Letters in Applied Microbiology 1997, 24, 405–409
Interactive effects of temperature and o-cresol co-disposal on the methanogenic fermentation of refuse Sulisti, E. Senior1 and I.A. Watson-Craik Department of Bioscience and Biotechnology, University of Strathclyde, Glasgow, UK, and 1International Centre for Waste Technology (Africa), University of Natal, Pietermaritzburg, South Africa 1261/96: received 19 August 1996 and accepted 9 October 1996
The influence of temperature on both o-cresol biodegradation and methanogenic refuse decomposition was investigated. Maximum o-cresol attenuation was recorded from 25 to 37°C. Mesophilic and thermophilic sulphate-reducing bacterial (SRB) activity was observed, but thermophilic methanogenesis was not recorded. Maximum methane release and SRB activity was recorded at 25–37°C, and −30°C, respectively. Optimum conditions for acetate utilization were similar to those for methanogenesis, but propionate degradation apparently depended on SRB activity. Propionate degradation was recorded under thermophilic conditions, even in the absence of methanogenesis, although the optimum temperature was 37°C. When SRB were inhibited, at temperatures ¾25°C, no significant propionate catabolism was observed.
S UL IS T I, E. S EN IO R AN D I . A. WA T SO N- C RA IK . 1997.
INTRODUCTION
The increasingly important commercial exploitation of methane from landfill sites (de Rome 1995) requires that the effects on methane release of environmental factors such as pH, temperature, moisture content and the presence of toxic materials should be considered (Barlaz et al. 1990). Temperatures in UK landfills have been reported to range from 25 to 43°C (Senior and Kasali 1990) and will be influenced by factors such as moisture content and flow, initial aerobic microbial activity, refuse composition, depth and density. With increasing emphasis in the UK on landfill sites as ‘flushing bioreactors’ (Robinson 1995), care must be taken that the increased moisture contents now recommended, achieved through either leachate recycle or rain water infiltration, do not result in decreased refuse temperatures. Of the bacterial groups that comprise methanogenic associations, methanogens have been considered the most sensitive to factors such as temperature and inhibitory compounds, although Peck et al. (1986) reported that bacteria which degraded volatile fatty acids (VFA), in particular propionate, were more sensitive to temperature than methanogens, and Watson-Craik and Senior (1989a) noted that in a multi-stage continuous culture system, inoculated with a methanogenic association enriched
from refuse, sulphate-reducing bacteria (SRB) were more sensitive than methanogens to the inhibitory effects of phenol. The degradation rates of xenobiotic molecules such as phenols will also be influenced by temperature. With the retention, in the EU Draft Landfill Directive, of co-disposal (the disposal of hazardous wastes with domestic refuse), it is particularly important to determine the effects of environmental factors such as temperature on both the indigenous refuse decomposition processes and biodegradation of the co-disposed hazardous waste molecule. Although previous studies have examined phenol co-disposal with domestic refuse (Watson-Craik and Senior 1989a, b), the effects of temperature and o-cresol co-disposal have not been addressed, although phenolic waste streams are generally characterized by a range of substituted phenols (Fedorak and Hrudey 1986). o-Cresol is of particular interest because of its reported recalcitrance under anaerobic conditions (Wang et al. 1988), although recent studies have demonstrated the anaerobic degradation in refuse of 4 mmol o-cresol l−1 (Sulisti et al. 1996a). MATERIALS AND METHODS Refuse preparation
Correspondence to : Dr Irene A. Watson-Craik, Department of Bioscience and Biotechnology, University of Strathclyde, 204 George Street, Glasgow G1 1XW, UK (e mail : I.A.
