- Email: [email protected]
Enhanced hydrolysis of carbohydrates in primary sludge under biosulfidogenic conditions
ARTICLE IN PRESS WAT E R R E S E A R C H
40 (2006) 1577 – 1582
Available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/watres
Enhanced hydrolysis of carbohydrates in primary sludge under biosulfidogenic conditions K.J. Whittington-Jonesa,, J.B. Molwantwab, P.D. Roseb a
Department of Biochemistry, Microbiology & Biotechnology, Rhodes University, Grahamstown, South Africa Environmental Biotechnology Research Unit, Rhodes University, Grahamstown, South Africa
b
art i cle info
A B S T R A C T
Article history:
The potential for using readily available and cost-effective complex carbon sources, such as
Received 26 August 2005
primary sludge (PS), for the bioremediation of sulfate-rich effluent streams, including acid
Received in revised form
mine drainage, has been constrained by the slow rate of solubilization and low yield of
1 February 2006
soluble products. Disposal of PS also remains a global problem. Recent studies of a patented
Accepted 17 February 2006
recycling sludge bed reactor have shown that the solubilization of PS is enhanced under
Available online 17 April 2006
biosulfidogenic conditions. The current study investigated the enhanced solubilization of
Keywords:
the carbohydrate fraction of PS under these conditions, using selective metabolic
Primary sludge
inhibitors. The mean maximum rate of reducing sugar production was significantly higher
Recycling sludge bed reactor
under sulfidogenic (167 mg L1 h1) than methanogenic (51 mg L1 h1) conditions and the
Enhanced hydrolysis
utlization of volatile fatty acids under sulfidogenic conditions was rapid. Analysis of VFA
Sulfate reduction
profiles indicated preferential utilization of longer chain acids under sulfidogenic
Carbohydrate
conditions and of acetate in the methanogenic systems and that the acetogenic step was
Rhodes BioSURE processs
unlikely to be rate-limiting in the solubilization of complex carbon. & 2006 Elsevier Ltd. All rights reserved.
1.
Introduction
Biological treatment processes are considered to offer a number of significant advantages over traditional physicochemical methods for the remediation of industrial and domestic wastewaters. These advantages include the potential for complete mineralization of contaminants and the reduced operational costs associated with purchase of chemicals and disposal of large volumes of contaminated sludge. Biological nutrient removal (BNR) is a particularly successful application of these systems (Brinch et al., 1994), and the use of biological systems for the desalination of sulfate-rich wastewaters, including acid mine drainage (AMD), has also received much attention (Maree and Hill, 1989; Lens et al., 1995; Riekkola-Vanhanen and Mustikkama¨ki, 1997).
One of the factors constraining development of applications in this area, particularly for large effluent volumes, is the availability of a suitable carbon source and electron donor. Simple electron donors, including methanol and ethanol, have been used successfully to drive the biological treatment of a range of industrial effluents (Maree et al., 1996; Fauville et al., 2004). However, these compounds are relatively expensive and are therefore not suitable for use in developing countries. A wide variety of relatively inexpensive agricultural and domestic wastes have been investigated as possible alternative sources of soluble carbon. These have included animal waste slurries (Ueki et al., 1988), waste-grown microalgal biomass (Boshoff et al., 1996), and primary sludge (PS) (Molipane, 1999; Hatziconstantinou and Efstathiou, 2003). The use of these complex organic substrates for biological sulfate reduction has met with limited success, primarily due to the limited accessibility of the carbon.
Corresponding author. Current address: Department of Environmental Science, Rhodes University, P.O. Box 94, Grahamstown 6140, South Africa. Tel.: +27 46 603 7006; fax: +27 46 622 9319. E-mail address: [email protected] (K.J. Whittington-Jones). 0043-1354/$ - see front matter & 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2006.02.017
ARTICLE IN PRESS 1578
WAT E R R E S E A R C H
40 (2006) 1577– 1582
Although a fraction of the total carbon in these materials may be soluble and therefore readily available to heterotrophic microbial populations, the largest proportion is trapped within complex organic molecules such as lignin and cellulose. These macromolecules must be subjected to digestion before soluble carbon can be accessed. Until recently, the digestion of complex organic matter under biosulfidogenic conditions has been largely ignored, although research has shown that the mineralization of lignocellulosic solid waste was improved significantly in the presence of biological sulfate reduction (Kim et al., 1997; Pareek et al., 1998; Molwantwa et al., 2004). Studies into the mechanism of enhanced hydrolysis of complex carbon under biosulfidogenic conditions indicated that fracturing of larger sludge flocs was facilitated in the presence of sulfide and it was proposed that this fracturing process was central to increased yields of soluble product observed (Whittington-Jones, 2000; Whittington-Jones et al., 2002). This understanding was used to develop the Recycling Sludge Bed Reactor (RSBR), a key process component central to the Rhodes BioSUREs process for the bioremediation of AMD, and tests on both laboratory- and pilot-scale systems indicated yields in excess of 50%. Preliminary studies by Molwantwa et al. (2004) provided evidence that both the rate and extent of solubilization of PS was enhanced significantly under sulfidogenic conditions. Results of experiments incorporating toluene as a selective inhibitor of sugar uptake by bacteria (Boschker et al., 1995) suggested that the observed enhanced hydrolysis of PS was at least partially due to solubilization of the carbohydrate fraction. These preliminary studies were based on changes in the concentration of particulate and soluble chemical oxygen demand (COD) and the purpose of the current study was to confirm the observations by following changes in the concentrations of reducing sugars and volatile fatty acids (VFAs) profiles.
2.
Methods and materials
2.1.
