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- Materials and methods
Two obligately anaerobic bacterial strains were isolated from the contents of a pilot scale, anaerobic digester treating slaughterhouse waste with a high protein and lipid content. The isolates, LIP1 and MW8, were characterized as spore-forming, Gram-positive rods, capable of fermenting glycerol. Isolate LIP1 was also observed to be lipolytic and was able to hydrolyse tallow and olive oil. Both isolates grew optimally at 37 °C and formed either acetate and formate (LIP1), or acetate and butyrate (MW8), as major glycerol fermentation products. Both isolates produced ethanol as the major reduced fermentation end-product. Neither MW8 nor LIP1 had growth and metabolism inhibited by the addition of stearic acid at concentrations normally considered bactericidal. Analysis of the 16S rRNA gene sequences, in conjunction with the phenotypic data, confirmed that the isolates are members of the genus Clostridium (sensu lato), clustering with species of clostridial clusters I (MW8) and XIVa (LIP1).
The wool, meat, dairy and food industries produce large volumes of waste water containing significant amounts of protein and lipid. It has been estimated that 15 200 tonnes of lipid waste are produced per year in New Zealand alone ( Cohen et al. 1994 ). This waste is considered non-recoverable and disposal is both an economic and environmental concern. Problems with current aerobic treatments are compounded by uncertainties regarding the future levels of spray irrigation and landfilling that will be possible, due to the availability of land necessary for these practices ( Cohen et al. 1994 ). Therefore, anaerobic processing of these wastes is seen as a viable alternative technique compared with the conventional aerobic waste treatment processes.
Anaerobic digestion processes require bacterial consortia consisting of three trophic (feeding) groups: (i) hydrolytic fermentative micro-organisms; (ii) syntrophic acetogenic bacteria; and (iii) methanogenic bacteria ( Thiele 1991). The bacterial consortia and therefore treatment capacities and stabilities of high-rate anaerobic waste water systems containing lipid are seriously hampered by biomass encapsulation and the inhibitory effects of long-chain fatty acids ( Rinzema et al. 1994 ). Certainly, data exist which demonstrate the inhibition of pure cultures of anaerobic rumen bacteria by long-chain fatty acids (LCFA) at concentrations between 0·005 and 0·1 g l−1 ( Maczulak et al. 1981 ). However, little information exists on the LCFA inhibition thresholds for pure cultures of hydrolytic fermentative bacteria from anaerobic digesters. It has been established that a concentration of 1 g l−1 LCFA in anaerobic digester sludge is sufficient to cause severe inhibition of the digestion process ( Hanaki et al. 1981 ). This inhibitory effect is compounded further by the presence of high levels of ammonia-nitrogen (NH3-N) in high protein content slaughterhouse waste waters ( Heinrichs et al. 1990 ). It has been established, as well, that the initial and severe concentration thresholds for NH3-N toxicity are 40–80 mg l−1 and 200 mg l−1, respectively ( Heinrichs et al. 1990 ). While data exist on the anaerobic treatment of waste waters containing diluted liquid neutral lipids ( Angelidaki et al. 1990 ; Velioglu et al. 1992 ), there is a lack of both operational and microbiological data on the anaerobic digestion of slaughterhouse waste waters containing high levels of solid neutral lipids.
The present paper reports the isolation and characterization of glycerol-fermenting bacteria from a pilot scale anaerobic digester, treating high lipid- and protein-content waste water from a slaughterhouse.
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- Materials and methods
Few data exist describing the bacteria present in anaerobic digesters treating high protein- and lipid-containing wastes. While it is well established that anaerobic digestion is essentially a microbiological process, conflicting views exist with respect to the characterization of the bacteria associated with the degradation of the organic material (carbohydrates, proteins and lipids) in anaerobic digesters ( Sahm 1984; Van Andel & Breure 1984; Thiele et al. 1988 ; Lowe et al. 1993 ).
This paper is the first published report of the isolation and characterization of a mesophilic lipolytic anaerobic bacterium (isolate LIP1) from an anaerobic digester ecosystem. It also presents the first data on the isolation and characterization of glycerol-fermenting bacteria from an anaerobic digester treating high protein- and lipid-content slaughterhouse waste.