[email protected]). © 1997 The Society for Applied Bacteriology
One-month-old pulverized refuse (Shewalton Landfill site, Cunninghame District Council) was stored in sealed poly-
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ethylene bags at 4°C. It was hand-sorted to remove large particles (−5 mm) of stones, wood, glass, metal and rigid plastic, then homogenized in a domestic blender (Osterizer) for 30 s. Batch cultures
Refuse (25 g) and growth medium (200 ml) (Coutts et al. 1987) containing 4 mmol o-cresol l−1 were decanted into bottles (500 ml), which were then closed with subaseals (Fisher Scientific). Duplicate bottles were incubated at 4, 20, 25, 30, 37, 45 and 55°C. Samples (10 ml) were taken at regular intervals and analysed for pH, o-cresol, sulphate, VFA and methane concentrations. Analytical methods
Table 1 Sulphate concentrations in cultures incubated at
temperatures between 4 and 55°C — ––––––––––––––––––––––––––––––––––––––––––––––––––––– Sulphate concentration (mmol l−1) — –––––––––––––––––––––––––– Incubation temperature (°C) Day 8 Day 22 — ––––––––––––––––––––––––––––––––––––––––––––––––––––– 4 0·9 1·1 20 0·9 1·1 25 0·9 0·8 30 0·1 ³0·1 37 bd bd 45 0·1 ³0·1 55 0·1 ³0·1 — ––––––––––––––––––––––––––––––––––––––––––––––––––––– bd, Below detection limits.
o-Cresol and VFA. Concentrations of o-cresol and VFA were
determined by use of a GC under the same operating conditions as described previously for phenol and leachate VFA, respectively (Watson-Craik and Senior 1989b). Methane and sulphate. The GC method for the assay of headspace methane concentrations and the standard barium chloride turbidometric methods for sulphate assay were used as described by Coutts et al. (1987). RESULTS AND D ISCUSSION
It was apparent that 37°C was the optimum temperature for the methanogenic degradation of both organic refuse components and o-cresol. By 49 d the o-cresol concentration was reduced by 95·3%. No further reduction was observed by 81 d (Fig. 1), when at incubation temperatures of 25 and 30°C
attenuation was 72·1 and 77·3%, respectively. Although ocresol attenuation (35·0–42·7%) was still recorded at 4, 45 and 55°C, this was possibly due to physico-chemical mechanisms such as adsorption (Sulisti et al. 1996b). At 37°C, removal of sulphate by 8 d (Table 1) suggested an active SRB population, and mean sulphate concentrations were ¾0·1 mmol l−1 in the cultures incubated at 30, 45 and 55°C. By 22 d, 21 mmol methane ml−1 were measured in the culture headspace (Fig. 2), confirming the establishment of an actively methanogenic population at 37°C, and supporting previous studies (Sulisti 1994) which established that 4 mmol o-cresol l−1 did not have an inhibitory effect on refuse biodegradation. The low concentrations (¾0·34 mmol l−1) of VFA, except acetate, by 49 d (Fig. 3) reflected rapid turnover of the acids at 37°C, 120
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Fig. 3 Changes in (a) acetate, (b) propionate and (c) butyrate concentrations, in cultures incubated at , 4, ž, 20, , 25, Ž, 30, r, 37, R, 45 and t, 55°C
facilitated by the activities of hydrogen-sink bacteria such as SRB and methanogens. Total methane release was greatest (105–112 mmol ml−1 headspace at 133 d) at incubation temperatures between 25 and 37°C (Fig. 2), although the lag phase increased progressively with temperature decrease. A temperature decrease to 30°C increased net concentrations of propionate (2·47 and 0·81 mmol l−1) and butyrate (0·92 and 0·52 mmol l−1) at 49 and 81 d, respectively (Fig. 3), which could be attributed to accumulation of these acids in the presence of reduced hydrogen-sink bacterial activity, as at this temperature a longer methanogenic lag phase was noted (Fig. 2). The degradation of propionate is particularly sensitive to increased partial pressures of hydrogen (Wiegant et al. 1986). Once a methanogenic population was established, however, total methane production at 30°C (88 d) was comparable to that at 37°C, supporting other studies which have suggested that a period is required to acclimate methanogens to this temperature (Bardulet et al. 1990). A further reduction at 25°C resulted in an extended lag period for methane production and temporary accumulations of acetate and butyrate (Fig. 3a, c). The inhibition of SRB activity (Table 1) possibly reduced early utilization of hydrogen, with concomitant effects on VFA catabolism. Other studies have demonstrated that decreased temperatures resulted in an accumulation of hydrogen that affected a metabolic shift of methanogens from utilizing acetate to hydrogen (Conrad et al. 1989), and this increased the contribution of hydrogen in methane production. In this study, the rate of methanogenesis was greater at 25 than at 30 or 37°C, although the lag phase was longer, suggesting a similar shift may have