Inhibitor studies
The only source of reducing sugars in the current system was via the hydrolysis of PS but in order to determine the rate at which these products are produced, and therefore obtain a more accurate estimate of the rate of hydrolysis, further processing of the products had to be inhibited. Boschker et al. (1995) reported that the inhibition of sugar uptake by bacteria could be achieved using toluene (3% vol/vol), and that this does not affect extracellular hydrolysis of polysaccharides in the sample. Boschker et al. (1995) reported a study where initial decomposition rates of naturally occurring polysaccharides could be measured. They applied a method of the selective inhibition of microbial carbohydrate uptake using toluene, without affecting the extracellular hydrolysis of the polysaccharides in the samples under study. The hydrolysis products would then accumulate in the reactors, and these were monitored over time. The experimental setup was the same as reported by Molwantwa et al. (2004), with all experiments being conducted in triplicate. The two controls
(methanogenic and sulfidogenic) and the two experiments to which toluene had been added (methanogenic+toluene and sulfidogenic+toluene) were set up in triplicate in 500 mL flasks. Each flask was innoculated (10% by volume) with populations of SRB and methane-producing bacteria (MPB), respectively, obtained from 5 L cell culture units that had been operating for 3 months prior to the current experimental program. Sieved (2 mm mesh) PS obtained from Grahamstown Municipal Works was used as a carbon source and was diluted with distilled water to a final COD of 2000 mg L1. The initial sulfate concentration within the sulfidogenic flasks was increased to 2000 mg L1 using analytical-grade Na2SO4 (Merck), thus giving a COD:SO4 ratio of 1:1. The initial pH of all the flasks was adjusted to 7 using 10% Sodium hydroxide (NaOH). The neck of each flask was sealed with a rubber stopper but gas was allowed to escape through a U-tube filled with a zinc acetate solution. In order to prevent ingress of oxygen into the flasks during sampling, samples were forced out of the flasks under positive pressure using nitrogen gas. A total of 400 mL of fresh sieved (2 mm mesh) PS was then added to each flask as the sole carbon source. The flasks were placed on a Labcon desktop shaker (100 rpm) and were allowed to acclimate for 2 days in a CE room at 25 1C before the addition of 15 mL of toluene (to give a final concentration of 3% vol/vol) to the six experimental flasks. The remaining six flasks served as the sulfidogenic and methanogenic controls and had no toluene added to them. Samples were collected (described earlier) at hourly intervals for 8 h (as the inhibition of toluene on the bacteria only lasts for 7–8 h). The first sample was taken at t ¼ 0 after which toluene was immediately added to the experimental flasks. The breakdown of reducing sugars results in the production of VFAs, which are then available to be utilized by SRB. In order to investigate the effect of SRB on the uptake and distribution of VFA and the hydrolysis process, their activity was selectively inhibited using molybdate (Lens et al., 1995). In the presence of molybdate, SRB are inhibited and thus the methanogenic microbes are able to grow. Therefore, those treatments referred to as ‘‘methanogenic’’ in the current study are flasks to which molybdate was added.
2.2.
Analytical procedures
All samples were analyzed in triplicate using the procedures outlined below. Prior to analysis, all samples were acidified with 32% HCl to pH 2 and agitated for 1 min in order to eliminate any sulfide interference with the assay (Whittington-Jones, 2000). A Mercks spectroquant test kit was used to determine the COD and sulfate concentrations were determined using an HPLC anion method applying a model 510 Waters HPLC and model 430 Waters conductivity detector fitted with a Hamilton PRPX 100 150 4.1 mm column. A 10-fold dilution of the sample was prepared using milli-Q water and then filtered through a 0.45 mm nylon filter before passing it through two Waters sep-paks light C18 cartridges to remove the organic contaminants. The samples were then injected into the HPLC and run at 2 mL min1. A 100 mg L1 Na2SO4 standard was prepared in order to determine the retention time of the
ARTICLE IN PRESS WAT E R R E S E A R C H
sulfate peak as well as to standardize the accuracy of the instrument. Reducing sugars were assayed using a modified Simogy– Nelson method (Saeman et al., 1945). Absorbance was determined using a Beckman DUR UV/vis spectrophotometer at 520 nm and the reducing sugar concentrations calculated from a glucose standard curve. The concentration of total VFA present was determined using a modified steam distillation and titration method (American Public Health Association (APHA), 1989). Differentiation of the four component VFAs in each sample was determined by gas chromatography using a 30 m 0.320 mm, 0.5 mm HP-Innowax column. VFA standards (Sigma) were used to determine the elution times of the fourcomponent VFAs measured in each sample. The results of the gas chromatography studies are reported as relative peak area, showing the change in proportion of each VFA through the course of the experiment. Statistica Version 6.0 was used for statistical analysis of the recorded data and to determine the standard deviations of the triplicate readings.
3.
Results and discussion
Comparison of the accumulation of reducing sugars in a control system with that in a system where the uptake of reducing sugars is inhibited, should enable accurate quantification of the rate of reducing sugar production, and therefore, a rate of hydrolysis of the parent complex carbohydrates. The rates of hydrolysis under methanogenic and sulfidogenic conditions could then be compared. Because of active biological sulfate reduction and subsequent production of bicarbonate ions, the pH in the sulfidogenic system increased from pH 7 to 7.6 while that of the methanogenic systems showed a decrease from pH 7 to 6.5 (data not shown). This pH data reflects the ideal pH for both the sulfidogeneic and methanogenic systems, respectively (Widdel, 1988). The addition of toluene to the experimental flasks had a significant impact on the uptake of reducing sugars under both methanogenic and sulfidogenic conditions (ANOVA, po0:001, n ¼ 3) (Fig. 1). A decrease in reducing sugars in both
control systems suggested that the gradual increase in soluble COD (CODfiltered) observed in control flasks by Molwantwa et al. (2004) was not due to the accumulation of reducing sugars as they did not accumulate over the experimental period in the current study. The controls showed a total decrease in the reducing sugars of 32% and 58% in the methanogenic and the sulfidogenic systems, respectively, relative to starting concentrations (Fig. 1). However, the experimental systems showed a significant increase in reducing sugars over the first 6 h, after which there was a decrease for the last 2 h, presumably due to utilization by bacterial populations upon the release of inhibition. The difference between concentrations in the inhibited and control systems indicated the rate of utilization of the sugars and was significantly higher under sulfidogenic than methanogenic conditions (ANOVA, po0:05, n ¼ 3). However, under both conditions, a decline in the concentration of reducing sugars in the control systems over the 8-h experimental period indicated that the rate of utilization was greater than production and suggested that the acidogenic step of carbohydrate degradation was not ratelimiting. The overall maximum percentage increase in reducing sugar concentration over the inhibition period was 41% and 65% for the methanogenic ðt ¼ 5Þ and sulfidogenic ðt ¼ 6Þ systems, respectively. The maximum rate of production of reducing sugars in the methanogenic system (51 mg L1 h1) and was found to be 60% less than that of the sulfidogenic system (167 mg L1 h1), supporting previous results (Molwantwa et al., 2004) that hydrolysis of complex carbohydrates was approximately three times faster under sulfidogenic than methanogenic conditions. The concentration of VFAs decreased in both the sulfidogenic and methanogenic systems (Fig. 2) indicating that the rate of utilization of these compounds exceeded production. After the 2-day acclimation period, the sulfidogenic systems showed a significantly higher removal than the methanogenic systems (ANOVA, po0:05, n ¼ 12). Although addition of toluene should not have had any direct affect on the concentration of VFAs in either system, indirect impacts were expected, as inhibition of soluble sugar uptake would
methanogenic+toluene sulfidogenic+toluene
methanogenic sulfidogenic
1800 1600 1400 1200 1000 800 600 400 200 0
VFA (mg/L)
Reducing Sugars (mg/L)
methanogenic sulfidogenic
1
2
3
4 5 Time (hrs)
6
7
8
Fig. 1 – Reducing sugar concentrations of the control and experimental flasks under methanogenic and sulfidogenic conditions (n ¼ 3). The highest standard deviation was 46 and as such were not easily visible on the figure.
