• , . ,, ELSEVIER 0960-8524(94)00037-9 Bioresource Technology 49 (1994) 261-265 © 1994 Elsevier Science Limited Printed in Great Britain. All rights reserved 0260-8774/94/$7.00 CHARACTERIZATION AND 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. At 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 to 261