been operative. Propionate turnover was also temperature sensitive, since net concentrations were relatively unaltered (2·7–3·5 mmol l−1) throughout the study (Fig. 3b). This ¨ ztu¨rk 1993). supports earlier studies on anaerobic digesters (O It was not clear, however, whether this sensitivity was direct or due to reduced SRB activity : the latter would result in increased hydrogen partial pressures, to which propionate degradation is particularly sensitive. Peck et al. (1986) considered that in anaerobic digesters acetoclastic methanogens, hydrogenotrophic methanogens and/or homoacetogens are able to reduce hydrogen partial pressures sufficiently to promote first butyrate degradation, then, if the hydrogen partial pressure continues to decrease, valerate and hexanoate, then propionate turnover. In the present study, where methanogenesis and acidogenesis were not apparently inhibited at 25°C, methanogens and/or homoacetogens may have played a similar role. Incubation at 20°C both delayed and reduced methane production, to 71 mmol ml−1 at 133 d, and inhibited ocresol degradation and sulphate reduction. The increased accumulation of propionate (4·0 mmol l−1 by 8 d) may have been due to this inhibition of hydrogen-sink activity. Although decreases in net acetate concentrations were similar to those at 25°C (Fig. 3a), total methane production was lower, which suggests that either both acetate production and consumption were reduced, or that hydrogenotrophic methanogenesis was selectively inhibited. In a multi-stage continuous culture system, inoculated with a methanogenic association enriched from refuse, a comparable reduction in methanogenesis was recorded in the last vessel (James et al.
© 1997 The Society for Applied Bacteriology, Letters in Applied Microbiology 24, 405–409
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1996), in which image analyses demonstrated changes in the numbers of acetoclastic Methanosarcina-type and Methanothrix-type cells relative to the total fluorescent population (Howgrave-Graham et al. 1994). Total inhibition of methane production, sulphate reduction and most VFA catabolism was recorded at 4°C where, because of low acid production, initial decreases in pH values were also less pronounced than at higher temperatures (Fig. 4). At 45°C, the presence of low headspace methane concentrations (¾5 mmol ml−1) at 22 d was attributed to methane initially present as dissolved methane in the refuse inoculum, since there was thereafter no evidence of thermophilic methanogenic activity, although the existence and activity of thermophilic methanogens, such as Methanobacterium thermoautotrophicum, is well documented (Stams et al. 1992 : Schmidt and Ahring 1995). Scottish landfills are rarely in the thermophilic range and James et al. (1996) also noted no thermophilic methanogenic activity in an association enriched from the Scottish landfill site sampled in the present study. At 45 and 55°C acetate concentrations were high (−11·0 mmol l−1) (Fig. 3a), which suggested that acetogenesis in the early stages was not significantly inhibited. The presence and activity of SRB (Table 1) presumably facilitated the turnover of acids such as butyrate and propionate (Stams et al. 1992 ; James et al. 1996) until the sulphate was depleted, at which point, in the absence of an active hydrogen-sink population, VFA accumulated (Fig. 3), resulting in reduced leachate pH values (Fig. 4). The results of this study indicate that at temperatures greater than 37°C, although SRB and acidogenic activity may continue in the presence of exogenous sulphate, methane release is progressively inhibited. Although there has, in 7·5 7·0 6·5
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recent years, been increased interest in thermophilic anaerobic digestion of food, paper and agricultural wastes (Schmidt and Ahring 1995), these results suggest that in landfills in temperate climates thermophilic methane production is not likely to be significant, and efforts to promote methanogenesis should focus on maintaining site temperatures within the mesophilic range and on optimizing other environmental conditions, such as moisture content.
ACKNOWLEDGEMENT
This research was funded by the Second Universities Development Project/World Bank XVII Loan to the Indonesian government, whose support is gratefully acknowledged.
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