methanogenic+toluene sulfidogenic+toluene
2100 2000 1900 1800 1700 1600 1500 1400 1300 0
0
1579
4 0 (200 6) 157 7 – 158 2
1
2
3 Time (hrs)
4
5
6
Fig. 2 – Total VFA concentrations in the methanogenic and sulfidogenic control and experimental systems, showing the effect of inhibition of reducing sugar uptake by toluene (n ¼ 3). All methanogenic flasks contained molybdate to inhibit biological sulfate reduction.
ARTICLE IN PRESS 40 (2006) 1577– 1582
Area ratio
Acetate
Propionate
Butyrate
Valerate
9 8 7 6 5 4 3 2 1 0
(a)
Area ratio
have prevented soluble carbohydrates from being converted to VFAs during the acidogenic step. Upon inhibition, the sulfidogenic system exhibited a 44% decrease in VFA concentration over a 6-h period, while the decrease in the methanogenic treatment was only 5%. The general decrease in total VFA concentration in all systems indicated that utilization of this organic fraction was rapid although the higher percentage and rate of removal under sulfidogenic conditions was attributed to the fact that SRB have been shown to utilize a wider range of VFA than MPB (Fauville et al., 2004). Conversely, the relatively slower utilization of VFAs by methanogenic populations reflects their limited ability to consume longer chain fatty acids. Although the above data showed that the rate of VFA utilization was more rapid under sulfidogenic than methanogenic conditions, profiles of the individual VFAs were required to confirm the expected differential utilization and accumulation of the various acids under different electron acceptor conditions. The distribution of VFA under methanogenic conditions, with and without inhibition of sugar uptake, was similar (Figs. 3a and b). In both cases, acetate was used preferentially, resulting in a period of accumulation of the other acids. After 2 h, once the acetate had been depleted or reached a low concentration, other VFAs were used. The pattern of VFA utilization was reversed in the sulfidogenic system (Figs. 3c and d). Acetate accumulated initially, particularly in the absence of the toluene inhibitor, with preferential utilization of the longer-chain fatty acids. Once these had decreased below a certain level, acetate was used. These results confirm previously reported results that MPB utilize acetate preferentially over other VFA, while SRB utilize others VFA over acetate (Smith and Klug, 1981; Qabiti et al., 1990). This observation supports the hypothesis that acetogenesis is not rate-limiting in the solubilization of PS, and that the rapid utilization of soluble products under sulfidogenic conditions is, at least partially, due to consumption of a range of VFAs. The results of the current study support the findings of enhanced hydrolysis of carbohydrates in sufidogenic environments (Khan and Trottier, 1978; Kim et al., 1997; Whittington-Jones, 2000). Furthermore, although there are differences in the production and consumption of VFAs between sulfidogenic and methanogenic systems, the most significant difference is at the level of hydrolysis, i.e. the production of reducing sugars. The exact mechanism by which either the SRB themselves or products of their activities affect enhanced hydrolysis is not yet clear. Whittington-Jones (2000) proposed that the sulfide acted indirectly via disruption of sludge flocs, thereby facilitating contact between hydrolytic enzymes and their substrates while Whiteley et al. (2004) suggest that sulpfide directly enhances the activity of the hydrolytic enzymes themselves. The descriptive model developed in this study is centered on the ability of SRB to utilize a wider spectrum of these VFAs, which was observed in this study, thus no accumulation of VFA was observed in the sulfidogenic system. As a result, the occurrence of a negative feedback inhibition is avoided and the rate of hydrolysis is not affected. A diagrammatic representation of this model is shown on Fig. 4.
0
2
3
4
5
6
1
2
3
4
5
6
0
2
3
4
5
6
1
2
3 4 Time (hrs)
5
6
9 8 7 6 5 4 3 2 1 0
(b)
Area ratio
WAT E R R E S E A R C H
8 7 6 5 4 3 2 1 0
(c)
Area ratio
1580
8 7 6 5 4 3 2 1 0
(d)
Fig. 3 – The profile of volatile fatty acids in methanogenic and sulfidogenic flask experiments. (a) methanogenic; (b) methanogenic+toluene; (c) sulfidogenic; (d) sulfidogenic+toluene. (n ¼ 3). All methanogenic flasks contained sodium molybdate to inhibit biological sufate reduction.
4.
Conclusion
Disposal of PS is a global environmental problem and conventional digestion technologies provide limited relief in terms of reduction of solid material requiring further disposal. However, this material is a potentially valuable and inexpensive source of energy provided that yields of soluble products can be improved and one application of
ARTICLE IN PRESS WAT E R R E S E A R C H
1581
4 0 (200 6) 157 7 – 158 2
COMPLEX ORGANIC COMPOUNDS (carbohydrates, proteins, lipids)
Negative feedback
Low rate of VFA utilization under methanogenic conditions leads to VFA accumulation and decrease in pH to below optimum for hydrolysis
HYDROLYSIS
SIMPLE ORGANIC COMPOUNDS (SUGARS, AMINO ACIDS, PEPTIDES)
High rate of VFA utilization in the presence of sulfate leads to enhanced hydrolysis by preventing pH decrease
ACIDOGENESIS
LONG-CHAIN FATTY ACIDS (propionate, butyrate etc.)
