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Bioresource Technology 49 (1994) 261-265 © 1994 Elsevier Science Limited

Printed in Great Britain. All rights reserved 0260-8774/94/$7.00

CHARACTERIZATION A N D ENUMERATION OF MICROORGANISMS ASSOCIATED WITH ANAEROBIC

DIGESTION OF TOMATO-PROCESSING WASTE

R. Sarada & Richard Joseph*

Microbiology and Bioengineering Department, Central Food Technological Research Institute, Mysore 570 013, India

(Received for publication 28 June 1994)

Abstract Different physiological groups of microorganisms; cel- lulolytics, xylanolytics, pectinolytics, proteolytics, lipoly- tics and methanogens were enumerated and monitored during the anaerobic digestion of tomato-processing waste (TPW). In the batch digestion lasting 110 days, the numbers of cellulolytics, xylanolytics, pectinolytics, pro- teolytics and lipolytics showed a steady increase up to 40 days, but declined thereafter. In the semicontinuous digestion, the numbers of cellulolytics, proteolytics and lipolytics were found to be greater at 24 and 32 days hydraulic retention times (HRT) than at 8 and 16 days HRT. On the other hand, the numbers of xylanolytics and pectinolytics varied very little at different HRT. A t the higher HR T, there were more methanogens than at the lower HRT. Differences in the types of bacteria iso- lated from digesters in the batch and semicontinuous processes were found. Bacteria belonging to Bacter- oides, Eubacteria, Fusobacteria, Lactobacillus, Pro- pionibacteria and Selenomonas were isolated. Two apparently novel isolates of cellulolytic bacteria belong- ing to the genus Eubacteria were also found.

Key words: Tomato processing waste, anaerobic micro- organisms, batch digestion, hydraulic retention time.

INTRODUCTION

Anaerobic degradation of organic matter to methane is carried out by microorganisms constituting several trophic levels (Zeikus, 1980). Studies on microflora in anaerobic digestion of organic matter have been mostly limited to animal manures and sewage sludge. It is known that the nature of the substrate will generally influence the development and character of the micro- flora and the methane yield (Zeikus, 1980), but no specific information is available on various substrates that could be used in digestions.

Tomato-processing waste (TPW), which is generated in substantial quantities by the food processing in- dustries, could serve as a good substrate for methane

*To whom correspondence should be addressed.

production (Sarada & Krishna Nand, 1989). These studies further suggested that the hydrolytic and fer- mentative stages of digestion were faster than the acetogenic and methanogenic stages (Sarada & Joseph, 1993). This is in contrast to earlier results showing that in the anaerobic digestion of many complex organic wastes it is the hydrolytic step that is rate-limiting for methane production (Hobson et al., 1974). For these reasons, and also for the fact that TPW is not a pre- digested material like sewage sludge and animal manures, it was of interest to examine the microflora that developed during the anaerobic digestion of TPW in qualitative and quantitative terms.

METHODS

Digesters Laboratory digesters were set up as described earlier (Sarada & Joseph, 1993) for both batch and semi- continuous processes. Batch digestion was carried out in 1 1 Buchner flasks containing 6% TPW slurry and 10% inoculum at 33 + 2°C. At 10 day intervals samples were withdrawn and various physiological groups of microorganisms were enumerated.

The semicontinuous process was carried out at 8, 16, 24 and 32 days hydraulic retention time (HRT) using 5"5 1 capacity bottles each containing 5 1 of slurry with 4"5% solids. When the digesters attained a steady- state, samples were drawn for enumeration of micro- organisms.

Media Anaerobic media were prepared as the procedure of Hungate (1969) and Holdemann and Moore (1972): 10 ml and 30 ml injection vials were used as roll tubes for isolation and growth of the microorganisms.

Enumeration of microorganisms Well-mixed digester slurry was withdrawn anaerobic- ally into a bottle and sealed immediately under nitrogen. The sample was serially diluted to 109 into a series of sealed bottles containing sterile dilution medium (Holdeman et al., 1977). Samples from 104 t o

261

262 R. Sarada, R. Joseph

107 dilutions were added to roll tubes containing the media. The substrates used for enumerating various groups of microorganisms are shown in Table 1. Bacterial counts were taken after 1 and 2 weeks of incubation at 35°C. Methanogens were enumerated by a most probable number (MPN) technique (de Man, 1975).

