Growth Rates of Microorganisms in a Periphyton Community

Jap. J. Limnol. 40, 1, 10-19 1979.

Growth Rates of Microorganisms in a Periphyton Community

Morihiro AIZAKI

Abstract

The growth rates of microorganisms were determined for a periphyton community grown on artificial

substrata submerged in the midstream of the Tamagawa River. The growth rates were measured

seasonally for bacteria, algae, the heterotrophic periphyton community and the whole periphyton com-

munity. The doubling times were as follows: bacteria, 3-10 hours; sessile algae, 12-28 hours; hetero-

trophic periphyton community, 15-104 hours; and periphyton community, 12-59 hours. The growth

rates showed a good correlation with water temperature, and the Q10 values were as follows: bacteria,

1.9; sessile algae, 1.6; heterotrophic periphyton community, 2.5; and periphyton community, 2.2.

1. Introduction

In recent years, considerable changes in

the biotic community of many rivers in or

near urbanized areas have been recorded.

These are mostly due to increasing pollu-

tion by municipal sewage. The tremendous

increase in the amount of periphyton on

river beds is the most predominant change

(SAKURAI, 1973 ; TEZUKA et al., 1974; TANAKA 1975; WATANABE et al., 1975;

AIZAKI, 197$). Such periphyton com-

munities consist of abundant bacteria, algae,

protozoa and other organisms. The mea-surement of the growth rates of these mi-

croorganisms and the examination of their

relation to the environmental conditions

are of great importance in understanding

the ecological situation of the biotic com-

munity in polluted aquatic habitats. Very

few reports have been made on the growth rates of microorganisms in the natural

periphyton community. BOTT (1975), and BOTT and BROOK (1969, 1970a, 1970b)

determined the growth rates of epilithic

bacteria in rivers. KEVERN et al. (1966)

have studied the whole periphyton com-munity. BROOK (1971) has reviewed many

practical and theoretical problems associated

with various approaches to the measure-ment of growth rates of periphyton micro-

bes in natural aquatic habitats. In this study, the growth rates of micro-

organisms were seasonally determined for

a periphyton community grown on artificial

substrata. Experiments were conducted

from March 1972 to January 1974 in the midstream of the Tamagawa River located

in the southwest part of Tokyo. The

length of the river is about 100km, and the drainage area is approximately 1, 200

km'. The midstream has been polluted by

municipal sewage, and organic and inorganic

nutrient content is considerably high in the

river water. The site of the experiments

was a river bed area of about 100m wide

and about 30cm deep under ordinary con-

ditions. Current velocity observed there was 0. 6-1. Om/second throughout the present

investigations.

The author is grateful to Drs. Y.

TEZUKA, S. TAKII and Mr. HAYASHI, and

of the other scientists of the Laboratory of Microbiological Chemistry, Tokyo Metro-

politan University, for their invaluable suggestions and stimulating discussions

during the course of this work.

Supported by a grant-maid from the Ministry of Education

AIZAKI 11

2. Methods

The majority of the experiments were

carried out using artificial substrata of

microscopic slides and polyvinylchloride

plates (5cm x 5cm). The surfaces of the latter were roughened with a rasp to minimize sample errors. These substrata

were fixed on an iron frame with clips or

wires and submerged parallel to the current

in the running river water. About twenty

slides and about thirty plates were used

for each experiment. One slide and/or two

plates for each sample time were removed from the frame at regular intervals and

brought to the laboratory. The slides were utilized for the determination of bacterial

growth rate, and the plates were used for the growth rates of sessile algae, hetero-

trophic periphyton community and the

whole periphyton community.

Bacterial growth rates were measured as

follows. About 30 large colonies were

randomly selected for each slide and the

number of cells per colony was counted

under a phase contrast microscope at 1000

fold. Doubling times were estimated by

plotting the average cell numbers per colony against immersion periods. Algal

growth rates were estimated from the change of the chlorophyll content with

submersion time on the upper surfaces of

the polyvinylchloride plates. Growth rates

of the heterotrophic periphyton community

were also estimated from the change with

time in the amount of organic carbon on

the back surfaces of the plates where little

algae grows because of the lack of light.

The increase rates of the standing crop of

the whole periphyton community was

estimated from the change of the amount

of organic carbon content on the upper

surfaces of the plates with submersion

time. The methods for preparation and

determination of chlorophyll and organic

carbon content were described in the pre-

vious paper (AIZAKI, 1978).

