Biotransformation potential of phytosterols under anoxic and anaerobic conditions.

The biotransformation potential of three phytosterols (campesterol, stigmasterol and ß-sitosterol) under denitrifying, sulfate-reducing and fermentative/methanogenic conditions was assessed. Using a group contribution method, the standard Gibbs free energy of phytosterols was calculated and used to perform theoretical energetic calculations. The oxidation of phytosterols under aerobic, nitrate-reducing, sulfate-reducing and methanogenic conditions was determined to be energetically feasible. However, using semi-continuously fed cultures maintained at 20-22 °C over 16 weekly feeding cycles (112 days; retention time, 21 days), phytosterol removal was observed under nitrate-reducing and sulfate-reducing conditions, but not under fermentative/methanogenic conditions. Under sulfate-reducing conditions, stigmast-4-en-3-one was identified as an intermediate of phytosterol biotransformation, a reaction more likely carried out by dehydrogenases/isomerases, previously reported to act on cholesterol under both oxic and anoxic (denitrifying) conditions. Further study of the biotransformation of phytosterols under anoxic/anaerobic conditions is necessary to delineate the factors and conditions leading to enhanced phytosterol biodegradation and the development of effective biological treatment systems for the removal of phytosterols from pulp and paper wastewaters and other phytosterol-bearing waste streams.

Syntrophic acetate oxidation in two-phase (acid-methane) anaerobic digesters.

The microbial processes involved in two-phase anaerobic digestion were investigated by operating a laboratory-scale acid-phase (AP) reactor and analyzing two full-scale, two-phase anaerobic digesters operated under mesophilic (35 °C) conditions. The digesters received a blend of primary sludge and waste activated sludge (WAS). Methane levels of 20% in the laboratory-scale reactor indicated the presence of methanogenic activity in the AP. A phylogenetic analysis of an archaeal 16S rRNA gene clone library of one of the full-scale AP digesters showed that 82% and 5% of the clones were affiliated with the orders Methanobacteriales and Methanosarcinales, respectively. These results indicate that substantial levels of aceticlastic methanogens (order Methanosarcinales) were not maintained at the low solids retention times and acidic conditions (pH 5.2-5.5) of the AP, and that methanogenesis was carried out by hydrogen-utilizing methanogens of the order Methanobacteriales. Approximately 43, 31, and 9% of the archaeal clones from the methanogenic phase (MP) digester were affiliated with the orders Methanosarcinales, Methanomicrobiales, and Methanobacteriales, respectively. A phylogenetic analysis of a bacterial 16S rRNA gene clone library suggested the presence of acetate-oxidizing bacteria (close relatives of Thermacetogenium phaeum, 'Syntrophaceticus schinkii,' and Clostridium ultunense). The high abundance of hydrogen consuming methanogens and the presence of known acetate-oxidizing bacteria suggest that acetate utilization by acetate oxidizing bacteria in syntrophic interaction with hydrogen-utilizing methanogens was an important pathway in the second-stage of the two-phase digestion, which was operated at high ammonium-N concentrations (1.0 and 1.4 g/L). A modified version of the IWA Anaerobic Digestion Model No. 1 (ADM1) with extensions for syntrophic acetate oxidation and weak-acid inhibition adequately described the dynamic profiles of volatile acid production/degradation and methane generation observed in the laboratory-scale AP reactor. The model was validated with historical data from the full-scale digesters.

Inhibitory effects of nitrate reduction on methanogenesis in the presence of different electron donors.

The preferential utilization of different electron donors and their effects on the nitrate reduction and methanogenesis in a mixed, mesophilic (35 degrees C) methanogenic culture were investigated. Batch methanogenic cultures were fed with dextrin/peptone (D/P), propionate, acetate, and H(2)/CO(2) at an initial COD of 500 mg/L and an initial nitrate concentration of 50 mg N/L. Immediate cessation of methane production was observed in all nitrate-amended cultures. Methane production completely recovered in the D/P- and acetate-fed cultures, and partially recovered or did not recover in the propionate- and H(2)/CO(2)-fed, nitrate-amended cultures, respectively. Accumulation of denitrification intermediates was observed in both the propionate- and H(2)/CO(2)-fed cultures, which resulted in inhibition of fermentation and/or methanogenesis. The fastest and the slowest nitrate reduction were observed in the acetate- and propionate-fed cultures, respectively.

Effect of didecyl dimethyl ammonium chloride on nitrate reduction in a mixed methanogenic culture.

The effect of the quaternary ammonium compound, didecyl dimethyl ammonium chloride (DDAC), on nitrate reduction was investigated at concentrations up to 100 mg/L in a batch assay using a mixed, mesophilic (35 degrees C) methanogenic culture. Glucose was used as the carbon and energy source and the initial nitrate concentration was 70 mg N/L. Dissimilatory nitrate reduction to ammonia (DNRA) and to dinitrogen (denitrification) were observed at DDAC concentrations up to 25 mg/L. At and above 50 mg DDAC/L, DNRA was inhibited and denitrification was incomplete resulting in accumulation of nitrous oxide. At DDAC concentrations above 10 mg/L, production of nitrous oxide, even transiently, resulted in complete, long-term inhibition of methanogenesis and accumulation of volatile fatty acids. Fermentation was inhibited at and above 75 mg DDAC/L. DDAC suppressed microbial growth and caused cell lysis at a concentration 50 mg/L or higher. Most of the added DDAC was adsorbed on the biomass. Over 96% of the added DDAC was recovered from all cultures at the end of the 100-days incubation period, indicating that DDAC did not degrade in the mixed methanogenic culture under the conditions of this study.

