This article was downloaded by: [University of Sydney]On: 31 August 2014, At: 07:23Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number:1072954 Registered office: Mortimer House, 37-41 Mortimer Street,London W1T 3JH, UK

Journal of EnvironmentalScience and Health .Part A: EnvironmentalScience and Engineeringand Toxicology: Toxic/Hazardous Substances andEnvironmental EngineeringPublication details, including instructions forauthors and subscription information:http://www.tandfonline.com/loi/lesa19

Filamentous bacteriaand protozoa found inthe rotating biologicalcontactorJae‐Chun Chung a & Peter F. Strom b

a Department of Environmental Science ,Yonsei University , 234 Maeji‐ri,Heungup‐meun, Wonju‐gun, Koreab Department of Environmental Science, CookCollege , Rutgers University , New Brunswick,New Jersey, 08854Published online: 15 Dec 2008.

To cite this article: Jae‐Chun Chung & Peter F. Strom (1997) Filamentousbacteria and protozoa found in the rotating biological contactor, Journalof Environmental Science and Health . Part A: Environmental Science andEngineering and Toxicology: Toxic/Hazardous Substances and EnvironmentalEngineering, 32:3, 671-686, DOI: 10.1080/10934529709376569

To link to this article: http://dx.doi.org/10.1080/10934529709376569

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J. ENVIRON. SCI. HEALTH, A32(3), 6 71-686 (1997)

FILAMENTOUS BACTERIA AND PROTOZOA

FOUND IN THE ROTATING BIOLOGICAL CONTACTOR

Key words : Filamentous bacteria, Protozoa, Rotating biological

contactor, Organic loading

Jae-Chun Chung1 , Peter F. Strom2

1Department of Environmental Science, Yonsei University, 234 Maeji-ri,

Heungup-meun, Wonju-gun, Korea2 Department of Environmental Science, Cook College, Rutgers University,

New Brunswick, New Jersey, 08854

Abstract

Slime samples from 66 rotating biological contactors(RBC) in 20 states in

USA were examined. For the filamentous bacteria, Beggiatoa, Sphaerotilus,

Type 0041, Type 1701 and Nocardia were observed in decreasing order of

frequency. For the protozoa, flagellates, Opercularia, Difflugia, Arcella,

Paramecium and Epistylis were present in decreasing order of frequency.

Organisms differed in abundance along the RBC stages. Using linear

regression, Beggiatoa, Type 0041 (negative), and flagellated protozoa showed

signigicant correlations(p<0.05) with organic loading, while Sphaerotilus and

Opercularia did not. In an attempt to establish an indicator organism system,

RBC organisms were grouped based upon the organic loading preference.

Beggiatoa and flagellated protozoa were grouped as the organism associated

with high organic loading. Type 0041, and Arcella and Difflugia were grouped

as the organism associated with moderately low organic loading.

The microbial population in the last stage was investigated in association

with the effluent quality. Flagellates and Opercularia were more frequently

671

Copyright © 1997 by Marcel Dekker, Inc.

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672 CHUNG AND STROM

observed in plants with effluent BOD's above 20mg/l, while Arcella and

Difflugia were more frequent in the plants with effluent BOD's below 20mg/l.

INTRODUCTION

The Rotating Biological Contactor(RBC) process is a secondary treatmentone utilizing attached microorganisms growing on the biodisc surface.Evolving from the trickling filter process, the RBC process has manyadvantages; no head loss, competitive treatment efficiency, small energyconsumption and simple operation. The biggest advantage would be its simpleoperation. Therefore, the adoption of the RBC process is highlyrecommendable in developing countries and in the rural area wherewell-trained operators are not usually available.

With the recognition of considerable advantages of the RBC process,quite a few research on the RBC has been done. However, most of the RBCpapers are engineering papers, providing microbiological data as supplementaryinformation[l-3]. Since the RBC process is microbiological in nature,microbiological study is very important to understand the rational behind thesystem and improve the RBC operation.

The purpose of this paper was to identify microorganisms in the RBCslime from a number of plants, determine the relative abundance and toexamine the association of microorganisms with organic loading.

