Jpn. J. Limnol., 48, 4, 249-256,1987.

Distribution of Lactate-, Propionate-, and Acetate-Oxidizing

Sulfate-Reducing Bacteria in Various Aquatic Environments

Manabu FUKUI and Susumu TAKII

Abstract

Distribution of sulfate-reducing bacteria (SRB) utilizing lactate (1-SRB), propionate (p-

SRB) and acetate (a-SRB) was examined along with some physico-chemical environmental

factors in various aquatic environments with various trophic levels and various salinities . In

marine environments a-SRB showed a tendency to dominate SRB, except for several samples .

On the other hand, in freshwater environments 1-SRB tended to dominate SRB . However, a-

SRB were found abundantly as well as 1-SRB in hypertrophic freshwater sediments. The

relationship between distribution of 1-, p- or a-SRB and environmental factors was discussed .

Key words : Sulfate-reducing bacteria, fatty acids, freshwater and seawater sediments , distribu-

tion.

1. Introduction

A part of organic matter produced in or

introduced into aquatic ecosystems is supplied to bottom sediments and decomposed by various mi-

croorganisms. In sediments various anaerobic de-

composition processes prevail because of the limited

supply of oxygen. Sulfate respiration as well as methanogenesis is an important process as the termi-

nal step of anaerobic mineralization.

Sulfate respiration is carried out by sulfate-reduc-

ing bacteria (SRB). It has been well known that SRB mainly utilize lactate and pyruvate as electron

donors and produce acetate and C02 in their sulfate

reduction. Recently, WIDDEL and PFENNIG (1977,

1981a, 1981b and 1982) isolated and classified vari-

ous SRB which were able to decompose acetate and or propionate as electron donors to C02. Many

researchers have since reported that SRB were sig-nificant for the oxidation of acetate and propionate

in anaerobic, marine and estuary sediments (SOREN

SEN et at., 1981; BANAT et at., 1981; SMITH and KLUG,

1981; CHRISTENSEN, 1984). However, there are few reports on the distribution of SRB utilizing acetate

and/or propionate (LAANBROEK and PFENNIG, 1981;

TAYLOR and PARKES, 1985; BATTERSBY et at., 1985). In the present study the distribution of SRB utiliz-

ing lactate (1-SRB), propionate(p-SRB) and acetate

(a-SRB) was examined in various aquatic environ-

ments with various trophic levels and salinities

along with some physico-chemical environmental

factors. The relationship between the distribution

of 1-, p- or a-SRB and environmental factors was

analyzed.

2. Materials and methods

2-1. Study sites and sampling

Surveys were carried out in five lakes, one river

and one marine canal. The lakes were as follows;

Lake Kizaki (freshwater, mesotrophic, Nagano

Prefecture), Lake Suwa (freshwater, eutrophic,

Nagano Prefecture), Lake Biwa, south basin (fresh-

water, eutrophic, Shiga Prefecture), Lake Tega-

numa (freshwater, hypertrophic, Chiba Prefecture),

and Lake Shinhama (seawater, eutrophic, Chiba

Prefecture). In the polluted Tama River, surveys

were carried out at three stations: Daishi-bashi

(brackish water), Gasu-bashi (brackish water) and

Chofu-seki (freshwater), 2.5 km, 5 km and 10.5 km

upstream from the river mouth, respectively. Ka-

tsushima, a highly polluted canal in Tokyo Bay, was

also surveyed.

Samples of sediments and bottom water were

collected using a polycarbonate plexiglass tube (~_

4.5 cm, length=30 cm). The sediment was cut off

into several layers.

2-2. Enumeration of bacteria

Wet sediment of 2.4 or 2.5 ml was dispersed in 100

ml of sterilized water by a mechanical shaker for 5

min. The sediment suspension for inoculation was

appropriately diluted with sterilized water. For

marine samples the diluent was added with NaCI (20

250 FUKUI and TAKII

g•1-1) and MgC12.6H2O (3 g•1-'). SRB were enumerated by the anaerobic petri dish

method (WAKAO and FURUSAKA, 1972). The enu-

meration medium used was C medium of BUTLIN et

at. (1949) added with vitamin solution (5 ml•1-~) instead of yeast extract, containing either lactate,

propionate or acetate. The medium by BUTLIN et at. (1949) contains (g•1-'): K2HPO4 (0.5), NH4Cl