Chemical analyses were made by the

following method : BOD by the Standard

Method (American Public Health Associa-tion, 1971) ; reactive phosphorus and total

phosphorus by the method of STRICKLAND and PARSONS (1965); NH4-N (ammonium

nitrogen) with Nessler reagent after distilla-

tion; N02-N (nitrite nitrogen) by colori-

metry with sulphanilamide and N-(1-

naphthyl) ethylendiamine ; N03-N (nitrate

nitrogen) by colorimetry after reduction to

nitrite (STRICKLAND and PARSONS, 1965).

3. Results

1. Water Quality

Some chemical qualities and physical

conditions of the water during the experi-

ments are shown in Table 1. Water tem-

perature was 4-10°C in winter, 10-20°C in

Table 1. Chemical and physical conditions of river water at the

study site at various months.

12 Growth Rates of Microorganisms in a Periphyton Community

spring and autumn, and 20-29°C in summer.

BOD was 5-13mg/l under ordinary condi-

tions. Concentrations of nutrients were as

follows : NH4-N, 0.7-4.08mg/l ; N02-N, 0.13-0.36 mg/i ; N03-N, 1.00--2.83 mg/l ;

P04-P, 0.29-1.22mg/l ; and total phos-

phorus, 0.52-1.46mg/l. 2. Growth Rate of Bacteria

Fig. 1 shows microcolonies of bacteria

grown on a slide submerged at the study site for 70 hours. Several forms of bacteria

were distinguished by microscopic obser-

vation. The growth curves of bacteria

grown on slides at different times of the

year are shown in Fig. 2. The cells grew on the same plane until the colonies reached

the 20 to 30-cell stage, but after this stage

they overlapped. Therefore, the values

obtained in the late stage of the experiments

do not seem suitable to estimate growth rate. Before reaching this critical stage,

the cell number per microcolony increased

exponentially with time. Doubling times

derived from the gradient of the cell

increase are given in Table 2. The doubling

time was about 3 hours in July, about 5

hours in May and about 10 hours in

January. The water temperatures in these three observation times were approximately

25°C, 17°C and 6°C, respectively.

The relationship between water tempera-

ture and doubling times of bacteria is shown in Fig. 3. A high correlation was

found between them. From these figures

Qio was estimated as about 1.9.

Fig. 1. Microcolonies adhering to a microscopic slide 70 hours after submerging in the running water of the Tamagawa River in Dec. 1973. (Dark field microscopy ; x 200).

Fig. 2. Seasonal changes in the growth curves of bacteria

at the study site.

AIZAKI 13

3. Growth Rate of Algae

Although the growth rate of algae has

been generally estimated from the change

of cell number and the rate of primary

production, in the present study it was

measured by the change of chlorophyll

amount, because of the relative ease of

chlorophyll determination. Fig. 4 shows

the increase curve of the chlorophyll

amount after submergence of artificial

substrata in the river water at different

times of the year. The doubling times of

sessile algae in each season calculated from

this figure are shown in Table 2. Chloro-

phyll content in algae is known to differ

due to the difference of algal species and

environmental conditions. Moreover, there

is heterogeneity of algal distribution on the

plates. Therefore, the present values must be regarded as approximate growth rates.

The chlorophyll content increased exponen-

tially during most of the early stages of

the observations except in August 1972,

and July and September 1973, when the

standing crop decreased due to the influence

of heavy rains. In the three exceptional

cases mentioned, the growth rates were

determined for the most rapid increase

period in the early stages. The shortest doubling time of about 12 hours was

obtained in August 1972 and July 1973,

and the longest of about 28 hours was

observed in January 1973.

The relationship between water tem-

perature and doubling times of algae is shown in Fig. 5. A high correlation was

found between them, and Qio derived from

these figures was about 1.6.

Microscopic observation revealed that the

dominant sessile algae throughout the year

were Stigeocloniurn and diatoms. In sum-mer, Spirogyra and Phormidium sometimes

appeared. Among the dominant diatom

genera, Gomplaonema, Synedra, Diatoma, Nitzschia, and Navicula were frequently

observed in spring and autumn. Melosira

Table 2, Doubling time (hr) of each component microorganism in periphyton community at the study site from March 1972 to January 1974.

Fig. 3, Relation between water temperature and

doubling times of bacteria.


appeared in summer, and a considerable

amount of Achnanthes was present in

winter.

