Anaerobic granule development for removal of pentachlorophenol in an upflow anaerobic sludge blanket (UASB) reactor

The granulation process using synthetic wastewater containing pentachlorophenol (PCP) in four 1.1 l laboratory scale upflow anaerobic sludge blanket (UASB) reactors was studied, and the anaerobic biotransformation of PCP during the granulation process investigated. After 110 days granular sludge was developed and up to 160 and 180 mg/l of PCP was added into the reactors R1 and R2, respectively, when they were inoculated with acclimated anaerobic sludge from an anaerobic digester of a citric acid plant. The inoculum was predominately composed of bacilli and filamentous bacteria. Granulation did not occur in reactors R3 and R4 which were inoculated with acclimated anaerobic sludge from aerobic sludge of the municipal sewage treatment plant which consisted mainly of cocci. Despite similar bacilli in the granule, the filamentous bacteria from reactor R1 were thicker than those of reactor R2. The granular sludge had a maximum diameter of 2.5 and 2.2 mm, and SMA of 1.44 and 1.32 gCOD/gTVS per day for reactors R1 and R2, respectively. Over 98% chemical oxygen demand (COD) removal rate and 99% of PCP removal rate were achieved when reactors R1 and R2 were operated at PCP and COD loading rates of 150 and 7.5 g/l per day, respectively. H₂-producing acetogens were the dominant anaerobes in the granular sludge.     

Improvement of the anaerobic treatment of potato processing wastewater in a UASB reactor by co-digestion with glycerol

The effect of three different types of glycerol on the performance of up-flow anaerobic sludge blanket (UASB) reactors treating potato processing wastewater was investigated. High COD removal efficiencies were obtained in both control and supplemented UASB reactors (around 85%). By adding 2 ml glycerol product per liter of raw wastewater, the biogas production could be increased by 0.74 l biogas ml⁻¹ glycerol product, which leads to energy values in the range of 810–1270 kWh_electric per m³ product. Moreover, a better in-reactor biomass yield was observed for the supplemented UASB reactor (0.012 g VSS g⁻¹ COD_removed) compared to the UASB control (0.002 g VSS g⁻¹ COD_removed), which suggests a positive effect of glycerol on the sludge blanket growth.     

The effect of calcium on the treatment of fresh leachate in an expanded granular sludge bed bioreactor

This research investigated the calcium effect on the anaerobic treatment of fresh leachate in an expanded granular sludge bed (EGSB) bioreactor under mesophilic conditions. The observations show that the bioreactor, inoculated with anaerobic granular sludge, can be started up only in about 40 days for the treatment of calcium-containing fresh leachate with chemical oxygen demand (COD) removal efficiency above 90% and organic loading rate up to 72.84 kg COD/m³ day. The calcium accumulation onto the granules was monotonically related to the calcium concentration, accounting for 17-18 wt.% of Ca in the suspended solid in the form of calcium carbonate, phosphates/phosphonates and carboxylates. The mineral formation significantly increased the granule settling velocity (by ∼50%) and the suspended solid concentration (by ∼100%). However, the effect of calcium precipitation on the specific methanogenic activity and the CH₄ production rate was complex, first positive during the start-up but later on negative.     

Anaerobic biodegradation of sugar beet pulp

Sugar beet pulp is a by-product of sugar production and consists mainly of cellulose, hemicellulose and pectin. Its composition is suitable for biological degradation. A possible alternative for the utilization of this material (besides cattle feeding) can be anaerobic methanogenic degradation. It has an additional advantage – biogas production. Beet pulp was treated by a two-step anaerobic process. The first step consisted of hydrolysis andacidification. The second step was methanogenesis. In this paper, observation ofthe process of anaerobic degradation and determination of optimal parameters is discussed. A laboratory-scale model for sugar beet pulp anaerobic biodegradation was operated. Results of model performance have shown very good pulp digestion characteristics. In addition, high efficiency removal of organic matter was achieved. Methane yield was over 0.360 m³ kg^-1 dried pulp and excess sludge production was 0.094 g per gram COD added.     

