ENUMERATION AND ISOLATION OF ANAEROBIC BACTERIA IN SEWAGE DIGESTOR FLUIDS: ISOLATION OF LACTATE-UTILIZERS

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1 J. Gen. App!. Microbiol., 26, (1980) ENUMERATION AND ISOLATION OF ANAEROBIC BACTERIA IN SEWAGE DIGESTOR FLUIDS: ISOLATION OF LACTATE-UTILIZERS ATSUKO UEKI,1 HAJIME MINATO, RYOZO AZUMA, AND TSUNEJI SUT02 National Institute of Animal Health, Yatabe, Ibaraki-ken , Japan (Received August 20, 1979) Addition of lactate to the medium for the non-selective colony counts in sewage digestor fluids resulted in remarkable increase in the counts. In the presence of lactate, percentages of strictly anaerobic, Gramnegative curved rods in the isolates were usually much higher than those in the absence of lactate. Conversion of lactic acid to acetic acid by these bacteria was thought to be a very important process in the fermentation in digestor fluids. The lactate-utilizing bacteria were thought to belong to Desulfovibrio. Some strains of lactate-utilizing, strictly anaerobic, Gram-negative rods were also isolated. As previously reported (1), for enumeration and isolation of non-methanogenic anaerobic bacteria in sewage digestor fluids by the anaerobic roll tube method (2), VLS agar, which contained 40% of the supernatant of autoclaved digestor fluid (1), with mixed gas phase (95% N2 and 500 C02) gave good results. In bacterial groups in the fluids of three digestors examined under this condition, facultatively anaerobic streptococci were often the most dominant group, which produced mainly lactic acid in the medium. Since lactic acid was also produced in the medium by some of other isolated bacteria, it seemed that this acid would be accumulated in the digestor fluid. However, we could not detect it in any digestor fluids (1). We considered that there existed lactate-utilizing bacteria in the sewage digestor fluids. Conversion of lactic acid to other compounds was thought to be significant in the process of fermentation in digestor fluids. In the rumen, bacteria such as Veillonella alcalesence, Megasphaera elsdenii, 1 Present address: Laboratory of Applied Microbiology, Faculty of Agriculture, Yamagata University, Tsuruoka 997, Japan. 2 Present address: Nippon Veterinary and Zootechnical College, Musashino, Tokyo 180, Japan. 15

2 16 UEKI, MINATO, AZUMA, and SUTO VOt.. 26 and Selenomonas ruminant/urn convert lactic acid to other compounds including acetic and propionic acids (3). A selective medium containing lactate for veillonellas has been devised (4), and Megasphaera elsdenii is also known to grow in this medium (5). Another medium for non-selective enumeration of anaerobic bacteria in the rumen, which contains lactate in addition to sugars, has also been devised (5, 6). Further, HoBSON and SHAW (7) modified this medium to study anaerobic bacteria in piggery waste anaerobic digestors. However, any experiments on the effect of lactate addition were not carried out by these authors. Addition of lactate as a sole carbon source markedly reduced the colony count of a fluid of swine waste digestor when compared with those obtained with other carbon soure or no addition (8). In this study, we examined "lactate-utilizers" in digestor fluids and the effect of lactate addition to the medium on the colony counts of anaerobic bacteria in the fluids. MATERIALS AND METHODS Anaerobic digestors. Three digestors (A, B, and C) for digestion of piggery waste used previously by UEKI et al. (1) were employed. Both (A) and (C) digestors consisted of differently connected 4 fermentors and the former was equipped with a reservoir (R) at the top of the line. (B) digestor consisted of 3 fermentors. Bacterial florae in the fluid of these 3 digestors investigated in the previous examination were rather different from each other, but such groups as facultatively anaerobic streptococci and strictly anaerobic Grampositive cocci and Gram-negative curved rods were often isolated as predominant groups. Concentration of organic acids and ph value in each digestor fluid were almost the same as previously reported (1), and lactic acid, again, was not at all detected any of the fluids. Sampling of digestor fluids and enumeration of anaerobic bacteria. Sampling of digestor fluids and enumeration of anaerobic bacteria by the modified (9) anaerobic roll tube method (2) were performed by the same procedure as described previously (1). Two media, lactate agar and VLS-lactate agar, were used to examine the effect of lactate in the medium. These two media were basically the same as VLS agar (1), which contained Trypticase, yeast extract, beef extract, the supernatant of digestor fluid (1), and four sugars (glucose, maltose, cellobiose, and starch) in addition to salt solutions and reducing agents. In lactate agar, the four sugars in VLS agar were replaced by 1 % sodium lactate and VLS-lactate agar contained sodium lactate together with the four sugars. VLS-lactate agar was mainly used to compare colony counts and composition of bacteria with those obtained with VLS agar. Mixed gas phase (95 % N2 and 5 % C02) was used. Isolation and characterization of bacteria. The methods of isolation and

