates of five plates were pouredfor each sample and for each medium. Counts presented in Tables 1, 2, and 3were taken from neopeptone-dextrose-rose bengal-Tetracycline agar plates. Inaddition, microscopic observations showed mycelial filaments in much of theactivated-sludge floe obtained on each of the seven sampling dates.

RESULTS

Numbers of bacteria and fungi recovered from samples obtained in the Lebanonsewage-treatment plant and in the tertiary-treatment pilot plant are given inTables 1 and 2. In addition, certain pertinent data concerning the sewage are

TABLE 1

Lebanon Sewage-Treatment Plant Data

Date

10-12-6710-30-6712-14-671-25-682-20-683-27-684-23-68

Code

ABCDEPG

ABCDEFG

ABCDEFG

ABCDEFG

ABCDEFG

SVI

121117112

SS

220225225

767864

1199

BOD

190192178194190169184

72747177707875

10101212151211

D O

2.41.9

.9

TOC Org. N. p H

1. RAW SEWAGE

12812266

125869865

2. PRIMARY

52583328434039.

15.215.68.4

11.211.22.3

10.4

SETTLED

10.46.40.95.26.87.6

5 7.0

7.357.5NTNTNTNT6.8

BacteriaTotal/ml

X105

60140NTNT463.9

29

' SEWAGE

7.57.1NTNTNTNT6.2

2934

NTN T511.2

17

3. MIXED LIQUOR INFLUENT(2.5)(2.9)c—)

4. :

3.93.94.0

10001000324648

1200343306

HIXED

680420103238

1000408450

115.0187.0

9.5133.0270.052.0

108.0

7.07.3N TN TNTNT6.1

8573

NTN T

2.45.9

180

LIQUOR E F F L U E N T145.0110.0

4.059.8

130.080.0

115.0

6.97.0NTNTNTN T6.3

5. FINAL PRODUCT

19.2 5.71192226291211

.0 12.2

.0 4.5

.5 4.9

.5 <0.05

.0 2.0

.0 1.9

7.27.0NTNTNTNT6.6

3428

NTNT

1.34.9

170.0

2.92.1N TNT0.680.164.3

Yeast-like

IN 7mlxio»

1010.233

100.1

0.10.20.20.14.31.00.001

1.01.0

20.02.9

30.0100.010.0

1.010.020.02.03.01.01.0

0.10.10.30.30.0010.10.1

Fungi

Filamentous

Colonies/mlX103

1.29.01.91.81.63.78.0

2.215.01.42.00.82.47.0

160.05.8

12.015.042.012.024,0

220.0520 0

6.417.017.010.024.0

1.323.00.642.2

0.90.260.23

Colonies/gmQDW**

xio«

6.40.092.12.01.80.611.3

1.0.111.031.40.560.260.73

37.01.31.60.249.50.170.8

76.01.82.35.85.40.520.8

1.20.190.00541.80.00750.0750.011

No. 3 FUNGI IN SEWAGE TREATMENT

TABLE 1—Continued

133

Fungi

Date Code SVI SS BOD DO TOC Org. N. pH Bacteria Yeast- FilamentousTotal/ml like

X105 IN*/ml Colonies/ml Colonies/gmX103 X103 ODW**

X106

6. RETURN SLUDGE

2000.0 332.0 6.71670.0925.01950.03250.0

19.0964.0

308.0119.0401.0440.0104.0275.0

7.1NTNTNTNT6.3

35.078.0NTNT4.35.9

380.0

7. PRIMARY SETTLED SLUDGE

10,500.012,300.016,000.015,900.0

NS18,000.01,400.0

386.01155.0lost1255.0NS930.0120.0

5.86.1NTNTNSNT6.8

230.01600.0

NTNTNS490.0600.0

1.02.0

300.0300.0

0.210.01.0

100.0300.020.020.0NS

100.010.0

3.7500.023.022.055.020.047.0

8300.0660.0200.0130.0NS250.041.0

5.80.783.63.48.60.0760.18

21.00.175.03.3NS5.80.96

*IN—Indicated Number."ODW-Oven Dry Weight.SVI—Sludge Volume Index.SS—Suspended Solids.NT or NS—Not sampled.