MATERIALS AND METHODS

Based upon 770 RBC plants listed in the 1987 EPA National NeedsSurvey[4], 377 letters were sent to 20 states requesting samples. A total of 74plants sent samples by first class mail and those from 66 plants wereanalyzed on September 1990. Samples from the 8 plants were severelybiodegraded to be examined.

In the laboratory, excess water in mailed sample bottles was decantedand the slimes were mildly agitated with a spatula. Vigorous mixing wasavoided to prevent breaking of filamentous forms in the slime. Two ml of thepreparation was pipetted into a plastic bottle with a wide-bore pipette and2ml of tap water was added and again mildly mixed with a spatula. Then, themixed diluted slimes were mounted and spread uniformly to make a thin layerover the slides for microscopic observation.

To determine relative abundance, 10 random microscopic fields werechosen and microorganisms were counted. A magnification of 1000X was usedfor filamentous bacteria and fungi and 100X for protozoa and metazoa. the

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ROTATING BIOLOGICAL CONTACTOR 673

field diameter was 1950/aii at 100X and 195/a at 1000X. For the stalkedciliated protozoa with multiple heads(Epistylis, Opercularia, Carchesium andZoothamnium), each head(zooid) was counted as 1 individual.

Filamentous microorganisms were identified to types using themicroscopic method of Eikelboom[5], Eikelboom and van Buijsen[6] asmodified by Strom and Jenkins[7], Protozoa were identified to genus using thekeys of Jahn and Jahn[8], and Lee et al[9]. The abundance of filamentousbacteria was determined using the method shown by Chung and StromflO].

RESULTS AND DISCUSSION

Operational Conditions

Among the 66 plants investigated, 36 plants were treating 100% domesticwastewater, 4 plants were treating purely industrial wastewater, and 26 plantswere treating domestic plus up to 30% industrial wastewater. Table 1 showsthe organic loading range, which was generally low.

Abundance of Filamentous Bacteria

Table 2 shows the frequency of occurrence of filamentous bacteria in the66 plants. Beggiatoa was frequently present. Sphaerotilus was the second andType 0041, Type 021N and Type 1701 were present in decreasing order offrequency. This trend was pretty much similar to the authers' previouspaperflO].

This frequency of occurrence differs with that of activated sludge. Inactivated sludge, Type 1701, Nocardia, Type 0041, Type 021N, Type 0092,Haliscomenobacter hydrossis and Sphaerotilus natans are most frequentlyobserved and Beggiatoa is a minor type[7].

The abundance of Beggiatoa in RBC's is usually ascribed to highorganic loading and low DO [11-12]. However, the relationship of Beggiatoawith sulfur reducing bacteria such as Desuljbvibrio appears to be the directcause [13]. In a heavily loaded RBC biofilm, Beggiatoa grows outermostaerobic layer forming a whitish coating, while sulfur reducing Desuljbvibriogrows inner anaerobic black layer producing sulfate which serves as energysource for Beggiatoa

Sphaerotilus was present with the second frequency. The reasons forSphaerotilus abundance in RBC's can be suggested as follows: (1) SinceSphaerotilus can grow in anaerobic conditions, it can assimilate nutrients evenfairly deep within the slime[14]; (2) Sphaerotilus can produce free-swimmingflagellated cells, which can rapidly recolonize the RBC surface after sloughing;

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674 CHUNG AND STROM

Table 1. Range of organic loading for plants studied (BOD g/m'/d).

O.L.

No. ofPlants*

Organic Loading (O.L.) on the

0-24.0

35

24.1-49.0

14

First Stage

49.1-73.0

6

73.1-98.9

2

£98.1

1

Overall Organic Loading

O.L.

No. ofPlants*

0-2.4

10

2.5-4.8

10

4.9-7.3

17

7.4-9.8

7

9.9-12.2

6

12.3-14.6

4

£14.7

4

* A total of 58 plants provided BOD organic loading data.

Table 2. Frequency of occurrence of filamentous bacteria.

Organism

BeggiatoaSphaerotilus

Type 0041

Type 021N

Type 1701

Unidentified (bead-shaped)

Filamentous bacilli

NocardiaSpirochetes

Number of Plants

53

43

33

11

8

4

3

1

4

%*

80

65

50

17

12

6

5

2

6

* Based on total of 66 plants.