3 1980 Anaerobic Bacteria in Sewage Digestors 17 characterization of bacteria were as previously described (1). To determine the lactate utilization, sodium lactate (0.5 %) was added to VLS liquid medium in place of sugars and consumption of lactate by bacteria was measured by gas chromatography. Catalase activity, production of H2S, and other characteristics were determined according to the methods described in Anaerobe Laboratory Manual (10), with VLS liquid medium without sugars as a basal medium. The mixed gas phase was also used except for the examination of the effect of gas phase on the growth of streptococci. RESULTS Lactic acid production of streptococci isolated from sewage digestor fluids As shown in Table 1, all the strains of streptococci examined, which had been previously isolated from the three digestors (1), mainly produced lactic acid and minorly acetic acid from glucose. Growth of these strains and the production of lactic acid were usually better under 100 % CO2 gas phase than under the mixed gas phase. Table 1. Acids produced by streptococci isolated from digestor fluids. Presence of lactate-utilizers in digestor fluids In the first place, presence of lactate-utilizers in (A) and (B) digestor fluids was examined by using lactate medium. As shown in Table 2, colony counts obtained with this medium were % of those obtained with VLS agar. Several distinctly different bacterial groups such as Gram-positive streptococci, Gram-negative rods, and Gram-negative curved rods were isolated with this medium. Some strains of the last group actively consumed lactate in the medium. They were commonly isolated from both digestors. Thus, the presence of lactate-utilizers in the fluids in a rather high proportion, which

4 18 UEKI, MINATO, AZUMA, and SUTO VOL. 26 Table 2. Comparison of colony counts obtained with VLS agar and lactate agar from the fluids of two digestors. were able to grow in the basically same medium This suggests the possibility of obtaining them enumeration in VLS-lactate agar. as VLS agar, efficiently by was clarified. non-selective Enumeration and isolation of anaerobic bacteria in digestor fluids with VLS-Iactate agar Enumeration of anaerobic bacteria in the fluids of (C) digestor was performed 3 times at intervals of about 2 months (Table 3). The addition of lactate to VLS agar was remarkably effective in increasing (12-90%) the colony counts. Compositions of bacteria isolated twice from F-II and F-IV are presented isolates from in Fig. 1. In all the samples, more than 40% of all the each of VLS agar and VLS-lactate agar were facultatively anaerobic streptococci. Addition of lactate to the medium did not appeared to affect the growth of this group so markedly. It is notable that the percentage of strictly anaerobic, Gramnegative curved rods increased in all the samples. The percentage or viable counts of this group were usually very low when isolated with VLS agar, but those with VLS-lactate agar were usually rather high. Especially in F-II, the increase ( % and % in two experiments) was remarkable. As Table 3. Comparison of colony counts in (C) digestor flu ids obtained or without lactate in the medium. with