Fig. 6. shows the increase curves of the

diatoms and chlorophyll in March 1972.

The doubling time derived from these

curves was about 37 hours for the former

and about 25 hours for the latter. Such a remarkable difference was probably due to

the dominance of Stigeoclonium sp. in the

periphyton community at this time. 4, Growth Rate of the Heterotrophic Pen-

phyton Community On the back surface of the plates, algal

growth did not occur due to the lack of light. The maximum chlorophyll-a amount

on the back surface of the plates was 20-30mg/m2 in the spring, the optimum season

for algal growth. The chlorophyll-a con-tents on the upper surface of the plates

were 300-400mg/m2 at that time. The ratio

of organic carbon to chlorophyll-a content

in periphyton communities grown on the upper and back surfaces of the plates in

Fig. 4. Growth curves of sessile algae in different months

of the year at the study site.

Fig. 5. Relation between water temperature and

doubling times of sessile algae.

AIZAKI 15

Fig. 6, Increase curves of chlorophyll and number of diatom cells

on artificial substrata after their placement at study site.

Experiment was carried out in May 1972.

Fig. 7. Growth curves of the heterotrophic periphyton community

grown on the back surface of plates submerged in different months of the year at the study site.


spring was about 40 in the former and

about 220 in the latter. Therefore, the

increase curve of the organic carbon amount

on the back surface of the plates may be

taken to represent the growth curve of the heterotrophic periphyton community.

Fig. 7 shows the changes of the organic

carbon with time on the back surface of

the plates. The increase is similar to that

for an algal standing crop. The doubling times estimated from the increase gradients

are given in Table 2. The shortest doubling

time of 15 hours was obtained in July 1973,

and the longest of 104 hours was observed in January 1973.


ture and doubling time of the heterotrophic

periphyton community is shown in Fig. 8. Although the figures reveal some scattering,

a considerable correlation exists between

them. From these figures, Quo was estima-

ted as about 2.5. The heterotrophic periphyton community

was mainly composed of a large number

of bacteria with a considerable amount of

protozoa such as Vorticella sp., Epistylis

sp., and small fiagellata. Although macro-

consumers such as Nematoda and Chiro-

nomts sp. were also present, these worms

seem to have had little effect on the growth

rates for the reason that they appeared

after the formation of thick periphyton layers.

3. Growth Rates of the Whole Periphyton Community

Increase curves of the organic carbon

for the whole periphyton community at

different times of the year were presented

in Fig. 9. The curves tend to resemble

those of the algae and the heterotrophic

periphyton community. Table 2 shows the doubling times derived from these curves.

The shortest doubling time was about 11

hours in July 1973, and the longest one was about 59 hours in March 1972. The

doubling times during the summer were

approximately the same as those of the

algae.


ture and doubling times of the whole

periphyton community is given in Fig. 10. Although the figures show some scattering

similar to that of the heterotrophic peri-

phyton community, a considerable correla-tion is found between temperature and time.

Qlo derived from these figures was about 2.2.

4. Discussion

As pointed out by BROOK (1971), the use

of artificial substrata for determining

growth rates of microorganisms involved several difficulties such as the adhesion of

detritus to the plates and the exfoliation

of attached substances. The increase of

the standing crop of microorganisms on

the plates be formulated as . follows

AS =g±i-e---f.

where JS=increase in standing crop of microorganisms per unit area

and unit time

g=increase in growth of microor-

Fig. 8. Relation between water temperature and

doubling times of heterotrophic periphy•

ton community.

AIZAKI 17

ganisms per unit area and unit

time

i =amount of adhesions of microor-

ganisms from the river water

per unit area and unit time

e=amount of exfoliation of micro-

organisms from the substrata

per unit area and unit time

f =amount of grazing of microor-

ganisms per unit area and unit time

The rate of adhesion and exfoliation must

be determined during the immersed period

to obtain the accurate growth rates of

microorganisms which partly reflect on the

change in the standing crop. BOTT and BROOK (1970a) determined quantitatively

the adhesion rate of detritus using a

technique involving ultraviolet radiation.

In the present study, bacterial growth

rates were estimated by counting the

number of cells per large microcolony at

different immersion times, and therefore,

the adhesion and exfoliation amounts could

be neglected. The doubling times of 3-10 hours observed were approximately the

same as those of BoTT and BROOK (1970b)

and BOTT (1975). In the former, the

doubling time is reported to be 5 hours in

Fig. 9. Growth curves of the whole periphyton community in

different months of the year at the study site.