Isolation and identification of microorganisms The microorganisms which degraded cellulose, hemi- cellulose, pectin, protein and lipid were isolated using the corresponding substrate as sole carbon source. The isolates were randomly selected from higher dilqtions for further identification. Isolated colonies were trans- ferred into the liquid medium containing the respective substrate. The purity of the isolates was ensured by re- isolation on roll tubes. The specific activities were further confirmed by identifying the degradation pro- ducts of the substrate and also observing the clearance zones surrounding the colonies. Identification of organisms was carried out according to Holdemann et al. (1977) and descriptions of the identified genera are given in Table 2.

Analyses The volatile acid fermentation products were analysed by gas chromatography by the method of Holdemann et al. (1977). Lactic acid and succinic acid were identi- fied by paper chromatography.

RESULTS

TPW contains cellulose, hemicellulose, pectin, protein and lipid as the major components (Sarada & Joseph, 1993). Therefore the microorganisms which hydrolyse these components were examined at various stages during the anaerobic digestion. The data obtained on the profile of various groups of microorganisms during the batch process are given in Table 3. Almost all the physiological groups increased their counts during the digestion up to 40-50 days. However, the extent of increase and peak values varied for the different groups. Cellulolytics and xylanolytics had attained 800- and 1250-fold increases over their initial counts on the 40th day. The maximum counts of pectinolytics (238-fold increase)and lipolytics (200-fold increase)

were noted on the 20th day of digestion, while a 42- fold increase in proteolytic count was obtained on the 10th day. There was no significant change in the counts of proteolytics, pectinolytics and lipolytics from 40 to 100 days, but the number of cellulolytics and xylano- lytics decreased gradually after 50 days of digestion and were lower than their initial counts at the end of digestion period of 110 days. The counts of methano- gens, unlike the other groups of microorganisms, were found to decrease by 3-4 orders of magnitude from the initial level of 106/ml.

The data obtained for various groups of micro- organisms from the four semicontinuous digesters are presented in Table 4. The number of cellulolytics were greater at 24 days than at 8 days HRT. No significant variations in the numbers of xylanolytics and pectino- lytics were found at different HRT. The numbers of proteolytics and lipolytics were 2- to 3-fold greater at 16 days HRT than at 8 days HRT. The higher the HRT, the higher was the number of methanogens counted. At 32 days HRT, this count was 3"3 x 106/ml, which was a five fold increase over the count at 8 days HRT.

The physiological and biochemical characters of the isolated organisms from TPW digester (from both batch and semicontinuous digesters) are shown in Table 2. Some of the salient features of the isolates are given below.

Fusobacterium The characteristic features of this genus are Gram-nega- tive, non-sporulating rod morphology and ability to produce butyric acid as the major fermentation pro- duct. Most of the isolates in the present work were found to be lipolytic. These isolates shared many com- mon characteristics with F. symbiosum but differed from it in the manner of fermentation of maltose, raf- finose, sorbitol and inulin.

Bacteroides These are Gram-negative, non-sporulating rods. Most of the Bacteroides isolated in the batch process were cellulolytic, while in the semi-continuous digestion lipolytics were also found. One group of isolates was found to be similar to B. uniformis and B. ruminicola ss. brevis. Another group resembled B. multiacidus.

Table 1. Substrates used for enumeration of different physiological groups of micro- organisms

Physiological group Substrate Reference

Cellulolytics Xylanolytics

Cellulose powder Xylan (larch wood)

Pectinolytics Apple pectin Proteolytics Skim milk powder Lipolytic s Tributyrin Methanogens Acetate, H 2 and CO 2

Hungate and Stack (1982) Vander Toorn and

Van Glylswyk (1985) Schink and Zeikus (1982) Fulghun and Moore (1963) Jones and Grainger (1983) Touzel and Albagnac (1983)

Microorganisms associated with TP W digestion 263

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Table 4. C.f.u. of different physiological groups of microorganisms in semicontinuous digestions enumerated by roll,tube methods

Physiological Counts/ml group HRT

8 days 16 days 24 days 32 days

Ceilulolytics 1.40 × 105 2.90 × 105 7.20 × 105 2.80 x 105 Xylanolytics 7.20 × 107 1.69 × 107 6.56 × 107 7.58 × 107 Pectinolytics 8.00 × 105 1.30 x 106 1.42 × 106 1.30 x 106 Proteolytics 1.64 × 106 4-80 × 106 3"32 × 106 4.15 x 106 Lipolytics 5.60 x 106 1.06 × 107 1.40 × 107 1.50 x 107 Methanogens (MPN) 7.00 x 105 1.70 × 106 3-00 × 106 3"30 x 106

Methanogens enumerated by most probable number method (MPN).