ACETOGENESIS
ACETATE
METHANOGENESIS
SUFATE REDUCTION
H2S, CO2
H2, CO2, CH4 Fig. 4 – Flow diagram summarizing the proposed mechanism of enhanced hydrolysis under sulfidogenic system showing the possible negative feedback inhibition.
these would be for sustainable bioremediation of AMD. Although the mechanism underlying enhanced hydrolysis of complex organic matter, including PS, is not understood, the above research indicates that digestion of this material in the presence of biological sulfate reduction offers significant advantages over conventional methanogenic conversion. Possible mechanisms include direct hydrolysis of ‘‘recalcitrant’’ compounds by SRB or the creation of a favourable environment for either chemical or enzymatic degradation or possibly also redox poising of the reagents, which could account for energy differences. These mechanisms therefore, should be the subject of further investigation.
Acknowledgments The authors acknowledge the funding of the Water Research Commission, Pretoria, South Africa for funding the research. R E F E R E N C E S
American Public Health Association (APHA), 1989. Standard Methods for the Examination of Water and Wastewater, 17th ed. American Public Health Association, Washington, DC. Boschker, H.T., Bertilsson, S.A., Dekkers, E.M.J., Cappenberg, T.E., 1995. An inhibitor-based method to measure initial decomposition of naturally occurring polysaccharides in sediments. Appl. Environ. Microbiol. 61, 2186–2192.
Boshoff, G., Duncan, J., Rose, P.D., 1996. An algal–bacterial intergrated ponding system for the treatment of mine drainage waters. Abstract J. Appl. Phycol. 8, 442. Brinch, P.P., Rindel, K., Kalb, K., 1994. Upgrading to nutrient removal by means of internal carbon from sludge hydrolysis. Water Sci. Technol. 29, 31–40. Fauville, A., Mayer, B., Frommichen, R., Friese, K., Veizer, J., 2004. Chemical and isotopic evidence for accelerated bacterial sulfate reduction in acid mining lakes of organic carbon: laboratory batch experiments. Chem. Geol. 204 (3/4), 325–345. Hatziconstantinou, G.J., Efstathiou, H., 2003. Sludge dewatering and disposal practices from small activated sludge wastewater treatment plants. J. Environ. Sci. Health, Part A—Toxic/ Harzardous Substances Environ. Eng. 38 (10), 2401–2413. Khan, A.W., Trottier, T.M., 1978. Effect of sulfur-containing compounds on anaerobic degradation of cellulose to methane by mixed cultures obtained from sewage sludge. Appl. Environ. Microbiol. 35, 1027–1034. Kim, S.K., Matsui, S., Pareek, S., Shimizu, Y., Matsude, T., 1997. Biodegradation of recalcitrant organic matter under sulphate reducing and methanogenic conditions in the landfill column reactors. Water Sci. Technol. 36, 91–98. Lens, P.N., De Poorter, M.P., Cronenberg, C.C., Verstraete, W.H., 1995. Sulphate reducing and methane producing bacteria in aerobic wastewater treatment systems. Water Res. 29, 871–880. Maree, J.P., Hill, E., 1989. Biological removal of sulphate from industrial effluents and concomitant production of sulphur. Water Sci. Technol. 21, 265–276. Maree, J.P., van Tonder, G.J., Millard, P., 1996. Underground neutralization of mine water with limestone. Report to the Water Research Commission, CSIR. WRC Report No. 609/1/96.
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Molipane, N.P. 1999. Biological sulphate reduction utilising hydrolysis of a complex carbon source. M.Sc. Thesis, Rhodes University, Grahamstown, South Africa. Molwantwa, J.B., Whittington-Jones, K.J., Rose, P.D., 2004. An investigation into the mechanism underlying enhanced hydrolysis of complex carbon in a biosulphidogenic recycling sludge bed reactor (RSBR). Water SA 30, 155–159. Pareek, S., Kim, S.K., Matsui, S., Shimizu, Y., 1998. Hydrolysis of (ligno) cellulosic materials under sulfidogenic and methanogenic conditions. Water Sci. Technol. 38, 193–200. Qabiti, A.J., Bories, A., Garcia, J.L., 1990. Effects of sulfate on lactate and C2-, C3- volatile fatty acid anaerobic degradation by a mixed microbial culture. Antonie van Leeuwenhoek 58, 241–248. Riekkola-Vanhanen, M., Mustikkama¨ki, U.P., 1997. In situ treatment of acid mine drainage by sulphate reducing bacteria in an open pit mine. In: Proceedings of Biomine International Conference, Sydney, Australia. E-ROM3.1-E-ROM3.8. Saeman, J.F., Buhl, J.L., Harris, E.E., 1945. Quantitative saccharification of wood and cellulose. Ind. Eng. Chem. Anal. 17, 35–37.
Smith, R.L., Klug, M.J., 1981. Reduction of sulfur compounds in the sediments of a eutrophic lake basin. Appl. Environ. Microbiol. 41, 1230–1237. Ueki, K., Kotaka, K., Itoh, K., Ueki, A., 1988. Potential availability of anaerobic treatment with digester slurry of animal waste for the reclamation of acid mine water containing sulphate and heavy metals. J. Ferment. Technol. 66, 43–50. Whiteley, C., Pletschke, B., Rose, P., Tshivhunge, S., Watson, S., Whittington-Jones, K., 2004. Activation of proteases in an anaerobic sulphidogenic bioreactor. Biotechnol. Lett. 26, 55–59. Whittington-Jones, K.J., 2000. Sulphide-enhanced hydrolysis of primary sewage sludge: implications for the bioremediation of sulphate-enriched wastewaters. Ph.D. Thesis, Rhodes University, Grahamstown, South Africa. Whittington-Jones, K.J., Rose, P.D., Corbett, C.J., 2002. The Rhodes ‘BioSURE’ Process, Part 2: Enhanced Hydrolysis of Organic Carbon Substrates—Development of the Reciprocating Sludge Bed Reactor. Water Research Commission, South Africa.