TABLE 2

Lebanon Tertiary Processes

Date

10-12-6710-30-6712-14-671-25-682-20-683-27-68

4-22-68

Code

ABCDEF

EG

TotalOrganicCarbon

9.0NTNT

15.216.612.0

15.87.0

OrganicNitrogen

Lime

1.6NTNT2.32.51.1

Bacteria

TotalCount/ml

X103

Pseudo-monasper mlX103

Clarifier Effluent

8.0N TNTNT

51.01.0

5.2NTNTNT

49.00.7

Lime-Clarifier Filtered Effluent

1.80.9

180.04.8

170.02.7

Numberof

Species

72

5184

86

Fungi

Colonies

Fila-mentous

105

172250

2210

per ml

Yeast-like

20

13500

870

G 6.6

Granular-Carbon-Column Feed

0.95 5.0 4.4 2564

134 WM. BRIDGE COOKE

TABLE 2—Continued

Vol. 70

Date Code

ABEG

ADG

ABFG

4- 3-68 WashingScraping

TotalOrganicCarbon

OrganicNitrogen

Bacteria

TotalCount/ml

X103

Pseudo-monasper ml

X103

Numberof

Species

Granular-Carbon-Column Effluent

3.0NT

<0.62.0

0.7NT0.10.4

1.7NT

70.00.69

1.6NT

40.00.39

2447

Powdered-Carbon-Column Effluent

9.08.02.8

1.2NT5.02.6

3.90.150.35

3.7NT2.0

28.0NT1.5

Electrodialysis-Unit Product

0.9NT0.90.25

3.0NT1.31.0

2.7NT0.40.7

8117

9263

Electrodialysis-Unit Filter Surface

NTNT

NTNT

280.03300.0

190.03200.0

<8<8

Fungi

Colonies per ml

Fila-mentous

304

4014

2402732

1503

4418

< 50,000< 50,000

Yeast-like

20

TABLE 3

A verages of Bacterial and Fungal Populations in Lebanon SamplesBased on 7 sets of samples, Oct.-Apr.

Sewage Treatment Stage

Average Numbersof Bacteria

per ml sampleX106

5.62.66.94.80.5

10.173.0

Average Numbersof Fungi

per mlsampleX103

3.94.4

38.7116.0

4.196.0

1,083.0

per Gramoven dryweight

X106

2.00.937.2

13.20.473.26.0

RatiosBacteria : Fungi

per mlsample

143060017941

13210567

1111111

per Gramoven dryweight

2.7 : 12.9

.95

.41.23.1

12.1

111111

Raw sewagePrimary Settled SewageAerator InfluentAerator effluentFinal ProductReturn SludgePrimary Settled Sewage

NO. 3 FUNGI IN SEWAGE TREATMENT 135

also given. From available plant records, data were obtained on sludge-volumeindex (SVI), suspended solids (SS), biochemical oxygen demand (BOD), anddissolved oxygen (DO) for the dates on which the samples were collected; theseare included in Table 1. Total organic carbon and organic nitrogen are givenfor all samples in parts per million. Total aerobic bacteria, and total numbers ofPseudomonas observed on membrane filters, have also been listed. While totalbacterial counts were originally reported in terms of numbers per 100 ml, thesehave been corrected to numbers of bacteria per ml in Tables 1 and 2, since parallelrecords of fungi are based on numbers per ml.

Numbers of yeasts and of filamentous fungi were obtained in the FungusStudies Laboratory, as were pH levels when opportunity permitted. Numbersof fungi are reported both as total colonies observed per ml of sample and as totalcolonies per gram dry weight of sample. In obtaining the latter value, the amountof oven-dry matter (at 100°C) was determined for the first set of samples only.It was assumed that samples obtained from comparable stages in the treatmentprocess on later dates did not differ greatly from the first samples collected in theseries. Averages of the values for the total count of bacteria per ml, and of thecorrected total number of fungus colonies per ml of sample and per gram dryweight for each stage in the treatment process sampled are presented in Table 3.Ratios given here are for bacteria in relation to fungi present, based both on valuesper ml and values per gram oven-dry-weight.

An alphabetical list of species of fungi, regardless of systematic position,isolated from all secondary and tertiary treatment samples, is given in Table 4.Without considering incompletely identified yeasts, 93 species of fungi are listed.Of the yeasts, 32 species of white yeasts and two of red yeasts were identified from

TABLE 4

Alphabetical list of species of fungi isolated from various sewage-treatmentprocesses at Lebanon, Warren Co., Ohio

Allescheria boydii ShearAlternaria alternata (Fries) KeisslerAspergillus spp.

flavipes (Bainier and Sartory) Thorn and Churchflavus Link ex Linkfumigatus Freseniusniger van Tieghem

cf. puniceus Kwon and Fennellsydowii (Bainier and Sartory) Thorn and Churchustus (Bainier) Thorn and Church var. ustusversicolor (Vuillemin) Tiroboshi

Aureobasidium pullulans (de Bary) ArnaudBotrytis cinerea PersoonCandida brumptii Langeron and Guerra

catenulata Diddens and Loddercurvata (Diddens and Lodder) Lodder and Kreger-van Rijguilliermondii (Castellani) Langeron and Guerrahumicola (Daszewski) Diddens and Lodderintermedia (Ciferri and Ashford) Langeron and Guerrakrusei (Castellani) Berkhoutmycoderma (Reess) Lodder and Kreger-van Rijparapsilosis (Ashford) Langeron and Talicepelliculosa Redaelliscottii Diddens and Loddertennis Diddens and Loddertropicalis (Castellani) Berkhoututilis (Henneberg) Lodder and Kreger-van Rij