(3) The water velocity in streams where the maximum growth ofSphaerotilus occurrs is 0.18—0.45m/sec[15], which generally conicides with theperipheral velocity of most operating RBC's [16, 17]. Inasmuch as Scfanerotiluscommonly occurrs in the slime in polluted streams, its frequent occurrence onRBC surfaces appers to be quite natural.

Ecological Roles of Filamentous Bacteria in RBC's

Alleman et al., [13] suggested that filamentous bacteria in RBC's couldserve as a backbone matrix of the slime layer, providing ample attachmentsiles for other bacteria. However, the majority of filamentous bacteria do not

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ROTATING BIOLOGICAL CONTACTOR 675

have attached bacteria when they are alive. This is true for Beggiatoa,Sphaerotilus, Type 1701, Type 021N and Nocardia since attached cells werenot observed in their trichomes (Note : Type 1701 often and S. natansoccasionally do have attached cells in activated sludge). The only exception inthis study was Type 0041, which usually had many attached cells on itstrichomes. Although most filamentous bacteria do not provide attachment siteswhen they are alive, they apparently do when they are degrading since a lotof attached cells were observed on degrading trichomes.

Although quite a few strains of Beggiatoa have been reported to beheterotrophic [18-20], some strains are chemoautotrophic [21, 22] and manystrains appear to be mixotrophic, requiring a small amout of organic matter[23-25]. Therefore, the contribution of Beggiatoa to the removal of organic inwastewater treatment seem to be inferior to that of other heterotrophicbacteria due to a smaller assimilation capability of organic matter.

Unlike Beggiatoa, Sphaerotilus seems to contribute considerably to thecarbon removal. Sphaerotilus-based slime in the natural environment removed0.5~7.4g organic C/m'/day [26]. In the laboratory, Sphaerotilus-based slimeefficiently assimilated glucose and acetate at concentrations as low as 1 mg/1[27], It seems that the fairly high carbon removal efficiency of Sphaerotilus ispartly due to its capability to store carbon as PHB granules inside the cell:Sphaerotilus can store PHB granules amounting to between 11% and 23% ofits dry weight [28].

Type 1701 appears to have a similar carbon assimilation capability toShaerotilus, and is closely related to it in morphology [29]. Type 021N, whichis associated with low DO in activated sludge [7, 30], may contributeconsiderably to the carbon removal because it has a high assimilationcapability for simple sugars (glucose) and organic acids such as lactate. Thehalf saturation coefficients(Ks) for glucose and lactate were less than 1 mg/l[30]

Abundance and Ecological Roles of Protozoa in RBC's

Table 3 shows the frequency of protozoa in RBC's flagellated protozoa,Opercularia, Dißugia, Arcella, Paramecium, Epistylis were present indecreasing order of frequency. The results of Sudo and Aiba[3] from 80domestic RBC plants in Japan are shown together. The two results aresimilar in that generally stalked ciliates such as Opercularia and Epistylis,and Sarcodina such as Arcella were the most frequently observed protozoaother than flagellates. The free swimming ciliated protozoa were relatively lesscommon than stalked ciliates. These two results also are in good agreement

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676 CHUNG AND STROM

Table 3. Frequency of Occurrence on Protozoa and Other Organisms

this Study

Organism

Protozoa(lf'Flangellated

Protozoa(2) Opercularia(3) Dißugia(4) Arcella(5) Paramecium(6) Epistylis(7) Trachelocerca(8) Litonotus

AspidiscaUronema

(9) VorticellaCarchesium

(10) ColpidiumChilodonella

(11) AcinetaTetrahymena

Sudo and Aiba(1984)**

Plants in Which

Present* / (%)

43

3824201276444332211

Organism

65 \(\f Epistylis sp.

5836301811

(2) Vorticella conuularia(3) Opercularia sp.(4) Euglypha sp(5) Arcella vulgaris(6) Vorticella microstoma

9 (7) Cinetochilum margaritaceum6 ¡(8) Vorticellla sp.6 \(9) Carchesium polypinum6 ;(10) Zoothamnium sp.5 !

5 13322 ;

* Based on total of 66 plants. **Based upon 8 plants in Japan.Frequency rank.

with the earlier investigations by Torpey et al., [32], Pescod and Nair [33],

Kahn and Raman [34], in that flagellated protozoa, Paramecium, Aspidisca,

Colpidium, Diffugia, Chilodonella, Litonotus, Vorticella, Opercularia,Carchesium and Epistylis were frequently observed.