Selenomonas These isolates are Gram-negative, non-sporulating rods and produce acetate and lactate as the major fermentation products. In the present work they were found to be cellulolytic. They closely resembled S. sputigena and S. ruminantium. However they gave a positive Voges-Proskauer test, unlike S. sputigena, and were incapable of utilizing mannitol and differed con- siderably from S. ruminantium in the nature of the fermentation products formed.

Lactobacillus These are Gram-positive, non-sporulating rods and produce lactic acid as the major product of fermenta- tion. In the present study they were pectinolytic and were unable to ferment most of the sugars tested, or hydrolyse starch or gelatin. In these characteristics they appeared similar to L. minutus. Another set of isolates resembled L. brevis, mainly in their ability to ferment galactose, mannose, mannitol and raffinose.

Eubacterium The Eubacteria are characteristically Gram-positive, non-sporulating rods. The isolates obtained from both the batch and semi-continuous digesters were pectino- lytic. In addition, in the semicontinuous process they were the major cellulolytic and lipolytic organisms. These isolates were not able to ferment mannitol, rhamnose and inositol but were able to hydrolyse gelatin and to some extent, starch, thus resembling E. rectale and E. aerofaciens.

Two other isolates of this genus were both cellul- olytic but could not be classified under any of the species already described. The data on their fer- mentation properties are given in Table 2. They could not ferment cellobiose, rhamnose, ribose, xylose, inosi- tol and trehalose. Starch and gelatin hydrolysis was partial. The isolates were Voges-Proskauer negative. They did not produce ammonia from arginine or indole from tryptophan. They did not reduce nitrate. These isolates were markedly different from other reported species of Eubacterium in their ability to fer- ment mannitol, sorbitol, raffinose and melibiose. Hence they could be considered as two new species of the genus Eubacterium.

Propionibacterium These are Gram-negative, non-sporulating rods. Pro- pionate is the major fermentation product with traces of lactate and succinate. In the present work, the iso- lates were found to be lipolytic and appeared close to Propionibacterium acidi-propionici.

DISCUSSION

All the physiological groups of.microorganisms increased in number during the first 50 days of diges- tion of the TPW, reflecting the availability of specific substrates for the various groups of the organisms. The initial count of methanogens was of the order of 106/ml which was from the inoculum. In the batch process, the methanogen count decreased, possibly due to the decrease in pH of the slurry during the anaerobic digestion. In the semicontinuous process, the counts of almost all the specific groups of microorganisms increased with increase in the HRT. This could be due to the low dilution rate of the digester slurry at higher HRTs. The numbers of methanogens were almost pro- portional to the HRT, and this seemed to be reflected in the methane content of the biogas. For example, 65% of methane was obtained at 24-32 days HRT but it was only 20-30% at 8 days HRT (Sarada & Joseph, 1993). The overall results of the semicontinuous process suggested that xylanolytics and lipolytics were predominant organisms. In lower numbers were the proteolytics, pectinolytics and cellulolytics (Table 4). The reported predominant microorganisms for other feedstocks under similar conditions were cellulolytics and hemicellulolytics in piggery waste (Hobson & Shaw, 1971), cellulolytics in cattle manure (Malik et al., 1989), lipolytics in piggery waste (Hobson et al., 1974). It is significant to note that pectinolytes were not reported in these papers.

Of the isolates examined from TPW digesters, bac- teria belonging to the genera Bacteroides, Eubacterium, Fusobacterium, Lactobacillus, Propionibacterium and Selenomonas were found in the present study. Clostri- dium, reported to be present in different natural anaerobic ecosystems by Smith and Iannotti (1984), were, significantly, not found in the present study. From sewage digesters, Bacteroides, Clostridium and Bifido-

Microorganisms associated with TP W digestion 265

bacterium were reported as predominant organisms (Bryant, 1976), whereas Streptococcus, Lactobaccilus, Bacteroides, Eubacterium, Peptococcus and Pepto- steptococcus were reported from swine waste (Iannotti et al., 1982). Fusobacterium, Propionibacterium and Selenomonas encountered in the present study were not reported earlier from any digesters, although Fuso- bacteria and Propionibacteria were found in mice in- testinal tracts (Harris et al., 1976). The majority of the isolates from the TPW digesters were Gram-positive, non-spore-forming rods. The predominant micro- organisms in the batch process belonged to Bacter- oides, Propionibacterium and Selenomonas, while in the semicontinuous process they belonged to Eubac- terium, Fusobacterium and Bacteroides. Lactobacilli were present in both the processes.

ACKNOWLEDGEMENT

One of the authors (R.S.) acknowledges the receipt of a Fellowship granted by the University Grants Commis- sion.

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