?zeylanoides (Castellani) Langeron and Guerra


TABLE 4. (Continued)

Cephalosporium spp.Chaetomium funicolum M. C. CookeCladosporium cladosporioides (Fresenius) de VriesConiothyrium fuckellii SaccardoCryptococciis spp.

diffluens (Zack) Lodder and Kreger-van Rijlaurentii (Keufferath Skinnerluteolus (Saito) Skinner

Epicoccum purpurascens Ehrenberg ex SchlechtendahlFusarium spp.

acuminatum Linkaquaeductuum (Radelmacher and Rabenhorst) Saccardooxysporum Schlechtendahlroseum Link ex Priessolani (Martius) Appel and Wollenweber

Geotrichum candidum Link ex PersoonGliocladium spp.

roseum (Link) BainierGliomastix murorum (Corda) Hughes var. felina (March) HughesMoniliales spp.Mucor spp.

hiemalis Wehmerplumbeus Bonorden

Paecilomyces marquandii (Mason) Hughesvarioti Bainier

Penicillium spp.chrysogenum Thornfuniculosum Thornjanthinellum Biourgelilacinum Thornmartensii Biourgeochro-chloron Biourgevariabile Soppvermiculatum Dangeard

Phoma spp.herbarum Westendorp

Prototheca stagnora W. B. CookePyrenochaeta sp.Khinocladiella mansonii (Castellani) Schol-SchwarzRhizopus spp.Rhodotorula spp.

glutinis (Fresenius) Harrison var. glutinismucilaginosa (Jorgensen) Harrison

Septoria sp.Torulopsis aeria (Saito) Lodder

Candida (Saito) Loddercolliculosa (Hartmann) Saccardofamata (Harrison) Lodder and Kreger-van Rijglabrata (Anderson) Lodder and de Vriesholmii (Jorgensen) Lodderinconspicua Lodder and Kreger-van Rij

?saki (Saito and Ota) Lodder and Kreger-van Rijversatilis (Etchells and Bill) Lodder and Kreger-van Rij

Trichoderma viride Persoon ex S. F. Gray sensu latissimoTrichosporon spp.

capitatum Diddens and Loddercutaneum (de Buermann, Gougerot and Vaucher) Ota var. cutaneumfermentans Diddens and Loddermargaritiferum (Stautz) Buchwald

Verticillium sp.lateritium Berkeley

White yeast spp.


secondary-process materials, while eight of these were also recovered from tertiary-treatment processes.

Of the 93 species of fungi listed for the samples studied, 90 species were recoveredfrom processes in the secondary-treatment plant, and 37 in the tertiary processes.Of those in the secondary plant, 13 filamentous fungi were found at least once ineach of the seven processes sampled on each of the seven dates on which sampleswere taken. Two of these fungi were found in all 49 possible combinations ofcircumstances. Six species were found in six of the processes on at least one of theseven sampling days, two were found in five, five in four, ten in three, ten in two,and ten in only one process on only one sampling date. The data for distributionof the 37 species in the tertiary systems sampled are presented in Table 5.

TABLE 5

Distribution of Fungi in Tertiary-Treatment Processes

Powdered Electrodialysis UnitCarbon

Species Lime Clarifier Granular Carbon Column FilterColumn

Product Filtered Feed Effluent Effluent Effluent Washings Scrapings

Moniliales spp. x x x x x x x xPhotna spp. x x x x x x x xPenicillium lilacinum x x x x x xRhinocladietta mansonii x x x x x xRhodotonda spp. x x x x x xWhite yeasts spp. x x x x x xGeolrichum candidum x x x x* xAspergillus versicolor x x x xRhodotorula mucilaginosa x x x xCephotospot•ium spp. x x x xTrichoderma viride x x x xFusarium oxysporum x x xCandida krusei x x xPenicillium ochrochloron x x xFusarium aquaeductuutn x x xCladosporium cladosporioides x x xCryptococcus sp. x xRhodotorula glulinis x xAureobasidium pullulans x xMucor hiemalis x xAspergillus flavus xAspergillus ustus xTrichosporon sp. xCandida intermedia xTorulopsis inconspicua xPenicillium spp. x x x x x xPenicillium janthinellum x x xTrichosporon cutaneum x x xTorulopsis glabrata xRhizopus sp. xSeptoria sp. xCandida utilis xCryptococcus luleolus xTalaromyces vermiculatutn x*Phoma herbarum xAspergillus niger xFusarium spp. x xMucor spp. x x

*—Slime from carbon granules yielded these species.

Species per habitat 26 16Number of Samples 4 2

91

215

146

124

71

71


The species of filamentous fungi are listed in Table 6 by their occurrences onthe seven sampling dates. The numbers given under each date identify thesewage-treatment process from which, in isolations that were made, the namedfungus was found. In the last column of Table 6, the total number of times thefungus was observed is listed. In Table 7, letters representing the collecting datesof each fungus are given for each process. Again, in the last column of Table 7,the total number of times the fungus was observed is listed.