Fig. 14. Relation between water temperature

and doubling time of the whole

periphyton community.


Trinity Springs and 4.5 hours in the Jordan

River. The water temperatures of these

rivers were 8-15°C and 22.5-24.5°C, res-

pectively. The latter auther also reports a doubling time of 4.2 hours in summer,

10.8 hours in spring, and 42 hours in

winter in the riffle section of the White

Clay Creek. YANAGIDA (1976) reviewed the

bacterial growth rate in several habitats in nature. He found that the doubling times

of bacteria varied from 2.3 hours to 281

hours in inland water, from 11.4 hours to

110 hours in sea water and from 4.2 hours to 150 hours within the intestine of several

animals. The slide immersion method tends

to show a rapid growth rate, suggesting

that attached bacteria in aquatic habitats have high growth activities.

4n the other hand, the growth rates

determined for sessile algae, heterotrophic

periphyton community, and periphyton community in this study seem to be signi-ficantly affected by the amount of adhesion

and exfoliation. This is especially the case

for the heterotrophic periphyton community

and the whole periphyton community. The

considerable scattering of the data of these

growth rates may be partially due to such influences.

Few determinations of algal growth rate

in natural aquatic habitat have been made

because of technical difficulties. As noted

by YOSHIDA (1976) as to the growth rate

of planktonic algae in aquatic habitat, the doubling time of planktonic algae ranged

from 5 to 100 hours, with the mean over

24 hours. The values obtained in the pre-

sent study were higher than those cited. Conditions in the midstream of the Tama-

gawa River may well have encouraged the

growth of sessile algae in several aspects

(i. e., nutrient supply and light penetration), thus explaining the high growth rate.

The most rapid growth rates of peri-

phyton community were obtained in July 1973, and the doubling times were calculated

to be 3.1 hours for bacteria, 12 hours for

sessile algae, 37 hours for the heterotrophic

periphyton community, and 11 hours for the whole Periphyton community. The growth

rate of the whole periphyton community

was almost the same as that of algae,

probably because the biomass of the peri-

phyton community in summer was mostly composed of sessile algae.

As mentioned above, in the midstream of

the Tamagawa River, nutrients supply and

light penetration seem to be sufficient for

algal growth. Therefore, water tempera-

ture will be the most important factor to

control the growth rate of the periphyton

community in this river. The data of Qio

obtained in this study reveal that the rate of growth depends on temperature. The

values of Qio were 1.9 in bacteria, 1.6 in sessile algae, 2.5 in the heterotrophic

periphyton community and 2.2 in the whole

periphyton community. The heterotrophic

periphyton community showed a high sen-sitivity to a change in water temperature. RICHARD et at. (1966), who studied the

relationship between water temperature and

photosynthetic activities of phytoplankton in a shallow bay in North Carolina, reported

a Quo value of 2.25. For reasons still

unclear, their datum was relatively higher

than the value for sessile algae obtained

in the present study.

摘要

スライドグラスおよび塩化ビニール製の人工付着板

を用い,都市廃水による汚濁の著しい多摩川中流域に

おける付着性微生物群集の増殖速度を測定した.付着

性微生物群集を構成する細菌類,藻類,従属栄養微生

物群集および全付着性微生物群集についてそれぞれの

増殖速度を求めた.その結果,現存量が2倍になるた

めに要する時間(倍加の時聞)はそれぞれ,細菌類,

3～10時問;藻類,12～28時間;従属栄L養的微生物

群集,15～104時間;全付着微生物群集,12～59時間

であった.また増殖速度は水温と高い相関を示し,そ

れぞれの生物集団のQl。は以下のようであった.細菌

AIZAKI 19

類約1.9;藻類,約1.6;従属栄養微生物群集,約

2.5;全付着微生群集,約2.2.

References

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(著者:相崎守弘,東京都立大学理学部生物学教室,

東京都世田谷区;現在,国立公害研究所水質土壌環境

部,茨城県筑波郡谷田部町館野; Morihiro AIZAKI,

Department of Biology, Faculty of Science, Tokyo

Metropolitan University, Setagaya-ku, Tokyo,

158; Present address, Water and Soil Environment

Division, The National Institute for Environmental

Studies, Ibaraki, 300-21, Japan.)

Accepted : 6 February 1979

Documents

Growth Rates of Microorganisms in a Periphyton Community