40 (2006) 1577 – 1582
Available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/watres
Enhanced hydrolysis of carbohydrates in primary sludge under biosulfidogenic conditions K.J. Whittington-Jonesa,, J.B. Molwantwab, P.D. Roseb a
Department of Biochemistry, Microbiology & Biotechnology, Rhodes University, Grahamstown, South Africa Environmental Biotechnology Research Unit, Rhodes University, Grahamstown, South Africa
b
art i cle info
A B S T R A C T
Article history:
The potential for using readily available and cost-effective complex carbon sources, such as
Received 26 August 2005
primary sludge (PS), for the bioremediation of sulfate-rich effluent streams, including acid
Received in revised form
mine drainage, has been constrained by the slow rate of solubilization and low yield of
1 February 2006
soluble products. Disposal of PS also remains a global problem. Recent studies of a patented
Accepted 17 February 2006
recycling sludge bed reactor have shown that the solubilization of PS is enhanced under
Available online 17 April 2006
biosulfidogenic conditions. The current study investigated the enhanced solubilization of
Keywords:
the carbohydrate fraction of PS under these conditions, using selective metabolic
Primary sludge
inhibitors. The mean maximum rate of reducing sugar production was significantly higher
Recycling sludge bed reactor
under sulfidogenic (167 mg L1 h1) than methanogenic (51 mg L1 h1) conditions and the
Enhanced hydrolysis
utlization of volatile fatty acids under sulfidogenic conditions was rapid. Analysis of VFA
Sulfate reduction
profiles indicated preferential utilization of longer chain acids under sulfidogenic
Carbohydrate
conditions and of acetate in the methanogenic systems and that the acetogenic step was
Rhodes BioSURE processs
unlikely to be rate-limiting in the solubilization of complex carbon. & 2006 Elsevier Ltd. All rights reserved.
1.
Introduction
Biological treatment processes are considered to offer a number of significant advantages over traditional physicochemical methods for the remediation of industrial and domestic wastewaters. These advantages include the potential for complete mineralization of contaminants and the reduced operational costs associated with purchase of chemicals and disposal of large volumes of contaminated sludge. Biological nutrient removal (BNR) is a particularly successful application of these systems (Brinch et al., 1994), and the use of biological systems for the desalination of sulfate-rich wastewaters, including acid mine drainage (AMD), has also received much attention (Maree and Hill, 1989; Lens et al., 1995; Riekkola-Vanhanen and Mustikkama¨ki, 1997).
One of the factors constraining development of applications in this area, particularly for large effluent volumes, is the availability of a suitable carbon source and electron donor. Simple electron donors, including methanol and ethanol, have been used successfully to drive the biological treatment of a range of industrial effluents (Maree et al., 1996; Fauville et al., 2004). However, these compounds are relatively expensive and are therefore not suitable for use in developing countries. A wide variety of relatively inexpensive agricultural and domestic wastes have been investigated as possible alternative sources of soluble carbon. These have included animal waste slurries (Ueki et al., 1988), waste-grown microalgal biomass (Boshoff et al., 1996), and primary sludge (PS) (Molipane, 1999; Hatziconstantinou and Efstathiou, 2003). The use of these complex organic substrates for biological sulfate reduction has met with limited success, primarily due to the limited accessibility of the carbon.
Corresponding author. Current address: Department of Environmental Science, Rhodes University, P.O. Box 94, Grahamstown 6140, South Africa. Tel.: +27 46 603 7006; fax: +27 46 622 9319. E-mail address: [email protected] (K.J. Whittington-Jones). 0043-1354/$ - see front matter & 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2006.02.017
ARTICLE IN PRESS 1578
WAT E R R E S E A R C H
40 (2006) 1577– 1582
Although a fraction of the total carbon in these materials may be soluble and therefore readily available to heterotrophic microbial populations, the largest proportion is trapped within complex organic molecules such as lignin and cellulose. These macromolecules must be subjected to digestion before soluble carbon can be accessed. Until recently, the digestion of complex organic matter under biosulfidogenic conditions has been largely ignored, although research has shown that the mineralization of lignocellulosic solid waste was improved significantly in the presence of biological sulfate reduction (Kim et al., 1997; Pareek et al., 1998; Molwantwa et al., 2004). Studies into the mechanism of enhanced hydrolysis of complex carbon under biosulfidogenic conditions indicated that fracturing of larger sludge flocs was facilitated in the presence of sulfide and it was proposed that this fracturing process was central to increased yields of soluble product observed (Whittington-Jones, 2000; Whittington-Jones et al., 2002). This understanding was used to develop the Recycling Sludge Bed Reactor (RSBR), a key process component central to the Rhodes BioSUREs process for the bioremediation of AMD, and tests on both laboratory- and pilot-scale systems indicated yields in excess of 50%. Preliminary studies by Molwantwa et al. (2004) provided evidence that both the rate and extent of solubilization of PS was enhanced significantly under sulfidogenic conditions. Results of experiments incorporating toluene as a selective inhibitor of sugar uptake by bacteria (Boschker et al., 1995) suggested that the observed enhanced hydrolysis of PS was at least partially due to solubilization of the carbohydrate fraction. These preliminary studies were based on changes in the concentration of particulate and soluble chemical oxygen demand (COD) and the purpose of the current study was to confirm the observations by following changes in the concentrations of reducing sugars and volatile fatty acids (VFAs) profiles.
2.
Methods and materials
2.1.