In addition to total numbers of colonies observed on the pour-plates, whichwere prepared, and which are reported above, two other types of observationswere made on fungus colonies. In the first of these, a portion of the dilute samplewas placed in yeast nitrogen base with either 1% or 20% glucose and shaken forthree or four days. At the end of this time, the growth which appeared could berecorded as an indicated number, and cells could be streaked onto Diamalt agarplates from which, after purification, identify able colonies could be recoveredand studied. Results of identification of these colonies are given in Table 8.

TABLE 6

Distribution of Fungi in Lebanon Secondary-Sewage-Treatment Plant by Date and Process(Numerals represent process stages indicated below)

Species10-12-67

A10-30-67 12-14-67

C1-25-68

D2-20-68

E3-27-68

F4-23-6

GNumberof times

appearing

Rhinodadiella mansonii 1234567 1234567 1234567White Yeasts spp. 1234567 1234567 1234567Geoirichum candidum 1234567 1234567 1234567Moniliales spp. 1234567 1234567 1234567Penicillium spp. 1234567 12 567 23 56Rhodotorula spp. 234567 12 7 123 5 7Penicillium lilacinum 1234 67 1234567Mucor hiemalis 34 67 234 67 1 4 67Fusarium oxysporum 1234567 67Fusarium aquaeductuum 2 4 7 1234Trichosporon spp. 12 567 12 5 7 12 567Trichosporon cutaneum 1 34 67 123456 12Cephalosporium spp. 1234567 1234567 2 7Phoma spp. 2 4 1Cladosporium dados porioides 2345 1 3 12 45Trichoderma viride 1 567 12 6Aspergillus flavus 4 1 7 3Phoma herbarum 234567 7 2 5Aspergillus niger 123456 1Cryptococcus spp. 3Penicillium ochrochloron 3 567 567

123456712345671 34567

3 561 34 67

3 5123 567

34 671 5

12 6

1234567

1234567

1234567

1234 67

1234 67

1234567

3 567

6

1234 67

1234 6

1 5

1 345 7

1 3 561

3 6

4 6

3 6

123 5

1234567

1234567

1234 67

m m on567

12 4 67123 567123456

31

12 7

2 4564

2 4

3

6

1 4 6

12345671234567123456712345671234 671234567123456234 67

567

123 56767

1

1 34567

1 3 5

Process stages:

1—Raw sewage

2—Primary settled sewage3—Aerator influent4—Aerator effluent

5—Final product6—Return sludge7—Primary settled sludge

Species present in 5 or fewer samples include:

5: Aureobasidium pullulans, Penicillium janthinellum, Pyrenochaeta sp., and Verticillium laterilium;4: Fusarium spp., Mucor spp., Rhizopus spp., Verticillium sp.;3: Aspergillus spp., A. jlavipes, A. cf. puniceus, A. versicolor, Coniothyrium fuckelli, Epicoccum purpurascens, Glio-

cladium spp., Gliomastix murorum v&r.felinum, Mucor plumbeus, Penicillium funiculosum;2: Aspergillus fumigalus, Fusarium roseum, Gliocladium roseum, Paecilomyces varioti, Penicillium chrysogenum,

Trichosporon ntargaritiferum;1: Alternaria alternata, Aspergillus sydowii, A. ustus, Botrytis cinerea, Chaetomium funicolum, Fusarium acuminatum,

Paecilomyces marquandii, and Penicillium martensii.

NfO

. 3

FU

NG

I IN

SE

WA

GE

TR

EA

TM

EN

T

139

TABLE 7

Distribution of Fungi in Lebanon Secondary-Sewage-Treatment Plant by Process and Date(Letters represent dates indicated below)

Raw Primary Aerator Aerator Final Return Primary NumberSpecies Sewage Settled Influent Effluent Product Sludge Settled of Times

Sewage Sludge Appearing

Rhinocladiella mansoniiWhite yeasts spp.Geotrichum candidumMoniliales spp.Penicillium spp.Rhodotorula spp.Penicillium lilacinumMucor hiemalisTrichosporon spp.Fusarium oxysporumFusarium aquaeductuumTrichosporon cutaneumCephalosporium spp.Phoma spp.Cladosporium cladosporioidesTrichoderma virideA spergillus flavusPhoma herbarumA spergillus nigerCryptococcus spp.Penicillium ochrochloron

Dates of sampling:A—10-12-67 B—10-30-67 C—12-14-67 D—1-25-68 E—2-20-68 F—3-27-68 G—4-23-68

Species appearing in 5 samples or fewer are listed under table 6.

140 W

M. B

RID

GE C

OO

KE

Vol.