(1), Na2SO4 (1), CaC12.2H2O (0.1), MgSO4.7H2O

(2), Na-lactate (3.5), FeSO4.7H2O (0.2), yeast extract (1), and agar (15) (pH, 7.2). The vitamin

solution (LAANBROEK and PFENNIG, 1981) contains

(mg• 1-') : biotin (2), nicotinic acid (20), j9 a amino-benzoic acid (10), thiamin (20), panthotenic acid (10), pyridoxiamin (50) , and cobalamin (10). For marine samples the medium was added with the

same salts at the same concentrations as the diluent. 1-SRB, p-SRB or a-SRB were enumerated by the

medium added with Na-lactate, Na propionate or

Na-acetate as the sole electron donor and carbon

source, respectively, at a concentration of 30 mM. Black colonies in the plate were counted after five

weeks incubation at 30°C. Triplicate plates were

used for enumeration. Aerobic heterotrophic bacte-

ria were enumerated by the pour plate method using the medium of SAKURAI (1967). The plates were

incubated at 20°C for two weeks.

2-3. Analysis of some physico-chemical factors

The pH, redox potential and chloride were deter-

mined by the electrode method using pH/ion meter

(Model 225, Iwaki Glass). Sulfate ion in pore water and total sulfide in sediment were determined by the

barium chromate method and by the method of

SHIGA (1983), respectively. Pore water in sediment was obtained by centrifugation at 10,000 rpm and

4°C for 10 min.

3. Results

3-1. Colony formation of 1-SRB, p-SRB and

a-SRB on agar plates

The colony formation process of SRB on agar

plates was observed for sediment samples collected from Lake Shinhama (Fig. 1). Colonies on plates

of the medium of BUTLIN et at. (1949) (medium A)

began to appear on the second day after inoculation. But the colonies on the medium B containing lactate

and the vitamin solution instead of yeast extract

were formed two days later than the case on the

medium A. The number of colonies on the medium B was 60% of that on the medium A after 35 days.

This difference may have been caused by certain

growth factors contained in yeast extract but not in

the vitamin solution.

On the medium C containing acetate or on the

medium D containing propionate, colonies began to

appear on the 11th day, and reached a stationary

phase on the 35th day. The colonies of 1-SRB, p-

SRB or a-SRB were, therefore, counted after the

35th day. The mean value and standard deviation

(n, the number of samples) of colonial diameter

(mm) on plates of medium A, B, C, or D at the 35th

day were 2.31±2.11 (n=49), 2.24±1.78 (n=131),

0.60±0.67 (n=69), and 0.32±0.26 (n=38), re-

spectively. The colonies of p-SRB and a-SRB were

much smaller than those of 1-SRB.

3-2. Distribution of l-SRB, p-SRB and a-SRB

in various aquatic environments

Table 1 shows the numbers of 1-SRB, p-SRB and

a-SRB at various sites. At the center of Lake

Kizaki (depth, 29.5 m), the numbers of 1- and a-SRB

in sediments were 102 and 10' per ml sediment,

respectively. At the center of Lake Suwa (water

depth; 6 m), the numbers of 1-, p- or a-SRB were

between 10° and 10' per ml in overlying water, and

between 102 and 103 per ml in sediment. The num-

ber of each SRB in littoral zone (water depth; 1.5

m) of Lake Suwa was larger than at the lake center.

Fig. 1. Colony formation process of SRB in the surface

sediment 0-3 cm) of Lake Shinhama on plates of

the medium C of Butlin et al. (1949) (A) and

the modified medium containing lactate (B),

acetate (C) or propionate (D).

Distribution of Lactate-, Propionate-, and Acetate Oxidizing Sulfate-Reducing Bacteria 251

At both lake sites 1-SRB dominated SRB. In the

littoral zone of the south basin of Lake Biwa (water

depth; about 40 cm), the number of each SRB was

larger than that of Lake Suwa. The dominant SRB

were also 1-SRB, both in overlying water and in

sediment. In the littoral zone of Lake Teganuma

Table 1. Distribution of 1-SRB, p-SRB, and a-SRB in various sites.