Removal of malachite green (MG) from aqueous solutions by native and heat-treated anaerobic granular sludge

The performance of native and heat-treated anaerobic granular sludge in removing of malachite green (MG) from aqueous solution was investigated with different conditions, such as pH, ionic strength, initial concentration and temperature. The maximum biosorption was both observed at pH 5.0 on the native and heat-treated anaerobic granular sludge. The ionic strength had negative effect on MG removal. Kinetic studies showed that the biosorption process followed pseudo-second-order and q_e for native and heat-treated anaerobic granular sludge is 61.73 and 59.17 mg/g at initial concentration 150 mg/L, respectively. Intraparticle diffusion model could well illuminate adsorption process and faster adsorption rate of native anaerobic granular sludge than heat-treated anaerobic granular sludge. The equilibrium data were analyzed using Langmuir and Freundlich model, and well fitted Langmuir model. The negative values of ΔG° and ΔH° suggested that the interaction of MG adsorbed by native and heat-treated anaerobic granular sludge was spontaneous and exothermic. Desorption studies revealed that MG could be well removed from anaerobic granular sludge by 1% (v/v) of HCl–alcohol solution.     

Thermophilic treatment of bulk drug pharmaceutical industrial wastewaters by using hybrid up flow anaerobic sludge blanket reactor

The hybrid up flow anaerobic sludge blanket reactor was evaluated for efficacy in reduction of chemical oxygen demand (COD) and biochemical oxygen demand (BOD) of bulk drug pharmaceutical wastewater under different operational conditions. The start-up of the reactor feed came entirely with glucose, applied at an organic loading rate (OLR) 1 kg COD/m³ d. Then the reactor was studied at different OLRs ranging from 2 to 11 kg COD/m³ d with pharmaceutical wastewater. The optimum OLR was found to be 9 kg COD/m³ d, where we found 65–75% COD and 80–94% of BOD reduction with biogas production containing 60–70% of methane and specific methanogenic activity was 320 ml CH₄/g-VSS d. By the characterization studies of effluent using GC–MS, the hazardous compounds like phenol, l,2-methoxy phenol, 2,4,6-trichloro phenol, dibutyl phthalate, 1-bromo naphthalene, carbamazepine and antipyrine were present. After the treatment, these compounds degraded almost completely except carbamazepine. Thermophilic methanothrix and methanosaetae like bacteria are present in the granular sludge.     

l-Lactate Production from Biodiesel-Derived Crude Glycerol by Metabolically Engineered Enterococcus faecalis: Cytotoxic Evaluation of Biodiesel Waste and Development of a Glycerol-Inducible Gene Expression System

Biodiesel waste is a by-product of the biodiesel production process that contains a large amount of crude glycerol. To reuse the crude glycerol, a novel bioconversion process using Enterococcus faecalis was developed through physiological studies. The E. faecalis strain W11 could use biodiesel waste as a carbon source, although cell growth was significantly inhibited by the oil component in the biodiesel waste, which decreased the cellular NADH/NAD⁺ ratio and then induced oxidative stress to cells. When W11 was cultured with glycerol, the maximum culture density (optical density at 600 nm [OD₆₀₀]) under anaerobic conditions was decreased 8-fold by the oil component compared with that under aerobic conditions. Furthermore, W11 cultured with dihydroxyacetone (DHA) could show slight or no growth in the presence of the oil component with or without oxygen. These results indicated that the DHA kinase reaction in the glycerol metabolic pathway was sensitive to the oil component as an oxidant. The lactate dehydrogenase (Ldh) activity of W11 during anaerobic glycerol metabolism was 4.1-fold lower than that during aerobic glycerol metabolism, which was one of the causes of low l-lactate productivity. The E. faecalis pflB gene disruptant (Δpfl mutant) expressing the ldhL1_LP gene produced 300 mM l-lactate from glycerol/crude glycerol with a yield of >99% within 48 h and reached a maximum productivity of 18 mM h⁻¹ (1.6 g liter⁻¹ h⁻¹). Thus, our study demonstrates that metabolically engineered E. faecalis can convert crude glycerol to l-lactate at high conversion efficiency and provides critical information on the recycling process for biodiesel waste.     