70

Unidentified White YeastsRhodotorula spp.Trichosporon spp.Trichosporon cutaneum,Candida intermediaRhodotorula mucilaginosaCandida parapsiolosisTorulopsis versatilisTorulopsis aeriaTorulopsis holmiiCandida kruseiCryptococcus spp.Torulopsis famataTorulopsis CandidaCandida guilliermondiiCandida pelliculosaRhodotorula glutinis

TABLE 8

Distribution of Yeasts in Lebanon Secondary-Sewage-Treatment Plant by Process and Date{Letters represent dates indicated below)

SpeciesRaw Primary Aerator Aerator Final Return Primary Number

Sewage Settled Influent Effluent Product Sludge Settled of TimesSewage Sludge Appearing

Dates of sampling:A—10-12-67 B—10-30-67 C—12-14-67 D—1-25-68 E—2-20-68 F—3-27-68 G—4-23-68

Species appearing in four or fewer samples include:4: Trichosporon capitalum, Cryplococcus laurentii;3: Candida curvata;2: Candida utilis, C. ?zeylanoides, C. tenuis, C. catenulata, Torulopsis colliculosa, Cryptococcus diffluens, Trichosporon margaritiferum;1: Torulopsis inconspicua, T. glabrata, T. ?saki, Candida scottii, C. tropicalis, C. brumptii, C. mycoderma, C. humicola, Trichosporon fer-

mentans, Cryptococcus luteolus, Prototheca stagnora.


The categories "Rhodolorula spp." (or red yeasts) and "white yeasts" representunidentified colonies which were observed. Where white yeasts appear on allseven dates in all seven processes, the individual species making up the complexmay have been isolated only once, or may have appeared on all seven dates in allseven processes.

Because Candida albicans is an organism which could be present in sewageand sewage-polluted water, and because it had been isolated previously only twoor three times from such waters, two different types of media used to screencultures of this fungus from similar-appearing fungi in the laboratory were triedin an attempt to recover this yeast from samples in this series. The first of thesewere two bismuth-sulphite media, in which C. albicans colonies should appearblack with or without a darkened halo. The second of these was Pagano-LevinBase agar, on which C. albicans colonies should appear cream to bright pink.Various species of yeast, yeast-like, and filamentous fungi appeared on these media,but in no case could a definite assignment to C. albicans be made. Coloniessuspected of being that species were picked for future study, but growth patternsin each case were sufficiently different to rule out the recognition of this species inthe series of samples from which isolations were made.

DISCUSSION

The Nature of SludgeThe composition of sewage is unknown, varying from place to place and from

time to time. The elemental composition probably can be determined, but notthe structure, if one should want to consider sewage as a chemical entity. Anylisting of elemental components would produce an inaccurate picture. One-thirdto one-half of the contents of the solid fraction of sewage is cellulosic in nature(Maki, 1954) and comes from a variety of sources. The BOD of sewage is thoughtto be about 300 ppm in "average" untreated sanitary sewage. The DO of thissewage approaches zero, according to the Winckler test. The glucose contentcannot be determined easily and is thought to approach zero. Parenthetically, itmay be noted that most common fungi of sewage grow well on hexose sugars, whilea few grow on pentose sugars, in contrast to the waste stream in the paper-pulpindustry where pentose-using yeasts are common. Depending on the conditionsof the use of the environment, the nature of the wastes discharged to the trunksewer, and other factors, traces of a variety of substances may be present in wastewaters, such as odor- and taste-producing compounds, pesticides, stereoids, toxins,antibiotics, and a variety of other compounds.

How these elements, ions, compounds, substances, etc., are put together, andtheir relation to each other, is unknown. Mingling, mixing, blending, and otherphenomena occur in the waste streams as they flow from their sources, merge, andincrease in volume. Oxygen in these waters, and in the air through which theypass, is quickly depleted as the requirements of oxygen-demanding fractions ofthese waste components are satisfied. This means that there is a minimum ofoxygen for the microorganisms living in these waters which require this essentialelement.

Sewage- Treatment-Plant PopulationsA variety of types of filamentous fungi and yeasts was obtained from the seven

monthly samples of sewages in various stages of treatment in the Lebanon, Ohio,sewage-treatment plant, a small activated-sludge plant, and in auxiliary tertiary-treatment processes using effluent from the secondary plant. The same kinds offungi have been found in the secondary-treatment plant at Dayton, Ohio, atrickling-fjlter-type plant (Cooke, 1959), and in waste-stabilization ponds usedbriefly several years ago at a dormitory on a penal honor farm not far fromLebanon (Cooke and Matsuura, 1963, 1969). The list of species is not greatly


different from that obtained in a study of 19 activated-sludge plants in the vicinityof Chicago, Illinois (Cooke and Pipes, 1969).

All of these plants were operated independently of each other. The samplingat Dayton and at each of the two Lebanon installations was separated by severalyears; an increased sophistication in sampling and isolation techniques characterizedthe later samplings.