Inhibitor studies
The only source of reducing sugars in the current system was via the hydrolysis of PS but in order to determine the rate at which these products are produced, and therefore obtain a more accurate estimate of the rate of hydrolysis, further processing of the products had to be inhibited. Boschker et al. (1995) reported that the inhibition of sugar uptake by bacteria could be achieved using toluene (3% vol/vol), and that this does not affect extracellular hydrolysis of polysaccharides in the sample. Boschker et al. (1995) reported a study where initial decomposition rates of naturally occurring polysaccharides could be measured. They applied a method of the selective inhibition of microbial carbohydrate uptake using toluene, without affecting the extracellular hydrolysis of the polysaccharides in the samples under study. The hydrolysis products would then accumulate in the reactors, and these were monitored over time. The experimental setup was the same as reported by Molwantwa et al. (2004), with all experiments being conducted in triplicate. The two controls
(methanogenic and sulfidogenic) and the two experiments to which toluene had been added (methanogenic+toluene and sulfidogenic+toluene) were set up in triplicate in 500 mL flasks. Each flask was innoculated (10% by volume) with populations of SRB and methane-producing bacteria (MPB), respectively, obtained from 5 L cell culture units that had been operating for 3 months prior to the current experimental program. Sieved (2 mm mesh) PS obtained from Grahamstown Municipal Works was used as a carbon source and was diluted with distilled water to a final COD of 2000 mg L1. The initial sulfate concentration within the sulfidogenic flasks was increased to 2000 mg L1 using analytical-grade Na2SO4 (Merck), thus giving a COD:SO4 ratio of 1:1. The initial pH of all the flasks was adjusted to 7 using 10% Sodium hydroxide (NaOH). The neck of each flask was sealed with a rubber stopper but gas was allowed to escape through a U-tube filled with a zinc acetate solution. In order to prevent ingress of oxygen into the flasks during sampling, samples were forced out of the flasks under positive pressure using nitrogen gas. A total of 400 mL of fresh sieved (2 mm mesh) PS was then added to each flask as the sole carbon source. The flasks were placed on a Labcon desktop shaker (100 rpm) and were allowed to acclimate for 2 days in a CE room at 25 1C before the addition of 15 mL of toluene (to give a final concentration of 3% vol/vol) to the six experimental flasks. The remaining six flasks served as the sulfidogenic and methanogenic controls and had no toluene added to them. Samples were collected (described earlier) at hourly intervals for 8 h (as the inhibition of toluene on the bacteria only lasts for 7–8 h). The first sample was taken at t ¼ 0 after which toluene was immediately added to the experimental flasks. The breakdown of reducing sugars results in the production of VFAs, which are then available to be utilized by SRB. In order to investigate the effect of SRB on the uptake and distribution of VFA and the hydrolysis process, their activity was selectively inhibited using molybdate (Lens et al., 1995). In the presence of molybdate, SRB are inhibited and thus the methanogenic microbes are able to grow. Therefore, those treatments referred to as ‘‘methanogenic’’ in the current study are flasks to which molybdate was added.
2.2.
Analytical procedures
All samples were analyzed in triplicate using the procedures outlined below. Prior to analysis, all samples were acidified with 32% HCl to pH 2 and agitated for 1 min in order to eliminate any sulfide interference with the assay (Whittington-Jones, 2000). A Mercks spectroquant test kit was used to determine the COD and sulfate concentrations were determined using an HPLC anion method applying a model 510 Waters HPLC and model 430 Waters conductivity detector fitted with a Hamilton PRPX 100 150 4.1 mm column. A 10-fold dilution of the sample was prepared using milli-Q water and then filtered through a 0.45 mm nylon filter before passing it through two Waters sep-paks light C18 cartridges to remove the organic contaminants. The samples were then injected into the HPLC and run at 2 mL min1. A 100 mg L1 Na2SO4 standard was prepared in order to determine the retention time of the
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sulfate peak as well as to standardize the accuracy of the instrument. Reducing sugars were assayed using a modified Simogy– Nelson method (Saeman et al., 1945). Absorbance was determined using a Beckman DUR UV/vis spectrophotometer at 520 nm and the reducing sugar concentrations calculated from a glucose standard curve. The concentration of total VFA present was determined using a modified steam distillation and titration method (American Public Health Association (APHA), 1989). Differentiation of the four component VFAs in each sample was determined by gas chromatography using a 30 m 0.320 mm, 0.5 mm HP-Innowax column. VFA standards (Sigma) were used to determine the elution times of the fourcomponent VFAs measured in each sample. The results of the gas chromatography studies are reported as relative peak area, showing the change in proportion of each VFA through the course of the experiment. Statistica Version 6.0 was used for statistical analysis of the recorded data and to determine the standard deviations of the triplicate readings.
3.
Results and discussion
Comparison of the accumulation of reducing sugars in a control system with that in a system where the uptake of reducing sugars is inhibited, should enable accurate quantification of the rate of reducing sugar production, and therefore, a rate of hydrolysis of the parent complex carbohydrates. The rates of hydrolysis under methanogenic and sulfidogenic conditions could then be compared. Because of active biological sulfate reduction and subsequent production of bicarbonate ions, the pH in the sulfidogenic system increased from pH 7 to 7.6 while that of the methanogenic systems showed a decrease from pH 7 to 6.5 (data not shown). This pH data reflects the ideal pH for both the sulfidogeneic and methanogenic systems, respectively (Widdel, 1988). The addition of toluene to the experimental flasks had a significant impact on the uptake of reducing sugars under both methanogenic and sulfidogenic conditions (ANOVA, po0:001, n ¼ 3) (Fig. 1). A decrease in reducing sugars in both
control systems suggested that the gradual increase in soluble COD (CODfiltered) observed in control flasks by Molwantwa et al. (2004) was not due to the accumulation of reducing sugars as they did not accumulate over the experimental period in the current study. The controls showed a total decrease in the reducing sugars of 32% and 58% in the methanogenic and the sulfidogenic systems, respectively, relative to starting concentrations (Fig. 1). However, the experimental systems showed a significant increase in reducing sugars over the first 6 h, after which there was a decrease for the last 2 h, presumably due to utilization by bacterial populations upon the release of inhibition. The difference between concentrations in the inhibited and control systems indicated the rate of utilization of the sugars and was significantly higher under sulfidogenic than methanogenic conditions (ANOVA, po0:05, n ¼ 3). However, under both conditions, a decline in the concentration of reducing sugars in the control systems over the 8-h experimental period indicated that the rate of utilization was greater than production and suggested that the acidogenic step of carbohydrate degradation was not ratelimiting. The overall maximum percentage increase in reducing sugar concentration over the inhibition period was 41% and 65% for the methanogenic ðt ¼ 5Þ and sulfidogenic ðt ¼ 6Þ systems, respectively. The maximum rate of production of reducing sugars in the methanogenic system (51 mg L1 h1) and was found to be 60% less than that of the sulfidogenic system (167 mg L1 h1), supporting previous results (Molwantwa et al., 2004) that hydrolysis of complex carbohydrates was approximately three times faster under sulfidogenic than methanogenic conditions. The concentration of VFAs decreased in both the sulfidogenic and methanogenic systems (Fig. 2) indicating that the rate of utilization of these compounds exceeded production. After the 2-day acclimation period, the sulfidogenic systems showed a significantly higher removal than the methanogenic systems (ANOVA, po0:05, n ¼ 12). Although addition of toluene should not have had any direct affect on the concentration of VFAs in either system, indirect impacts were expected, as inhibition of soluble sugar uptake would
methanogenic+toluene sulfidogenic+toluene
methanogenic sulfidogenic
1800 1600 1400 1200 1000 800 600 400 200 0
VFA (mg/L)
Reducing Sugars (mg/L)
methanogenic sulfidogenic
1
2
3
4 5 Time (hrs)
6
7
8
Fig. 1 – Reducing sugar concentrations of the control and experimental flasks under methanogenic and sulfidogenic conditions (n ¼ 3). The highest standard deviation was 46 and as such were not easily visible on the figure.