An extension of the Anaerobic Digestion Model No. 1 to include the effect of nitrate reduction processes.

Nitrate reduction processes were incorporated into the IWA Anaerobic Digestion Model No. 1 (ADM1) in order to account for the effect of such processes on fermentation and methanogenesis. The general structure of the ADM1 was not changed except for modifications related to disintegration and hydrolysis of complex organic matter and decayed biomass. A fraction of butyrate/valerate and propionate degraders was assumed to be the fermentative denitrifiers carrying out fermentation in the absence of N-oxides. Nitrate reduction proceeded in a stepwise manner to nitrite, nitric oxide, nitrous oxide and nitrogen gas using four substrates as electron and/or carbon source. The utilization of the four substrates and N-oxides was based on stoichiometry and kinetics. The inhibitory effect of N-oxides on the methanogens was accounted for by the use of non-competitive inhibition functions. Model simulations were compared with experimental data obtained with a batch, mixed fermenting and methanogenic culture amended with various initial nitrate concentrations.

Anaerobic biodegradability of Tween surfactants used as a carbon source for the microbial reductive dechlorination of hexachlorobenzene.

Three structurally-related, nonionic, polysorbate surfactants (Tween 60, 61, and 65) were used as the sole carbon source to sustain the microbial, sequential reductive dechlorination of hexachlorobenzene (HCB) in a mixed, methanogenic culture derived from a contaminated estuarine sediment. The surfactants were partially degraded and fermented to methane with no measurable accumulation of volatile fatty acids, indicating that methanogenesis was rapid relative to the rates of hydrolysis and acidogenesis. Addition of the methanogenesis inhibitor 2-bromoethanesulfonic acid resulted in acetate accumulation without impact on the sequential dechlorination of HCB. An anaerobic biodegradability assay was performed and the following data were obtained for the Tween 60, 61, and 65, respectively: 53, 62, and 62% COD destruction; 35, 57, and 48% COD to methane conversion; and 38, 38, and 45% COD to acetate conversion. These data suggest that the hydrophobic moiety (stearate) of the surfactants was preferentially degraded, most likely through beta-oxidation, to acetate and ultimately to methane and carbon dioxide. Between 38 and 47% of the initial surfactant COD remained after 46 d incubation, which most likely corresponds to the hydrophilic polyoxyethylene moiety. An anaerobic biodegradation pathway of the Tween surfactants is proposed.

Anaerobic biodecolorization of textile reactive anthraquinone and phthalocyanine dyebaths under hypersaline conditions.

The biological decolorization of two industrial, spent textile reactive dyebaths was investigated using a suspended-growth, halophilic mixed culture fed with glucose. Dyebath I contained mainly Reactive Blue 19 (RB19), an anthraquinone dye, whereas dyebath II contained mainly Reactive Blue 21 (RB21), a phthalocyanine dye. Batch assays under anaerobic conditions with the two neutralized dyebaths resulted in 87 and 37% extent of decolorization for dyebaths I and II, respectively. The rate of glucose utilization and the extent of acetate production were impacted in the presence of each dyebath as compared to the control culture. However, dyebath decolorization occurred despite moderate culture inhibition. Reuse of a biologically renovated RB19-containing dyebath in the dyeing process resulted in reproducible but not identical cotton fabric shades as compared to a standard dyeing (i.e., control) using fresh water. This difference is attributed to a variable degree of RB19 aggregation during the dyeing process and is not related to the efficiency of the biodecolorization process. Further improvement of the redyeing efficiency will lead to the development of an in-plant, closed-loop decolorization system resulting in significant water conservation and minimization of textile pollutants such as salt and dyes.

A distributed model of solid waste anaerobic digestion: sensitivity analysis.

A distributed model of anaerobic digestion of solid waste was developed to describe the balance between the rates of polymer hydrolysis and methanogenesis during the anaerobic conversion of rich and lean wastes in batch and continuous-flow reactors. Waste, volatile fatty acids (VFAs), methanogenic biomass and sodium concentrations are the model variables. Diffusion and advection of VFAs inhibiting both polymer hydrolysis and methanogenesis were considered. A sensitivity analysis by changing the key model parameter values was carried out. The model simulations showed that the effective distance between the areas of hydrolysis/acidogenesis and methanogenesis is very important. An initial spatial separation of rich waste and inoculum enhances the methane production and waste degradation at high waste loading if relatively low VFA diffusion into the methanogenic area is taking place. When both hydrolysis and methanogenesis are strongly inhibited by high levels of VFA, fluctuations in biomass concentration are thought to be responsible for initiating the expansion of methanogenic area over the reactor space.