Bacteria and fungi are reducing organisms and as such form the most importantmembers of the populations in biological waste-water sewage treatment. Theyabsorb nutrients which are either readily available, or made available throughthe action on waste components of exoenzymes they secrete into the habitat.The majority of bacteria and all the fungi are aerobic organisms. While oxygen isreadily and always available to them in normal habitats, in sewage and in digestingsludges, free-oxygen supplies are not usually measurable by chemical means.Where free oxygen is not readily demonstrated by chemical techniques, survivaland growth depend on recovery, by the organism, of trace amounts of oxygenpresent as dissolved oxygen, or resulting from decomposition processes (Tabak andCooke, 1968b).

All the common reducers of sewage and sewage-treatment systems may beconsidered facultative anaerobes, on the basis of studies by Tabak and Cooke(1968a, 1968b). They may survive, metabolize, and grow in the presence ofdissolved oxygen at levels as low as — 40 Eh' (a measure of the oxidation-reductionpotential). During the processes of metabolism and growth, these organisms,added in the waste-water stream or air-spore fallout, or present as natural membersof populations in systems of conduction and treatment of sewage, reduce readilyavailable compounds which thus serve as nutrients. While at their death theymay add to BOD through dead cells and cytoplasm, in life they may add to BODonly through release of excess exoenzymes. During life they reduce BOD abun-dantly through metabolic processes, contributing to the biological treatment ofwaste water. On the other hand, these organisms add to the complexity of thepopulations present in sewage-treatment systems. While they include diseaseproducers in man and animals, potential disease producers in man, animals, andvascular crop plants, and producers of toxins of a variety of types, their activitywithin the treatment system, in the secondary-treatment process, is of greaterimportance than their potential nuisance outside the system, where they can beeliminated by disinfection processes.

It is known that at least one fungus, one streptomycete, and several species ofbacteria, which are true or facultative filament formers, cause bulking in activatedsludge (an undesirable condition). It is also known that one or more filamentousfungi cause clogging of trickling filters when rocks forming the filter bed are smallenough to produce only small-sized spaces between each other. It is assumed thatbulking of activated sludge, or clogging of trickling filters, is triggered by an excessof wastes, including nutrients which are capable of allowing rapid growth ofbulking organisms. On the basis of experiments in pure culture with presumedcausal organisms in the presence of presumed nutrient compounds as sole sourcesof nutrients, bulking or clogging can be demonstrated. In mixed culture, however,the trigger effect of small amounts or even traces of certain types of degradabledomestic or industrial wastes in a waste stream is still unknown.

As producers, algae added to this system, inadvertently or through deliberateprocesses, as in oxidation ponds and channels, add oxygen to the system throughmetabolic processes which include both normal photosynthesis and certain hetero-trophic phenomena comparable to those of bacteria and fungi. Protozoans, asconsumers, occur in the system as a result of additions with sewage input and asnatural inhabitants. These organisms may act as reducers through accidentalingestion of whatever compounds may be passing through the system. Moreimportantly, they have been shown to exert a type of natural control over the


bacterial population. When in balance, bacteria are ingested by protozoans andmetazoans. This results in maintenance of the bacterial population in a con-tinuous logarithmic phase of growth; if the plateau stage, at approximately asteady state, were reached, less activity could be expected from them.

Fungi in Sewage-Treatment PlantsThe Lebanon sewage treatment plant is operated on the activated-sludge

principle. Within the series of habitats found in this plant, the species of fungi,or decomposer organisms, which are present may be considered to be accidentalinhabitants. They reach the plant in the raw sewage, or as fall-out precipitatedfrom the air, or washed out of the air by rain. They reach the sewer from house-hold plumbing from the bathroom, the kitchen, the laundry, or the basementdrain. They include organisms which have been a part of the natural populationsin man's respiratory and digestive tracts, as well as organisms from the soil washedfrom muddy hands or clothing, organisms washed from the surface of the body,man's clothing, the vegetables he eats, the walls and woodwork of his house, soiltracked into the basement or utility room, and other sources.

Fifty-six of the categories of fungi listed in Table 4 represent that portion ofthose colonies observed on isolation plates which could be relatively readily identi-fied to species, or to genus in some cases. One of the more interesting of these wasFlammulina velutipes, a mushroom-producing fungus usually associated withstanding dead elms and other deciduous trees. In the "Moniliales" have beenplaced all unidentifiable molds, in the "white yeasts" all unidentifiable whiteyeasts. Completed identifications among the white yeasts have yielded 32 species,none of which can be distinguished by relatively simple techniques of observation.