Enhanced hydrogen and 1,3‐propanediol production from glycerol by fermentation using mixed cultures

The conversion of glycerol into high value products, such as hydrogen gas and 1,3‐propanediol (PD), was examined using anaerobic fermentation with heat‐treated mixed cultures. Glycerol fermentation produced 0.28 mol‐H₂/mol‐glycerol (72 mL‐H₂/g‐COD) and 0.69 mol‐PD/mol‐glycerol. Glucose fermentation using the same mixed cultures produced more hydrogen gas (1.06 mol‐H₂/mol‐glucose) but no PD. Changing the source of inoculum affected gas production likely due to prior acclimation of bacteria to this type of substrate. Fermentation of the glycerol produced from biodiesel fuel production (70% glycerol content) produced 0.31 mol‐H₂/mol‐glycerol (43 mL H₂/g‐COD) and 0.59 mol‐PD/mol‐glycerol. These are the highest yields yet reported for both hydrogen and 1,3‐propanediol production from pure glycerol and the glycerol byproduct from biodiesel fuel production by fermentation using mixed cultures. These results demonstrate that production of biodiesel can be combined with production of hydrogen and 1,3‐propanediol for maximum utilization of resources and minimization of waste. Biotechnol. Bioeng. 2009; 104: 1098–1106. © 2009 Wiley Periodicals, Inc.     

Biogas production from supernatant of hydrothermally treated municipal sludge by upflow anaerobic sludge blanket reactor

The supernatant of hydrothermally treated sludge was treated by an upflow anaerobic sludge blanket (UASB) reactor for a 550-days running test. The hydrothermal parameter was 170 °C for 60 min. An mesophilic 8.6 L UASB reactor was seeded with floc sludge. The final organic loading rate (OLR) could reach 18 kg COD/m³ d. At the initial stage running for 189 days, the feed supernatant was diluted, and the OLR reached 11 kg COD/m³ d. After 218 days, the reactor achieved a high OLR, and the supernatant was pumped into the reactor without dilution. The influent COD fluctuated from 20,000 to 30,000 mg/L and the COD removal rate remained at approximately 70%. After 150 days, granular sludge was observed. The energy balance calculation show that heating 1.0 kg sludge needs 0.34 MJ of energy, whereas biogas energy from the supernatant of the heated sludge is 0.43 MJ.     

Two-stage biological treatment of olive mill wastewater with whey as co-substrate

Olive mill wastewater (OMW) is a highly polluting wastewater, caused by a high organic load and phenol content. These characteristics suggest that it may be suitable for aerobic treatment and anaerobic bacterial digestion. Aerobic treatment coupled with anaerobic bacterial digestion may be economically feasible as the methane produced is a valuable energy source while simultaneously purifying the OMW. In an attempt to improve the overall performance of the process, the addition of a co-substrate such as whey to the aerobic treatment pre-treatment of OMW by the yeast Candida tropicalis was studied.The two-stage system operated satisfactorily up to an organic loading rate (OLR) of 3.0 kg COD L⁻¹ day⁻¹ with a biogas production rate of 1.25 L_biogas L_reactor⁻¹ day⁻¹ and a total COD reduction in excess of 93% (62% COD reduction in aerobic pretreatment and 83% COD reduction in anaerobic digestion). Fifty-four percent of the phenol was biodegraded during the aerobic treatment stage, and biogas with between 68% and 75% methane was produced during anaerobic digestion.     

Performance of upflow anaerobic sludge blanket (UASB) reactor treating wastewaters containing carbon tetrachloride

The anaerobic biodegradation of carbon tetrachloride (CT) was investigated during the granulation process by reducing the hydraulic retention time, increasing the chemical oxygen demand (COD) and CT loadings in a 2l laboratory-scale upflow anaerobic sludge blanket (UASB) reactor. Anaerobic unacclimated sludge and glucose were used as seed and primary substrate, respectively. Granules were developed 4 weeks after start-up, which grew at an accelerated rate for 8 months, and then became fully grown. The effect of operational parameters such as influent CT concentrations, COD, CT loading, food to biomass ratio and specific methanogenic activity (SMA) were also considered during granulation. The granular sludge cultivated had a maximum diameter of 2.1 mm and SMA of 1.6 g COD/g total suspended solid (TSS) day. COD and CT removal efficiencies of 92 and 88% were achieved when the reactor was firstly operating at CT and COD loading rates of 17.5 mg/l day and 12.5 g/l day, respectively. This corresponds to hydraulic retention time of 0.28 day and food to biomass ratio of 0.5 g COD/g TSS day. Kinetic coefficients of maximum specific substrate utilization rate, half velocity coefficient, growth yield coefficient and decay coefficient were determined to be 2.4 × 10^–3 mg CT/TSS day^–1, 1.37 mg CT/l, 0.69 mg TSS/mg CT and 0.046 day^–1, respectively for CT biotransformation during granulation.     