The major important function of protozoa in wastewater treatment

probably is to remove dispersed bacteria [35-37]. By ingesting weaker and

less active bacteria, protozoa may help maintain healthy bacterial populations

[38]. Also, by grazing on bacteria they might enhance mineralization and

respiration due to the selection for rapidly growing heterotrophs [39]. Other

roles have also been proposed, but appear to be of minor importance or less

convincing. Protozoa might help flocculation of bacteria by secreting

polysaccharides to form floes and by adsorbing bacteria onto metabolites of

protozoa [31, 40, 41]. Sometimes, protozoa may directly take up organic matter

such as simple sugars [31, 37, 42].

The reasons for stalked ciliated protozoan abundance in the RBC slime

are not fully understood. It can be partially explained as follows: (1) Stalked

ciliated protozoa have relatively high growth rates ranging from 1.6-3.3/day

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ROTATING BIOLOGICAL CONTACTOR 677

[31, 43]; (2) The reproduction of stalked ciliated protozoa may be efficient

since single zooid (head) could form an individual colony; (3) The RBC may

provide a good habitat for stalked ciliated protozoa since the available food is

constantly provided by the rotation.

Abundance of Microorganisms bv Stape

Abundance of filamentous bacteria by stage is shown in Table 4.

Beggiatoa occurred more frequently and in larger numbers in the first stage.

Sphaerotilus was observed more frequently in the first stage than the last but

the average number per field was same in both stages. Type 0041 occurred

more frequently in the last stage than the first with larger numbers. Types

1701 and 021N occurred in both stages with similar frequencies and numbers.

The other filamentous bacteria were present in small with low frequencies.

Table 5 shows common protozoa in RBC plants by stage. Flagellates,

Aspidisca, Epistylis, Opercularia and fungi were more frequently present in

the first stage with higher numbers. Arcella were more frequently present in

the last stage. The results of Sudo et al., [44] from 20 domestic RBC plants

in Japan are shown together for comparison. The general trend is similar in

that stalked ciliated protozoa occur more frequently in the earlier stages and

Arcella occurrs more frequently in the last stages. The frequency of

occurrence of protozoa was generally lower in this study, possibly due to the

time lag between sampling and observation. Normally, it took 3 ~ 7 days to

get samples by mail.

Occurrence of Protozoa bv Effluent Quality

Table 6 shows the abundance of protozoa in the last stage by effluent

quality. The RBC plants were divided into two groups according to the

effluent BOD concentration using 20mg/l as the dividing point. Flagellated

protozoa appeared more frequently and in higher numbers in the higher

effluent concentration group. Type 1701, Arcella and Difflugia were observed

more frequently in the plants with low effluent concentration. Uronema was

more frequently present in high effluent concentration group. The other

organisms appeared to give ambiguous responses with regard to occurring

more frequently or in higher numbers in one effluent concentration group or

the other.

The results form Curds and Cockburn[45] for activated sludge effluent

quality are shown together. Their results are similar to those of this study. It

can be said that Arcella, Difflugia, Vorticella, Opercularia, and Carchesim are

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678 CHUNG AND STROM

Table 4. Filamentous Bacteria in RBC Plants by Stage.

Organism

I\Beggiatoa'¿¡¡jhaerotilusType 1701Type 021ΝType 0041NocnrdiaFilamentous

bacilliSprochetes

First

Frequency*

48

3577

151

0

2

Stage

Average No.

per Field**3.7

2.7

1.9

0.60.3

0.7

0

2.3

Last Stage(Mostlv 4th)

Frequency*

362585

241

1

3

Average No.

per Field**

1.52.72.50.40.80.7

0.7

6.6

* Number of plants which have the organisms on the first or last stage.

** At 1000X; averages for plants in which present.

more frequently associated with higher effluent BOD concentration. Considering

that stalked ciliated protozoa(perhaps different species) frequently occurred in

the earlier stages, they appear to occur over a wide range of organic loadings.

Sudo[47] also did research on the association of protozoan populations

with effluent quality and found that the effluent improved with increasing

protozoan populations. Sudo and Aiba[31] proposed group of possible protozoan

candidates for assessing performance or judging effluent quality of biofilm

processes based upon the results from Pike and Curds[46], and Sudo[47].