These fungal and bacterial reducer organisms form most of the living materialin the larger particulate matter and solids settled out of the raw sewage. Evenin the soluble impurities, colloidal materials, and small suspended particles in theliquid which is channeled to an aerator after a brief period in the primary settlingtank, a quantity of bacterial and fungal cells is found. Just before entering theaerator, return sludge is mixed with primary settled sewage, thus adding a popula-tion of microorganisms already acclimated to a habitat of this type and developedin such a habitat over a period of time. Air, yielding a quantity of oxygen to themixed liquor, is pumped through a series of diffuser tubes into the mixed liquoras the sewage travels through the aerator tank. As the mixed liquor leaves theaerator tank, it still contains a large amount of organic matter, but this is nowlargely in the form of activated-sludge floe, a loose mass of microorganisms includingbacteria, fungi, and protozoans.

In order to remove this mass of organic matter, the mixed liquor is piped toa final, or secondary settling tank. Here the microbial floe is settled out, becomingreturn sludge, and the partially clarified supernatant becomes the effluent whichis discharged into the receiving stream, as the final product of the sewage-treatmentplant. At Lebanon, a portion of the final product is allowed to run into adjacentTurtle Creek, while the remainder, at the time of this study, was used in severaltertiary-treatment processes by a pilot-plant of the Advanced Waste TreatmentResearch Laboratory, Federal Water Pollution Control Administration, U. S.Department of the Interior.

In one process, the secondary effluent from the Lebanon treatment plant issent through a powdered activated-carbon system. Here organic matter andmicroorganisms are removed by the activated carbon by adsorption and sedi-mentation methods, much as taste- and odor-causing chemicals are removed in awater-treatment plant from raw waters in a purification process that utilizesactivated carbon.

In another instance, the secondary effluent is run through a lime-clarifier, whichremoves virtually all of the suspended organic matter and some of the micro-


organisms. The product is then processed through a granular-activated-carboncolumn, which removes soluble organics. The product of this process is thendemineralized by an electrodialysis unit. The final product of this process shouldneed only chlorination to make it near-potable drinking water.

Samples were obtained for testing for the presence of disseminules—spores orpieces of mycelium—from each of these processes. In all cases, fungus colonieswere obtained on agar plates. The numbers were small, but the colonies werepresent. Granular-carbon particles and electrodialysis membranes were testedfor the presence of fungus cells and these were found sometimes in larger numbersthan expected, therefore too numerous to count. Effluents from the granular-carbon column, powdered-carbon process, and lime-clarifier contained fungus cellsin low numbers, as did the effluent from the electrodialysis unit. In the first ofthese processes, the effluents contained viable cells, whether they were passedthrough sand filters or not. The effluent from the electrodialysis unit could havecontained spores as a result of picking these up from growths on the outer surfaceof the filter membrane.

There are problems in the interpretation of the meaning of the numbers offungal colonies given in Tables 1, 2, and 3. Bacteria, filamentous fungi, andyeasts are all saprobic organisms requiring preformed organic matter. At least,no photosynthetic or chemosynthetic bacteria are known to be operative in thesystem being considered here. Bacteria and yeasts usually form colonies whichcan be broken up readily into single cells, although some bacteria produce regularor irregular clumps of cells which are not readily broken up, and some yeastsproduce a true mycelium which usually remains in filaments. On the other hand,filamentous fungi usually produce growths which are made up of highly branchedfilaments which are not readily broken up into the individual cells of which theyare formed. Of the filamentous fungi, only Geotrichum candidum breaks up readilyin such a way that each cell presumably becomes a spore or a disseminule. Becauseof these growth characteristics, the interpretation of the meaning of a colony countis difficult. Does a colony arise from a single cell, from a one-celled spore, froma two-to-many-celled spore, from a piece of mycelium two to ten or more cellslong, or from a clump of mycelium which may include one or more complex branch-ing systems? Time has not been available for developing data on which to baseanswers to these questions. In any one sample, all these types of starting pointsfor colonies are possible.

In the method used in this study for counting colonies, no consideration waspaid to the source of the colony; thus, regardless of whether it arose from one cellor from many cells, the individual colony is valued at unity, the same as eachbacterial colony counted in the bacteriology laboratory, or the yeast colony in theFungus Studies Laboratory. Yeast colonies, too, could have originated frommore than one cell in some instances; the parental disseminule could have been aclump of cells from a pseudomycelium, or an elongated piece of mycelium composedof several cells. On the basis of the life form of the fungi listed in Tables 4-8, itis therefore very possible that the estimated numbers of fungal colonies listed inTables 1-3 are low.

The volumes of fungal cells, compared with those of bacterial cells, are muchgreater, so that in many cases the space occupied by one fungus cell could havebeen occupied by 100 bacterial cells. On this basis, the ratio of bacterial cellsper ml of sample to fungal cells per ml of sample yield interesting comparisons, eventhough these are based on colony counts which may be biased against the fungi.Here, on the average, there are many more bacteria than fungi in the raw-sewageinfluent to the plant. Again, on the average, in primary settled sewage enteringthe aerators, there is still a higher number of bacteria in relation to fungi, evenallowing for the difference in size. Within the aerator influent channel, wherereturn sludge has been mixed with primary settled sewage, the ratio decreases;


in the aerator effluent, there are relatively fewer bacteria, while in the final product,in the return sludge, there is nearly a 1:1 ratio of bacteria to fungi.