5 1980 Anaerobic Bacteria in Sewage Digestors 19 Fig. 1. Bacterial composition in (C) digestor fluids obtained with (/) or without (LI) lactate. Bacterial groups: 1-4, strictly anaerobes; 1, Gram-negative rods; 2, Gram-negative curved rods; 3, Gram-positive rods; 4, Gram-positive cocci; 5-8, facultative anaerobes; 5, Gram-positive cocci; 6, Gram-negative rods; 7, Gram-positive rods; 8, Grampositive streptococci. Percentages were calculated as follows: No. of isolates of the group without lactate of the group without lactate= - No. of total isolates without lactate x 100; No. of isolates of the group with lactate of the group with lactate=- No. of total isolates with lactate x colony count of the sample with lactate x 100. colony count of the sample without lactate See Table 3 for the colony count of each sample. for the other groups, including strictly anaerobic, Gram-negative rods and Gram-positive cocci, however, particular tendency of change in the percentage could not be pointed out. From these results, it may be concluded that the addition of lactate results in increase of the colony counts mainly due to the strictly anaerobic, Gramnegative curved rods. The same experiment was carried out with (A) digestor (Table 4). By the addition of lactate, the colony counts in the fluids of reservoir (R) and three fermentors (F-I, F-II, and F-IV) increased by %. The bacterial corn-

6 20 UEKI, MINATO, AZUMA, and SUTO VOL. 26 Table 4. Comparison of colony counts in (A) digestor fl uids obtained or without lactate in the medium. with position in the fluid of F-II is presented in Fig. 2. The facultatively anaerobic streptococci were also dominant in the presence or absence of lactate. The percentage of strictly anaerobic Gram-negative curved rods also increased from 33 % to 52% by the addition of lactate. The percentage (33 %) of this group in the isolates with VLS agar was relatively high compared with those in (C) digestor. Some minor groups isolated with VLS agar disappeared in the presence of lactate. Characteristics of lactate-utilizers Representative bacteria of each group isolated from (A) and (C) digestors were checked for their lactate-utilization by using lactate liquid medium. Of the strains examined, most of strictly anaerobic, Gram-negative curved rods and some of strictly anaerobic, Gram negative rods clearly showed lactateutilizing ability. This result agreed well with the consistent increase in the percentage of the former group by the addition of lactate and also the increase of the latter in F-II of (C) digestor. Lactate utilization was not confirmed in other groups including the strictly anaerobic, Gram-positive cocci. Some properties of lactate-utilizing strains are shown in Table 5. All the lactate-utilizing curved rods failed to grow in the glucose medium and produced

7 Table 5. Some characteristics of lactate-utilizing bacteria isolated from digestors.

8 22 UEKI, MINATO, AzUMA, and SuTO VOL. 26 acetic acid and H2S in the lactate medium. On the other hand, the Gramnegative rods actively fermented sugars such as glucose, maltose, fructose, and mannose, but not arabinose, lactose, cellobiose, trehalose, raffinose, rhamnose, or mannitol. They did not grow on the bile medium (10). They hydrolyzed esculin and did not produce H2S. They produced acetic, propionic, lactic, and succinic acids from glucose, and acetic and propionic acids from lactate, increasing the latter in the molar ratio compared with that from glucose. Based on these characteristics, the Gram-negative curved rods and rods were thought to belong to Desulfovibrio (I1) and Bacteroides (10), respectively. DISCUSSION The bacterial composition in the digestor fluids of piggery waste investigated with VLS agar was almost the same as that in the previous report with respect to the dominancy of facultatively anaerobic streptococci (1). These bacteria certainly produced lactic acid in the medium, but we could not detect it in the fluids in the same manner as in the previous examination (1). These results further suggested the presence of lactate-utilizers in the fluids. We think that improvement of culture conditions to catch more diverse species in the medium is one of basic and important studies on the bacterial ecosystem. If the proportion of lactate-utilizers to the total bacterial population is high enough and they can grow in the basically same medium as VLS agar, the addition of lactate to VLS agar may result in increasing the colony counts. On the contrary, if their proportion is not so high, the addition will have no significant effect on the count. Further, if the lactate-utilizers present in the fluids are not able to grow in the basically same medium as VLS agar, the lactate addition to this medium is also meaningless. To enumerate and isolate them, it is necessary to use a selective medium as for veillonellas in the rumen. The increase in the colony counts and the isolation of lactate-utilizers indicated the effectiveness of the addition of lactate to VLS agar. As shown by the examination with lactate agar, lactate-utilizers were also present in the fluids of (B) digestor. It is thought that they are generally present in digestor fluids in a rather high proportion. However, when VLS-lactate agar was used for counting the bacteria in the effluent of (A) digestor (data not shown), the count obtained was significantly low compared with that with VLS agar. This seems to indicate an inhibitory effect of lactate to some bacteria. In addition, nonlactate-utilizers also changed in their proportion sometimes by the addition of lactate. Thus, more investigation may be necessary to elucidate the correct effect of lactate, especially when this medium is applied to ecosystems other than digestor fluids. Some strains in the group of strictly anaerobic, Gram-negative curved rods