Their summary is as follows :

(1) Protozoan species occurring under normal operational conditions •'

Carchesium, Zootamnium, Epistylis, Opercularia, Vorticella, Aspidisca,

Oxytricha, Arcella, Euglypha. and Euplotes.

(2) Protozoa present under abnormal (bad) operational conditions :

Oikomonas, Bodo, Cercobodo. Paramecium, Colpiddium, Holophrya,

Glaucoma, Metopus, and Caenomorpha

(3) Protozoan species present under intermediate conditions :

Pleuromonas, Cinetochilum. Trachelophyllum, Spirostomum, Amoeba.

Litonotus, Loxophilum, and Amphileptus.

The results from this study are in fairy good agreement with the above

scheme in that stalked ciliated protozoa were frequently observed when the

effluent BOD was below 20mg/l.

Relationship, of Microbial Populations to BOD Organic Loading

To further investigate the associations of microbial populations with BOD

organic loading, linear regression methods were used. In this analysis, the

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ROTATING BIOLOGICAL CONTACTOR 679

Table 5. Common Protozoa in RBC Plants by Stage.

Organism

Flagellated

protozoaArcellaDiffugiaTrachelocercaLitonotusChilodonellaParameciumAspidiscaColpidiumVorticellaOperculariaEpistylisCarchesium

First

Frequency(%)*

59

88522

11825

4595

This StudyStage

Average No.per field

at 100X**

9.6

0.80.30.10.60.60.30.62.14.4

15.625.046.9

Last Stage(Mostly 4th)

Frequency(%)

35

27276329023

3060

Average No.per field

at 100X**

6.0

0.70.90.30.10.10.3

014.715.010.42.3

0* Based on total of 66 plants.** Averages for plants in which observed.

Organism

Monas sp.Pleuromonas jaculansArcella vulgarisEuglypha sp.Amoeba spp.Trachelophyllum pusillumCinetochilum margaritaceumLitonotus ficiolaVorticella conuulariaVorticella sp.Opercularia spp.Episylis sp.

Sudo et al. [44]First and Second StageOccurrencefrequency

605554590508575555050

Maximumnumber

presenti/cm1)2,000

6501,200

7101,600

3901,700

6806,700

13,0001,500

80 30,000

Third andOccurrencefrequency

(%)*606585957045451525505030

Forth StageMaximumnumber

present(/c[rf)73,0003,700

8601,200

400210680160

1,3005,1002,100

| 450

* Based on total of 20 domestic RBC plants in Japan.

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680 CHUNG AND STROM

Table 6. Abundance of Protozoa

Organism

Flagellated

protozaArcellaDifflugiaUronemaParameciumOberculariaCarchesium

by Effluent Quality.This Study (RBC

Effluent BOD

Frequency (%)*

31

22339

1122

7

: < 20mg/lAverage No.

per fieldat 100X*

7.0

0.40.83.90.65.4

47.0

s)Effluent BOD

Frequency (%)*

60

7200770

: > 20mg/lAverage No.

per fieldat 100X*

16.0

0.61.0

00.5

10.70

»Based on 45 plants. **Based on 15 plants.Averages for plants in which present.

Frequency of Occurence(%) of Protozoa inActivated Suldge Curds and Cockburn[45]

Organism0-10

BOD Range(mg/1)11-20 21-30 30

Flagellated ProtozoaAspidisca cicadaEuplotes patellaVorticella convulariaVorticella jromentelliCarchesium polipinum

07538633819

08025733347

375024371212

45560

2200

»Based on 53 English plants.

microbial populations and BOD organic loading for the first stage were usedfor each plant, since loading on other stages is not known and overall loadingis inappropriate for this purpose.

Table 7 summarizes the results of the linear regression analysis. Thecorrelation for Beggiatoa (r = +0.48) was significant (p < 0.05) but that forSphaerotilus (r = 0.20) was not. Type 0041 showed a significant negativecorrelation with organic loading (r = -0.27). Flagellated protozoa were the onlyother group to show a significant correlation (r = +0.52), if the regressionincluded only those plants in which they were present. If all plants wereincluded, and for all other groups, the correlations were not significant.