Thermophilic anaerobic treatment of sulphur rich forest industry wastewater

Thermophilic anaerobic treatment of sulphur-rich paper mill wastewater (0.8-3.1 gCOD/l, 340–850 mgSO₄/l; COD:SO₄ 3.4-5.3) was studied in three laboratory-scale, upflow anaerobic sludge blanket (UASB) reactors and in bioassays. The reactors were inoculated with non-adapted thermophilic granular sludge. In the bioassays, no inhibition of the inoculum was detected and about 62% COD removal (sulphide stripped) was obtained. About 70 to 80% of the removed COD was methanised. In the reactors, up to 60–74% COD removal (effluent sulphide stripped) was obtained at loading rates up to 10–30 kgCOD/m³d and hydraulic retention times down to 6 to 2 hours. The effluent total sulphide was up to 150–250 mg/l. Sulphide inhibition could not be confirmed from the reactor performances. The results from bioassays suggested that both the inoculum and sludge from the UASB reactor used acetate mainly for methane production, while sulphide was produced from hydrogen or its precursors.     

Improved glycerol to ethanol conversion by <Emphasis Type="Italic">E. coli</Emphasis> using a metagenomic fragment isolated from an anaerobic reactor

Crude glycerol obtained as a by-product of biodiesel production is a reliable feedstock with the potential to be converted into reduced chemicals with high yields. It has been previously shown that ethanol is the primary product of glycerol fermentation by Escherichia coli. However, few efforts were made to enhance this conversion by means of the expression of heterologous genes with the potential to improve glycerol transport or metabolism. In this study, a fosmid-based metagenomic library constructed from an anaerobic reactor purge sludge was screened for genetic elements that promote the use and fermentation of crude glycerol by E. coli. One clone was selected based on its improved growth rate on this feedstock. The corresponding fosmid, named G1, was fully sequenced (41 kbp long) and the gene responsible for the observed phenotype was pinpointed by in vitro insertion mutagenesis. Ethanol production from both pure and crude glycerol was evaluated using the parental G1 clone harboring the ethanologenic plasmid pLOI297 or the industrial strain LY180 complemented with G1. In mineral salts media containing 50 % (v/v) pure glycerol, ethanol concentrations increased two-fold on average when G1 was present in the cells reaching up to 20 g/L after 24 h fermentation. Similar fermentation experiments were done using crude instead of pure glycerol. With an initial OD₆₂₀ of 8.0, final ethanol concentrations after 24 h were much higher reaching 67 and 75 g/L with LY180 cells carrying the control fosmid or the G1 fosmid, respectively. This translates into a specific ethanol production rate of 0.39 g h^?1 OD^?1 L^?1.     

Kinetic behaviour of waste tyre rubber as microorganism support in an anaerobic digester treating cane molasses distillery slops

The kinetics of anaerobic digestion of cane molasses distillery slops was investigated using a continuous-flow bioreactor which contained waste tyre rubber as support, to which the microorganisms became immobilized. Hydraulic retention times (HRT) ranging from 1 to 10 days were investigated at an average influent chemical oxygen demand (COD) concentration of 47.7?g/l. The maximum substrate utilization rate, k, and half saturation coefficient, K _L, were determined to be 1.82?kg COD_removed/kg VSS day and 0.33?kg COD/kg VSS day. The yield coefficient, Y, and sludge decay rate coefficient, K _d, were also determined to be 0.06?kg VSS/kg COD_removed and 0.05?day^-1, respectively. Methane production was maximum (6.75?l/l day) at a 2 day HRT corresponding to a biomass loading rate of 2.578?kg COD/kg VSS day. Biogas yield ranged between 0.51?l/g COD (HRT=2 days) and 0.25?l/g COD (HRT=1?day). In addition, the methane percentage in the biogas varied between 70.5% (HRT=10?days) and 47.5% (HRT=1?day). The close relationship between biomass loading rate and specific substrate utilization rate supported the use of Monod equations. Finally, the experimental values of effluent substrate concentration were reproduced with deviations equal to or less than 10% in every case.     