* Colony forming unit

252 Fu Ku I and TAKH

vegetated by Phragmites communis, the number of

each SRB was about 105 per ml sediment, which was

the largest in the various lakes surveyed. In con

trast to other lakes, the number of a-SRB in this

lake was slightly larger than that of 1-SRB in the

sediment. In freshwater lakes with various nutrient

levels, the number ratio of a-SRB to 1-SRB (a/1)

may increase with the progress of eutrophication

(Table 2).

At three stations of the polluted Tama River, the

numbers of 1- and a-SRB in surface sediment were

as large as 105-106 per ml sediment, and a-SRB were

almost comparable in number to 1-SRB. At Daishi

-bashi , a-SRB were more abundant than 1-SRB in

deeper sediment, and at both Gasu-bashi and Daishi

-bashi the number of p-SRB was almost equal to

that of a-SRB.

In the littoral zone vegetated by Phragmites corn

munis (water depth, 40-60 cm) of a seawater lake,

Lake Shinhama, the numbers of 1-, p- and a-SRB in

overlying water and sediment were 101-102 per ml

and 104-105 per ml, respectively, and 1-, p- and a-

SRB were similar in number. In a polluted canal in

Tokyo Bay, the number of each SRB was 105 per ml

sediment (Fig. 2). Although l-SRB slightly domi

nated SRB in the surface layer, the proportion of a-

SRB to 1-SRB increased with depth (Fig. 2) as in the

case of Daishi-bashi. Redox potential was very low

(-140 mV) even at the sediment surface. Sulfate

concentration in pore water and also the ratio of

sulfate to chloride decreased with depth, and all

layers of the sediment had high total sulfide con-

Table 2. Comparison of the number ratio of a-SRB to 1-SRB in the water

and sediment of freshwater lakes.

Note ; (C) : Center of lake, (L) : Littoral zone *1 , SAKURAI and WATANABE (1974) *2 ; SAKAMOTO et at . (1975) *3 ; JIBP-PF Research Group of Lake Biwa (1975) *4 ; KOBAYASHI (1982a , 1982b)

Fig. 2. Vertical distributions of 1-, p- and a-SRB (A) and some environmental factors CBS in

Katsushima Canal in Tokyo Bay.

Distribution of Lactate-, Propionate-, and Acetate-Oxidizing Sulfate-Reducing Bacteria 253

tents. These results indicate that sulfate reduction

proceeded actively in the upper layers (JORGENSEN and COHEN, 1977; JRGENSEN, 1977, 1978a, 1978b, 1982; MOUNTFORT et al., 1980; DEVOL and AHMED,

1981; CRILL and MARTENS, 1983).

4, Discussion

Many species of SRB have been isolated in the last

decade. Based on the review by PFENNIG et al.

(1981) and the recent literatures (BAR and WIDDEL, 1986a, 1986b; SZEWZYK and PFENNIG,1987), SRB can

be classified as follows into six groups in terms of

the ability to oxidize three fatty acids, lactate,

propionate, and acetate as electron donors. Species oxidizing lactate, propionate and acetate; Desul-

fococcus multivorans, Desulfonema limicola, Desul-

fosarcina variabilis and Desulfotomaculum catecholi-cum. Species oxidizing propionate and acetate; Desulfonema magnum and Desulfobacterium in-

dolicum. Species oxidizing lactate and acetate;

Desulfobacter postgatei. Species oxidizing acetate;

Desulfotomaculum acetoxidance, Desulfovibrio baarsii

and Desulfobacterium phenolicum. Species oxidiz-ing lactate; many species of the genus Desulfovibrio

and the genus Desulfotomaculum. Therefore, the

number enumerated as 1-, p-, or a-SRB by our

method, may be partially overlapping. The number of SRB enumerated with the medium

B, in which yeast extract in the medium A was

replaced by the vitamin solution, was 60% of that with the medium A for sediment samples from Lake

Shinhama. However, the number of SRB in the

present study was almost equal to those of SRB enumerated by conventional media in the literature for the same or nearby sites; i. e., Lake Suwa

(FUKUHARA and SAKAMOTO, 1987), the Tama River (TEZUKA, 1979), and Tokyo Bay (TEZUKA, 1979).