methanogenic+toluene sulfidogenic+toluene
2100 2000 1900 1800 1700 1600 1500 1400 1300 0
0
1579
4 0 (200 6) 157 7 – 158 2
1
2
3 Time (hrs)
4
5
6
Fig. 2 – Total VFA concentrations in the methanogenic and sulfidogenic control and experimental systems, showing the effect of inhibition of reducing sugar uptake by toluene (n ¼ 3). All methanogenic flasks contained molybdate to inhibit biological sulfate reduction.
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Area ratio
Acetate
Propionate
Butyrate
Valerate
9 8 7 6 5 4 3 2 1 0
(a)
Area ratio
have prevented soluble carbohydrates from being converted to VFAs during the acidogenic step. Upon inhibition, the sulfidogenic system exhibited a 44% decrease in VFA concentration over a 6-h period, while the decrease in the methanogenic treatment was only 5%. The general decrease in total VFA concentration in all systems indicated that utilization of this organic fraction was rapid although the higher percentage and rate of removal under sulfidogenic conditions was attributed to the fact that SRB have been shown to utilize a wider range of VFA than MPB (Fauville et al., 2004). Conversely, the relatively slower utilization of VFAs by methanogenic populations reflects their limited ability to consume longer chain fatty acids. Although the above data showed that the rate of VFA utilization was more rapid under sulfidogenic than methanogenic conditions, profiles of the individual VFAs were required to confirm the expected differential utilization and accumulation of the various acids under different electron acceptor conditions. The distribution of VFA under methanogenic conditions, with and without inhibition of sugar uptake, was similar (Figs. 3a and b). In both cases, acetate was used preferentially, resulting in a period of accumulation of the other acids. After 2 h, once the acetate had been depleted or reached a low concentration, other VFAs were used. The pattern of VFA utilization was reversed in the sulfidogenic system (Figs. 3c and d). Acetate accumulated initially, particularly in the absence of the toluene inhibitor, with preferential utilization of the longer-chain fatty acids. Once these had decreased below a certain level, acetate was used. These results confirm previously reported results that MPB utilize acetate preferentially over other VFA, while SRB utilize others VFA over acetate (Smith and Klug, 1981; Qabiti et al., 1990). This observation supports the hypothesis that acetogenesis is not rate-limiting in the solubilization of PS, and that the rapid utilization of soluble products under sulfidogenic conditions is, at least partially, due to consumption of a range of VFAs. The results of the current study support the findings of enhanced hydrolysis of carbohydrates in sufidogenic environments (Khan and Trottier, 1978; Kim et al., 1997; Whittington-Jones, 2000). Furthermore, although there are differences in the production and consumption of VFAs between sulfidogenic and methanogenic systems, the most significant difference is at the level of hydrolysis, i.e. the production of reducing sugars. The exact mechanism by which either the SRB themselves or products of their activities affect enhanced hydrolysis is not yet clear. Whittington-Jones (2000) proposed that the sulfide acted indirectly via disruption of sludge flocs, thereby facilitating contact between hydrolytic enzymes and their substrates while Whiteley et al. (2004) suggest that sulpfide directly enhances the activity of the hydrolytic enzymes themselves. The descriptive model developed in this study is centered on the ability of SRB to utilize a wider spectrum of these VFAs, which was observed in this study, thus no accumulation of VFA was observed in the sulfidogenic system. As a result, the occurrence of a negative feedback inhibition is avoided and the rate of hydrolysis is not affected. A diagrammatic representation of this model is shown on Fig. 4.
0
2
3
4
5
6
1
2
3
4
5
6
0
2
3
4
5
6
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2
3 4 Time (hrs)
5
6
9 8 7 6 5 4 3 2 1 0
(b)
Area ratio
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8 7 6 5 4 3 2 1 0
(c)
Area ratio
1580
8 7 6 5 4 3 2 1 0
(d)
Fig. 3 – The profile of volatile fatty acids in methanogenic and sulfidogenic flask experiments. (a) methanogenic; (b) methanogenic+toluene; (c) sulfidogenic; (d) sulfidogenic+toluene. (n ¼ 3). All methanogenic flasks contained sodium molybdate to inhibit biological sufate reduction.
4.
Conclusion
Disposal of PS is a global environmental problem and conventional digestion technologies provide limited relief in terms of reduction of solid material requiring further disposal. However, this material is a potentially valuable and inexpensive source of energy provided that yields of soluble products can be improved and one application of
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4 0 (200 6) 157 7 – 158 2
COMPLEX ORGANIC COMPOUNDS (carbohydrates, proteins, lipids)
Negative feedback
Low rate of VFA utilization under methanogenic conditions leads to VFA accumulation and decrease in pH to below optimum for hydrolysis
HYDROLYSIS
SIMPLE ORGANIC COMPOUNDS (SUGARS, AMINO ACIDS, PEPTIDES)
High rate of VFA utilization in the presence of sulfate leads to enhanced hydrolysis by preventing pH decrease
ACIDOGENESIS
LONG-CHAIN FATTY ACIDS (propionate, butyrate etc.)