Nitrifying granules cultivation in a sequencing batch reactor at a low organics-to-total nitrogen ratio in wastewater

It is possible to cultivate aerobic granular sludge at a low organic loading rate and organics-to-total nitrogen (COD/N) ratio in wastewater in the reactor with typical geometry (height/diameter = 2.1, superficial air velocity = 6 mm/s). The noted nitrification efficiency was very high (99%). At the highest applied ammonia load (0.3 ± 0.002 mg NH₄⁺–N g total suspended solids (TSS)⁻¹ day⁻¹, COD/N = 1), the dominating oxidized form of nitrogen was nitrite. Despite a constant aeration in the reactor, denitrification occurred in the structure of granules. Applied molecular techniques allowed the changes in the ammonia-oxidizing bacteria (AOB) community in granular sludge to be tracked. The major factor influencing AOB number and species composition was ammonia load. At the ammonia load of 0.3 ± 0.002 mg NH₄⁺–N g TSS⁻¹ day⁻¹, a highly diverse AOB community covering bacteria belonging to both the Nitrosospira and Nitrosomonas genera accounted for ca. 40% of the total bacteria in the biomass.     

Characterization of sulfate-reducing bacteria dominated surface communities during start-up of a down-flow fluidized bed reactor

An anaerobic down-flow fluidized bed reactor was inoculated with granular sludge and started-up with sulfate containing synthetic wastewater to promote the formation of a biofilm enriched in sulfate-reducing bacteria (SRB), to produce biogenic sulfide. The start-up was done in two stages operating the reactor in batch for 45 days followed by 85 days of continuous operation. Low-density polyethylene was used as support. The biofilm formation was followed up by biochemical and electron microscopy analyses and the composition of the community was examined by 16S rDNA sequence analysis. Maximum immobilized volatile solids (1.2 g IVS/L_support) were obtained after 14 days in batch regime. During the 85 days of continuous operation, the reactor removed up to 80% of chemical oxygen demand (COD), up to 28% of the supplied sulfate and acetate was present in the effluent. Sulfate-reducing activity determined in the biofilm with ethanol or lactate as substrate was 11.7 and 15.3 g COD/g IVS per day, respectively. These results suggested the immobilization of sulfate reducers that incompletely oxidize the substrate to acetate; the phylogenetic analysis of the cloned 16S rDNA gene sequences showed high identity to the genus Desulfovibrio that oxidizes the substrates incompletely. In contrast, in the granular sludge used as inoculum a considerable number of clones showed homology to Methanobacterium and just few clones were close to SRB. The starting-up approach allowed the enrichment of SRB within the diverse community developed over the polyethylene support.     

Production of arabitol from glycerol: strain screening and study of factors affecting production yield

Glycerol is a major by-product from biodiesel production, and developing new uses for glycerol is imperative to overall economics and sustainability of the biodiesel industry. With the aim of producing xylitol and/or arabitol as the value-added products from glycerol, 214 yeast strains, many osmotolerant, were first screened in this study. No strains were found to produce large amounts of xylitol as the dominant metabolite. Some produced polyol mixtures that might present difficulties to downstream separation and purification. Several Debaryomyces hansenii strains produced arabitol as the predominant metabolite with high yields, and D. hansenii strain SBP-1 (NRRL Y-7483) was chosen for further study on the effects of several growth conditions. The optimal temperature was found to be 30°C. Very low dissolved oxygen concentrations or anaerobic conditions inhibited polyol yields. Arabitol yield improved with increasing initial glycerol concentrations, reaching approximately 50% (w/w) with 150 g/L initial glycerol. However, the osmotic stress created by high salt concentrations (≥50 g/L) negatively affected arabitol production. Addition of glucose and xylose improved arabitol production while addition of sorbitol reduced production. Results from this work show that arabitol is a promising value-added product from glycerol using D. hansenii SBP-1 as the producing strain.     