Beggiatoa seems to be associated with high organic loading since it isusually present in large numbers in the first stage. The linear regression

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ROTATING BIOLOGICAL CONTACTOR 681

Table 7. Correlation between Microbial Populations and Organic Loading

on the First Stage

Organism

Beggiatoa

Sphaerotilus

Type 1701

Type 0041

Type 021N

FlagellatedProtozoaOpercularia

Nematodes

Rotifers

No. of Plants'

5847583258

6581258

65836582558435820

Equation from

Linear Regression

Y=0.58+0.33XY=1.30+0.37XY=1.06+0.15XY=2.60+0.13XY=0.58+0.09XY=4.94-0.37XY=0.39-0.03XY=1.72-0.27XY=0.33-0.03XY=2.54-0.09XY=3.69-0.50XY=-1.08+0.5IXY=5.17+0.13XY=10.86+0.51XY=0.80-0.04XY=1.01-0.05XY=0.42-0.04XY=1.65-0.03X

Correlation

Coefficient(r)

+0.48*+0.42*+0.20+0.17+0.12-0.38-0.27*-0.48-0.16-0.55+0.19+0.52*+0.05+0.09-0.19-0.16-0.14-0.06

Critical

Values ofΙ Γ Γ α =0.05

0.2590.2880.2590.3490.2590.8110.2590.5760.2590.8110.2590.3290.2590.3810.2590.3010.2590.444

Upper number : total number of plants which provided organic loading (BOD)

data. Linear regression was performed on all 58 plants whether the plants had

the organism or not.

Bottom numbers ·' Number of plants in which the organism was observed.

Linear regression was performed only for those plants in which observed.

* The correlation coefficient is statistically significant at 5% level.88 source : Zar [48]

analysis (r = +0.48) supported this since it showed a significant correlation (p

< 0.05) with organic loading. However, Inasmuch as the key factor in

determining Beggiatoa abundance is considered to be the relationship with the

sulfur reducing bacteria[13], organic loading appears to exert an indirect effect

on the abundance of Beggiatoa.

Sphaerotilus showed little correlation(not significant) with organic loading

(r = +0.20). This seems consistent with the results from activated sludge[7]

where S. natans is associated with low DO rather than any specific organic

loading. It should also be noted that if Sphaerotilus is favored by high

loadingsibecause of lower DO), but not by very high loadings, this would

result in a poor resolution with linear regression.

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682 CHUNG AND STROM

Table 8. Association of RBC Organisms with Organic Loading.

Organic Loading

High

Moderately Low

Low

Organism

Filamentous BacteriaBeggiatoa

Type 0041

Protozoa

Flagellated Protozoa

ArcellaDiffugia

The linear regression analysis for Type 1701 was based on too few data

points, in which Type 1701 was present. However, this type may have similar

characteristics to Sphaerotilus since these types are closely related [29].

Type 0041, which is associated with low, organic loading in activated

sludge[7, 29], also seems to be associated with low organic loading in RBC's .

It was usually present in the last stages and in the plants with low organic

loadings. The linear regresssion analysis also indicated a moderate, significant

negative correlation with organic loading (r = -0.27).

Type 021N did not appear to be associated with organic loading,

although it was present in too few plants to make any definitive comment. In

activated sludge this type is associated with low DO.

Flagellated protozoa appear to be associated significantly with high

organic loading. They were usually present in higher numbers in the first

stage. Arcella and Diffugia seem to be associated with lower organic loadings

since they were more frequently observed in the later stages.

Rotifers seem to be associated with moderately low orgnic loading

judging by the results from this studies. However, this correlation was not

high enough to be significant (p < 0.05).

Based upon the data obtained from this study, filamentous bacteria,

protozoa and metazoa may be grouped according to organic loading (Table 8).

It would be possible to establish a system of indicator organisms with the

investigation of a larger number of plants.

Although it is worthwhile to establish indicator organisms according to

organic loading, some limitations exist. Besides organic loading, other

physicobiochemical factors such as DO, temperature, wastewater composition,

and prédation also affect the occurrence and abundance of microorganisms.

Therefore, the suggested groupings shown in Table 8 should be considered

only as a general tendency or guideline.

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Date Received: August 23, 1996Date Accepted: Oct. 8, 1996

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