Distribution of 1-, p- and a-SRB in sediments

described in previous reports is summarized in Table

3. Bacterial numbers shown in these reports were enumerated by the method of agar shake tube, using

media similar to the medium B, C and D. The

bacterial numbers of Ems-Dolland estuary are simi-

lar to those of Lake Shinhama, and those of the North-East Atlantic and Loch Eil were lower than

our results for the marine sites. The difference in

the numbers of SRB may reflect the difference in

nutrient levels of aquatic environments (GORLENKO et al., 1983). In the freshwater sediment (Ems-

Dolland estuary) 1- and p-SRB dominated SRB,

whereas in seawater sediments (North-East At-

lantic and Loch Eil) a-SRB were found to be nearly

equal in number to 1-SRB. This tendency agrees fairly well with our results.

Figure 3 shows the relationship between the num-

ber ratio of 1-SRB to p-SRB (1/p) and that of a-SRB to p-SRB (a/p) reported by previous inves-

tigators and the present study. In marine environ-

ments, a-SRB tended to dominate SRB, except in

samples of overlying water and surface layer (0-3

cm) of sediment in Tokyo Bay and at Daishi-bashi of the Tama River. KADOTA and MIYOSHI (1964)

reported that the upper layer of marine sediments

might be abundant in lactate which was utilized by 1-SRB as carbon or energy sources. They supposed

that distribution of SRB and their activities were

controlled mainly by the supply of lactate. On the

other hand, in freshwater environments 1-SRB tend-

ed to dominate SRB. However, it is notable that both a-SRB and 1-SRB were abundant in hypertro-

phic freshwater sediments, such as Lake Teganuma and Chofu-seki of the Tama River, unlike their

presence in seawater sediments. The relationship between a/l and some environ-

Table 3. Comparison of the numbers of 1-, p- and a-SRB in various aquatic sediment.

freshwater area *z ; seawater

*3 ; The sample was incubated at 20°C for 12 days .

254 FuKCI and TAKII

mental factors was tested (T test). In freshwater

environments, only the relationship between a/1 and

chloride ion was significant (r = 0.881, n14, p <

0.01). The a/i ratios in sediments were high, espe-

cially in Lake Teganuma (1.15) and Chofu-seki of the Tama River (3.38). There maybe some factors

stimulating the growth of a-SRB in highly polluted

environments, because the chloride concentration is

thought to reflect the degree of pollution in fre-shwater environments (HANYA,1960). On the other

hand, in marine environments there is no significant

relationship between the a/1 ratio and the environ-

mental factors examined. Our results suggest that factors affecting the relative dominancy of 1-, p- or

a-SRB are not simple. Distribution of 1-, p- and

a-SRB may be controlled by both the quality and

quantity of available substrates produced by fer-mentation, as well as the quantity of sulfate. It is

necessary to examine the concentrations and tur-

nover rates of available substrates for each SRB and

also to investigate the possible interaction between

SRB and other anaerobic bacteria such as both

fermentative and methanogenic bacteria.

摘 要

水界 にお ける乳酸,プ ロピオ ン酸お よび酢酸を

利用する硫酸還元菌の分布

い くつかの水域の水および底泥 におけ る乳酸,プ ロ

ピオン酸お よび酢酸を利用 する硫酸還元菌(以 下それ

ぞ れ1-SRB,p-SRB,a-SRBと 略す)の 分布 を調査

し,い くつ かの環境 要因 との関係 につ いて解析 を行

なった。その結果,海 水域では数試料を除 いてa-SRB

が優 占す る傾 向があ った。一方,淡 水域では レSRBが

優占する傾 向が得 られたが,汚 染 の著 しい淡水域底泥

で は1-SRBと 同 程 度 にa-SRBが 検 出 さ れ た。1-

SRB,p-SRBお よびa-SRBの 分布 と環境要 因 との関

係が議論 された。

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(著 者:福 井 学 ・滝 井 進,東 京都 立 大学 理 学部 生

物 学 教 室,〒158東 京 都 世 田 谷 区 深 沢2-1-1;Mana-

bu FUKUI and Susumu TAKII, Department of Biology,

Faculty of Science, Tokyo Metropolitan University,

Fukazawa 2-1-1, Setagaya-ku, Tokyo 158)

Received : 8 May 1987

Accepted : 18 September 1987