ACETOGENESIS
ACETATE
METHANOGENESIS
SUFATE REDUCTION
H2S, CO2
H2, CO2, CH4 Fig. 4 – Flow diagram summarizing the proposed mechanism of enhanced hydrolysis under sulfidogenic system showing the possible negative feedback inhibition.
these would be for sustainable bioremediation of AMD. Although the mechanism underlying enhanced hydrolysis of complex organic matter, including PS, is not understood, the above research indicates that digestion of this material in the presence of biological sulfate reduction offers significant advantages over conventional methanogenic conversion. Possible mechanisms include direct hydrolysis of ‘‘recalcitrant’’ compounds by SRB or the creation of a favourable environment for either chemical or enzymatic degradation or possibly also redox poising of the reagents, which could account for energy differences. These mechanisms therefore, should be the subject of further investigation.
Acknowledgments The authors acknowledge the funding of the Water Research Commission, Pretoria, South Africa for funding the research. R E F E R E N C E S
American Public Health Association (APHA), 1989. Standard Methods for the Examination of Water and Wastewater, 17th ed. American Public Health Association, Washington, DC. Boschker, H.T., Bertilsson, S.A., Dekkers, E.M.J., Cappenberg, T.E., 1995. An inhibitor-based method to measure initial decomposition of naturally occurring polysaccharides in sediments. Appl. Environ. Microbiol. 61, 2186–2192.
Boshoff, G., Duncan, J., Rose, P.D., 1996. An algal–bacterial intergrated ponding system for the treatment of mine drainage waters. Abstract J. Appl. Phycol. 8, 442. Brinch, P.P., Rindel, K., Kalb, K., 1994. Upgrading to nutrient removal by means of internal carbon from sludge hydrolysis. Water Sci. Technol. 29, 31–40. Fauville, A., Mayer, B., Frommichen, R., Friese, K., Veizer, J., 2004. Chemical and isotopic evidence for accelerated bacterial sulfate reduction in acid mining lakes of organic carbon: laboratory batch experiments. Chem. Geol. 204 (3/4), 325–345. Hatziconstantinou, G.J., Efstathiou, H., 2003. Sludge dewatering and disposal practices from small activated sludge wastewater treatment plants. J. Environ. Sci. Health, Part A—Toxic/ Harzardous Substances Environ. Eng. 38 (10), 2401–2413. Khan, A.W., Trottier, T.M., 1978. Effect of sulfur-containing compounds on anaerobic degradation of cellulose to methane by mixed cultures obtained from sewage sludge. Appl. Environ. Microbiol. 35, 1027–1034. Kim, S.K., Matsui, S., Pareek, S., Shimizu, Y., Matsude, T., 1997. Biodegradation of recalcitrant organic matter under sulphate reducing and methanogenic conditions in the landfill column reactors. Water Sci. Technol. 36, 91–98. Lens, P.N., De Poorter, M.P., Cronenberg, C.C., Verstraete, W.H., 1995. Sulphate reducing and methane producing bacteria in aerobic wastewater treatment systems. Water Res. 29, 871–880. Maree, J.P., Hill, E., 1989. Biological removal of sulphate from industrial effluents and concomitant production of sulphur. Water Sci. Technol. 21, 265–276. Maree, J.P., van Tonder, G.J., Millard, P., 1996. Underground neutralization of mine water with limestone. Report to the Water Research Commission, CSIR. WRC Report No. 609/1/96.
ARTICLE IN PRESS 1582
WAT E R R E S E A R C H
40 (2006) 1577– 1582
Molipane, N.P. 1999. Biological sulphate reduction utilising hydrolysis of a complex carbon source. M.Sc. Thesis, Rhodes University, Grahamstown, South Africa. Molwantwa, J.B., Whittington-Jones, K.J., Rose, P.D., 2004. An investigation into the mechanism underlying enhanced hydrolysis of complex carbon in a biosulphidogenic recycling sludge bed reactor (RSBR). Water SA 30, 155–159. Pareek, S., Kim, S.K., Matsui, S., Shimizu, Y., 1998. Hydrolysis of (ligno) cellulosic materials under sulfidogenic and methanogenic conditions. Water Sci. Technol. 38, 193–200. Qabiti, A.J., Bories, A., Garcia, J.L., 1990. Effects of sulfate on lactate and C2-, C3- volatile fatty acid anaerobic degradation by a mixed microbial culture. Antonie van Leeuwenhoek 58, 241–248. Riekkola-Vanhanen, M., Mustikkama¨ki, U.P., 1997. In situ treatment of acid mine drainage by sulphate reducing bacteria in an open pit mine. In: Proceedings of Biomine International Conference, Sydney, Australia. E-ROM3.1-E-ROM3.8. Saeman, J.F., Buhl, J.L., Harris, E.E., 1945. Quantitative saccharification of wood and cellulose. Ind. Eng. Chem. Anal. 17, 35–37.
Smith, R.L., Klug, M.J., 1981. Reduction of sulfur compounds in the sediments of a eutrophic lake basin. Appl. Environ. Microbiol. 41, 1230–1237. Ueki, K., Kotaka, K., Itoh, K., Ueki, A., 1988. Potential availability of anaerobic treatment with digester slurry of animal waste for the reclamation of acid mine water containing sulphate and heavy metals. J. Ferment. Technol. 66, 43–50. Whiteley, C., Pletschke, B., Rose, P., Tshivhunge, S., Watson, S., Whittington-Jones, K., 2004. Activation of proteases in an anaerobic sulphidogenic bioreactor. Biotechnol. Lett. 26, 55–59. Whittington-Jones, K.J., 2000. Sulphide-enhanced hydrolysis of primary sewage sludge: implications for the bioremediation of sulphate-enriched wastewaters. Ph.D. Thesis, Rhodes University, Grahamstown, South Africa. Whittington-Jones, K.J., Rose, P.D., Corbett, C.J., 2002. The Rhodes ‘BioSURE’ Process, Part 2: Enhanced Hydrolysis of Organic Carbon Substrates—Development of the Reciprocating Sludge Bed Reactor. Water Research Commission, South Africa.