9 1980 Anaerobic Bacteria in Sewage Digestors 23 could not utilize lactate, but most of the strains in this group intensively grew in the lactate medium, producing acetic acid. They could not grow in the medium which contained sugars in place of lactate. The relatively high percentage of this group in the isolates from (A) digestor fluid even without lactate was in accordance with the previous result (1), which showed their high percentages in some fluids of (A) and (B) digestor with the use of VLS agar. Most of strictly anaerobic curved rods isolated with VLS agar also used lactate as the strains isolated in the presence of lactate. Their growth on the slants of VLS agar after the isolation from roll tubes was usually very poor and easy to be lost during transfers, but they actively grew on the slants of VLS-lactate agar. In the roll tubes, lactic acid produced by streptococci or other bacteria may be able to be consumed by the lactate-utilizers. Thus, the addition of lactate to the medium is also necessary to avoid the loss of lactate-utilizers during transfers, even though they can be caught in the roll tubes in the absence of lactate. The remaining lactate-utilizers, Gram-negative rods, intensively grew on sugars such as glucose and maltose. Since strictly anaerobic, Gram-negative rods isolated from digestor fluids other than F-II of (C) digestor did not utilize lactate, distribution of lactate-utilizing rods seemed to be not so broad as that of curved rods. Furthermore, their percentage in the isolates from (C) digestor fluid was lower than that of curved rods. Thus, it is thought that the curved rods take an important part in converting lactic acid, even though it is possible that some other lactate-utilizers are present in the fluids. This seems to be a different aspect of the fermentation process in the digestor fluids from that in the rumen (3). It is known that acetate-utilizing methanogenesis occurs in sewage digestor fluids and other ecosystems such as bottom deposits of lakes (12-15). In the fluids we examined, the concentration of acetic acid, the main acid detected, decreased with the progress of fermentation, suggesting the presence of this methanogenic process (1). The conversion of lactic acid to acetic acid may be very important for the subsequent methanogenic process in digestor fluids. REFERENCES 1) 2) 3) 4) 5) 6) 7) A. UEKI, E. MIYAGAWA, H. MINATO, E. AZUMA, and T. SUTO, J. Gen. App!. Microbial., 24, 317 (1978). R. E. HUNGATE, In Methods in Microbiology., 3B, ed by J. R. NORRIS and D. W. RIBBONS, Academic Press, London (1969), p R. E. HUNGATE, In The Rumen and Its Microbes, Academic Press, New York (1966). M. ROGOSA, J. Bacteriol., 72, 533 (1956). P. N. HOBSON, In Methods in Microbiology, 3B, ed by J. R. NORRIS and D. W. RIBBONS, Academic Press, London (1969), p Y. KURIHARA, J. M. EADIE, P. N. HOBSON, and S. 0. MANN, J. Gen. Microbiol., 51, 267 (1968). P. N. HOBSON and B. G. SHAW, Water Res., 8, 507 (1974).

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