Based on the phenotypic and phylogenetic data, isolate MW8 is almost identical to Clostridium butyricum, a member of cluster I in the clostridial taxonomic group ( Collins et al. 1994 ). Interestingly, examination of glycerol dissimilation by four reference strains of Cl. butyricum showed that 1,3 propanediol, acetate and butyrate were the major fermentation products and only trace amounts (1–2 mmol l−1) of ethanol were formed ( Biebl et al. 1992 ). These data contrast with the results of this study, where it was established that ethanol, rather than propanediol is the major reduced end-product formed late in the fermentation process. However, it is also known that some strains of Cl. butyricum do produce ethanol as a fermentation end-product ( Cato et al. 1986 ), and it has been established that the production of ethanol is a common feature of many saccharolytic, mesophilic, butyrate-producing clostridia (such as Cl. butyricum) in late stages of the fermentation process ( Jones & Wood 1989). The isolate MW8 was observed to be saccharolytic, but was unable to hydrolyse lipids. However, isolate MW8 was able to use glycerol as the sole carbon substrate for growth. While being related phylogenetically to strains of Cl. butyricum ( Fig. 3), including a pectinolytic strain (DSM 2478) isolated from lake sediment ( Schink & Zeikus 1982), isolate MW8 exhibited a number of phenotypic differences, including the ability to ferment rhamnose and sorbitol and the inability to utilize mannitol or hydrolyse esculin. Therefore, isolate MW8 probably represents a new strain of Cl. butyricum, different to those previously described.
On the basis of 16S rRNA gene sequence comparisons, isolate LIP1 was shown to be phylogenetically related (98·3%) to the type strain of Cl. clostridiiforme (DSM 933T). However, Cl. clostridiiforme ferments lactose and is not capable of hydrolysing gelatin or lipids ( Kaneuchi et al. 1976 ), whereas isolate LIP1 cannot ferment lactose but exhibits gelatinolytic and lipolytic activities. Based on these data, isolate LIP1 probably represents a lipolytic strain of a new species belonging to clostridial cluster XIVa ( Collins et al. 1994 ).
The taxonomic organization of the genus Clostridium is undergoing significant revisions ( Collins et al. 1994 ) and the majority of species currently classified within this genus will ultimately be separated and reclassified within newly described, distinct genera. The isolate LIP1 described in this paper was observed to cluster with species of cluster XIVa. This phenetically heterogeneous group comprises described species currently classified as Clostridium, as well as several other recognized genera ( Collins et al. 1994 ). It is clear that all Clostridium species within cluster XIVa will be reclassified in the future as species in newly recognized genera. Thus, it is prudent, at this time, to recognize the isolate LIP1 of the current study simply as ‘Clostridium sp. (strain LIP1)’, rather than attempting to describe a new species epithet.
As there was comparatively little carbohydrate present in the anaerobic digester feed used in the current study, it is conceivable that isolate LIP1 may be a member of the lipid utilizing/glycerol-fermenting niche, either as an opportunist or as an indigenous member. Clostridium butyricum (strain MW8) exhibited a rapid growth rate on glycerol and may therefore be able to compete successfully for this growth substrate in the anaerobic digester ecosystem. Interestingly, a strain of Cl. butyricum has been isolated from the anaerobic digester ecosystem in a previous study ( Xiuzhu et al. 1991 ), although that strain was isolated and studied based on its ability to degrade galactomannan, rather than the ability to ferment glycerol.
The results from the LCFA toxicity studies demonstrated that isolates MW8 and LIP1 were capable of active growth and metabolism at LCFA concentrations 20–460 times higher than the levels reported to be toxic for other anaerobic bacteria ( Maczulak et al. 1981 ). The lipolytic isolate, LIP1, was observed to undergo a physiological change, resulting in a different glycerol fermentation end-product profile, with decreased VFA production being accompanied by a concomitant equimolar increase in the formation of reduced solventogenic end-products. This resulted in an alteration in the ratio of oxidized and reduced fermentation products in favour of the latter. One explanation for this may be related to the inhibition of hydrogenase activity and the regeneration of reduced co-factors, which is known to lead to the carbon flow of species within the genus Clostridium being directed to the formation of more reduced end-products, such as ethanol and butanol ( Jones & Wood 1989). Whether these effects are responsible for the results observed for the clostridial isolate LIP1 were not tested. The fact remains that both isolates were metabolically and physiologically active at LCFA concentrations that were well in excess of established toxic thresholds and which could be considered to represent extreme growth conditions. It is conceivable that the results obtained in vitro may reflect the initial extremophilic conditions present in the anaerobic digester which served as a source of the two isolates. Indeed, the digester had an NH3-N concentration (3 g l−1) well in excess of established severe toxicity threshold values ( Heinrichs et al. 1990 ) and had a feed containing almost 50% lipid within the solids phase. Therefore, it is not surprising that we have been able to isolate LCFA toxicity-resistant anaerobic bacteria, which, based on the results of the LCFA toxicity studies, could be considered to be included in the diverse group of bacteria termed ‘extremophiles’ ( Lowe et al. 1993 ).