Yeasts were present in all samples taken in the secondary plant, and in manyof those from the tertiary processes. On the basis of indicated numbers (Table 4),values for yeasts or yeast-like fungi reached as high as 200,000 cells per ml ofsample in primary settled sludge, or as low as 10 to 200 cells per ml of sample inthe final product. Thus yeast cells were available to various tertiary-treatmentprocesses where, in one instance (on February 20, 1968), 3000 cells per ml wererecovered from effluent from the lime clarifyer. In Table 1 the indicated valuesare based on inspection of shaken primary isolation flasks, rather than on actuallyidentified species present in the flask. That is, when 200 cells were reported, thedilution from which they were recovered was 1:100, and two observably differenttypes of yeast species were present.

It was of interest that, in trying to isolate the pathogenic yeast, Candidaalbicans, on each of two types of media presumably selective for it, it did not appear.In the medical mycology laboratory, in which it is important to have a rapid testfor the presence of this species among a number of other yeasts and yeast-likefungi in pathological specimens, the use of acid bismuth yeast medium (ABY), orbismuth-GGY medium (BiGGY), and the use of Pagano-Levin Base agar (PLB)can aid in the determination of the presence of this yeast. Since in this study,few such colonies appeared and of these none developed other growth patterns ofC. albicans, it is concluded that in samples collected between October 12, 1967,and April 22, 1968, Candida albicans was not present.

In those tertiary processes available for sampling in the period covered by thisreport, filamentous fungi and yeasts were present (Table 2). Neither bacteria norfungi were as abundant as in the secondary processes, but their presence wascertain, as a result of plating techniques using samples which appeared clear.Some effluents carried more fungus cells than others, but within the processesthemselves, especially on the granular carbon in the carbon columns (or tubes),and on the membranes in the electrodialysis unit, growth was good to abundantand cumulative until the unit was taken down for repair or cleaning.

CONCLUSION

The growth of fungi, filamentous and yeast-like, in those structures devoted tothe secondary and tertiary treatment of sewage, indicates that the liquors beingtreated contain substances available to these fungi as food and that the fungi areable to utilize such substances as food. As living organisms, these fungi formpart of the biological population the treatment plant was designed to attract andput in use as the basic unit of the biological treatment system. These fungi are,therefore, performing a useful and necessary function in the biological treatmentof sewage being presented to them as a substrate on or in which to grow.

ACKNOWLEDGEMENTS

Bacteriological counts were made by B. A. Kenner. Total organic carbon andorganic nitrogen determinations were made by R. T. Williams and his staff.R. V. Villiers, Advanced Waste Treatment Research Laboratory, in charge of thetertiary-treatment pilot-plant at the Lebanon waste-water treatment plant, wasmost helpful in aiding in sample collection and in explaining various parts of thetertiary-treatment process. He prepared the flow charts in Figures 1 and 2.

REFERENCESCooke, W. B. 1959. Fungi in sewage and polluted water. IV. The occurrence of fungi in a

trickling filter-type sewage treatment plant. Proc. 13th. Ind. Waste Conf., Purdue Univ.,Engineering Bull. 43: 26-45.

. 1963. A Laboratory Guide to Fungi in Polluted Waters, Sewage, and Sewage Treat-ment Systems. PHS Publ. 999-WP-l. Cincinnati.


. 1965. The enumeration of yeast populations in a sewage treatment plant. My-cologia 57: 696-703.— and G. Matsuura. 1963. A study of yeast populations in a waste stabilization pondsystem. Protoplasma 57: 163-187.•— and —• —. 1969. Distribution of fungi in a waste stabilization pond system. Ecology50: 689-694.

and W. O. Pipes. 1969. The occurrence of fungi in activated sludge. Proc. 23rd.Ind. Waste Conf., Purdue Univ., Engineering Bull. 53(2): 170-182.

Difco, 1962. Difco Supplementary Literature, pp. i-viii, 1-316. (pp. 58-59). Detroit. 1962.Maki, L. R. 1954. Experiments on the microbiology of cellulose decomposition in a municipal

sewage plant. Antonie van Leeuwenhoek, Jour. Microbiol. and Serol. 20: 185-200.Nickerson, W. J. 1953. Reduction of inorganic substances by yeasts. I. Extracellular

reduction of sulphite by species of Candida. Jour. Infect. Dis. 93: 43-56.Tabak, H. and W. B. Cooke. 1968a. The effects of gaseous environments on the growth and

metabolism of fungi. The Botanical Review 34: 126-252.and . 1968b. Growth and metabolism of fungi in an atmosphere of nitrogen.

Mycologia 60: 115-140.

the ohio journal of sciencethe ohio journal of science vol. 70 may, 1970 no. 3 fungi associated with...

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