Treatment of food processing wastewater in a full-scale jet biogas internal loop anaerobic fluidized bed reactor

A full-scale jet biogas internal loop anaerobic fluidized bed (JBILAFB) reactor, which requires low energy input and allows enhanced mass transfer, was constructed for the treatment of food processing wastewater. This reactor has an active volume of 798 m³ and can treat 33.3 m³ wastewater per hour. After pre-treating the raw wastewater by settling, oil separating and coagulation-air floating processes, the reactor was operated with a relatively shorter start-up time (55 days). Samples for the influent and effluent of the JBILAFB reactor were taken and analyzed daily for the whole process including both the start-up and stable running periods. When the volumetric COD loading fluctuated in the range of 1.6–5.6 kg COD m⁻³ day⁻¹, the COD removal efficiency, the volatile fatty acid(VFA)/alkalinity ratio, the maximum biogas production and the content of CH₄ in total biogas of the reactor were found to be 80.1 ± 5%, 0.2–0.5, 348.5 m³ day⁻¹ and 94.5 ± 2.5%, respectively. Furthermore, the scanning electron microscope (SEM) results showed that anaerobic granular sludge and microorganism particles with biofilm coexisted in the reactor, and that the bacteria mainly in bacilli and cocci were observed as predominant species. All the data demonstrated that the enhanced mass transfer for gas, liquid and solid phases was achieved, and that the formation of microorganism granules and the removal of inhibitors increased the stability of the system.     

The influence of anaerobic pretreatment on the nitrogen removal from biosynthetic pharmaceutical wastewaters

The C:N ratio of the pharmaceutical wastewaters is usually suitable for a combination of the anaerobic pretreatment with the high COD removal and aerobic posttreatment with the efficient biological N removal. This kind of anaerobic-aerobic process was tested in semipilot scale by using a UASB reactor and an activated sludge system with a predenitrification (total volume 100 1). It was found that at a total HRT of 2.3 days an average of 97.5% of COD and 73.5% of total N was removed. The UASB reactor was operated at 30°C with a volumetric loading rate of 8.7 kg.m^-3.d^-1, the efficiency of COD removal was 92.2%. The processes, which take part in the biological removal of nitrogen, especially the nitrification, were running with lower rates than usually observed in aerobic treatment systems.Abbreviations AAO anaerobic anoxic oxic configuration - AOO anaerobic oxic oxic configuration - B _V volumetric organic loading rate (kg COD.m^-3. d^-1) - dB _x specific COD removal rate (mg COD. g^-1 VSS. d^-1) - DNR denitrification rate (mg N–NO₃. g^-1 VSS. h^-1) - E_COD efficiency of COD removal (%) - HRT hydraulic retention time (d) - NR nitrification rate (mg N–NO₃. g^-1 VSS. h^-1) - R recirculation ratio (%) - SBP specific biogas production (m³.kg^-1 removed COD) - SRT solids retention time; sludge age (d) - SS suspended solids (g.1^-1) - UASB upflow anaerobic sludge blanket reactor - VSS volatile suspended solids (g.1^-1)     

Inorganic composition and microbial characteristics of methanogenic granular sludge grown in a thermophilic upflow anaerobic sludge blanket reactor

An upflow anaerobic sludge blanket reactor was operated under thermophilic conditions (55° C) for 160 days by feeding a wastewater containing sucrose as the major carbon source. The reactor exhibited a satisfactory performance due to the formation of well-settling granulated sludge, achieving a total organic carbon (TOC) removal of above 80% at an organic loading rate of 30 kg total organic C m^–3 day^–1. Structural and microbial properties of the methanogenic granular sludge were examined using scanning electron microscope X-ray analyses and serum vial activity tests. All the thermophilic granules developed showed a double-layered structure, comprised of a black core portion and a yellowish exterior portion. The interior cope portion contained abundant crystalline precipitates of calcium carbonate. Calcium-bound phosphorus was also present more prominently in the core portion than in the exterior portion. Methanogenic activities of the thermophilic granules both from acetate and from H₂ increased with increasing vial-test temperature in the range of 55–65° C [from 1.43 to 2.36 kg CH₄ chemical oxygen demand (COD) kg volatile suspended solids (VSS)^–1 day^–1 for acetate and from 0.85 to 1.11 kg CH₄ COD kg VSS^–1 day^–1 for H₂]. On the other hand, propionate-utilizing methanogenic activity was independent of vial-test temperature, and was much lower (0.1–0.12 kg CH₄ COD kg VSS^–1 day^–1) than that from either acetate or H₂. Acetate consumption during vial tests was considerably inhibited by the presence of H₂ in the headspace, indicating that a syntrophic association between acetate oxidizers and H₂-utilizing methane-producing bacteria was responsible for some portion of the overall acetate elimination by the theromophilically grown sludge.