Kinetics and inhibition during the decolorization of reactive anthraquinone dyes under methanogenic conditions.

The objective of this study was to assess the biological decolorization of two reactive anthraquinone dyes (Reactive Blue 4, RB 4; Reactive Blue 19, RB 19) under methanogenic conditions. Using a mixed, methanogenic culture, batch assays were performed to evaluate both the rate and extent of color removal as well as any potential inhibition. The effect of initial dye, biomass, and organic feed concentration, as well as the effect of repetitive dye addition on color removal kinetics and culture inhibition were assessed. Overall, a lower rate and extent of color removal was observed in RB 4-amended cultures as opposed to the RB 19-amended cultures. For an incubation time of ca. 15 days and an initial dye concentration of 2000 mg/L, the extent of color removal was 50 and 95% for RB 4 and RB 19, respectively. Inhibition of acidogenesis and to a larger degree of methanogenesis, resulting in accumulation of volatile fatty acids, was observed in both RB 4- and RB 19-amended cultures. Although the degree of inhibition varied among the two dyes tested (RB 19 was more inhibitory than RB 4), an increase of inhibition was observed with increasing initial dye concentration. At an initial dye concentration of 500 mg/L or higher, methane production was lower than 6% of that of the control culture for both RB 4 and RB 19. However, color removal occurred despite culture inhibition.

The IWA Anaerobic Digestion Model No 1 (ADM1).

The IWA Anaerobic Digestion Modelling Task Group was established in 1997 at the 8th World Congress on Anaerobic Digestion (Sendai, Japan) with the goal of developing a generalised anaerobic digestion model. The structured model includes multiple steps describing biochemical as well as physicochemical processes. The biochemical steps include disintegration from homogeneous particulates to carbohydrates, proteins and lipids; extracellular hydrolysis of these particulate substrates to sugars, amino acids, and long chain fatty acids (LCFA), respectively; acidogenesis from sugars and amino acids to volatile fatty acids (VFAs) and hydrogen; acetogenesis of LCFA and VFAs to acetate; and separate methanogenesis steps from acetate and hydrogen/CO2. The physico-chemical equations describe ion association and dissociation, and gas-liquid transfer. Implemented as a differential and algebraic equation (DAE) set, there are 26 dynamic state concentration variables, and 8 implicit algebraic variables per reactor vessel or element. Implemented as differential equations (DE) only, there are 32 dynamic concentration state variables.

Decolorization of a reactive copper-phthalocyanine dye under methanogenic conditions.

The objective of this research was to assess the biological decolorization of the copper-phthalocyanine dye Reactive Blue 7 (RB7) under methanogenic conditions using a mixed, methanogenic culture in a repetitive dye addition batch assay. The initial rate of decolorization was 13.2 mg/L-d and 5.7 mg/L-d for the first and second dye addition, respectively. For an initial RB7 concentration of ca. 300 mg/L, the extent of decolorization remained constant (about 62%) for each repetitive RB7 addition and resulted in a residual color build up. Declining absorbance ratio values (A664/A620) with increasing incubation time confirmed that the observed color removal was due to transformation as opposed to adsorption on the biomass. Chemical decolorization assays using sodium dithionite as the reducing agent resulted in similar absorbance spectra to that obtained after biological decolorization. In addition, in both the chemical and biological decolorization assays, partial oxidation of the reduced dye solution upon exposure to air resulted in higher residual color, indicating that the reduction and decolorization of RB7 are partially reversible. These results also suggest that RB7 reduction and decolorization both chemically and biologically most likely followed a similar reduction mechanism.

Development of hexachlorobenzene-dechlorinating mixed cultures using polysorbate surfactants as a carbon source.

The use of three nonionic polysorbate surfactants--Tween 60, 61 or 65--as the sole carbon source to sustain methanogenesis and dechlorination, as well as the effect of long-term exposure of enriched cultures to these surfactants, was investigated through the development of three sediment-derived cultures. Over a one-year period, the carbon source in these cultures was gradually switched from glucose and methanol to surfactant only, while the surfactant concentration was increased from an initial concentration of 100 mg/L to 400 mg/L. In each feeding cycle, the surfactants were partially degraded and converted to methane. Transition from glucose to Tween surfactants as the electron donor did not affect the rate, extent, and pathway of HCB transformation. These surfactants sustained the reductive dechlorination of HCB even after one year of continuous addition to the enriched cultures. This study demonstrated that reductive dechlorination of HCB sustained by the fermentation of Tween surfactants is feasible. The results support the use of anaerobically degradable Tween surfactants for the biotransformation of polychlorinated organic compounds. In principle, these surfactants could be used to simultaneously increase the bioavailability of subsurface contaminants while serving as the carbon and electron source for microbial reductive dechlorination.

Kinetics and modeling of autotrophic thiocyanate biodegradation.

The biokinetic parameters for autotrophic systems are difficult to obtain and are often mistakenly determined because the size of the autotrophic population in mixed (i.e., heterotrophic and autotrophic) cultures cannot be accurately estimated. This article presents a systematic approach, combining bioenergetic calculations and experimental data, to obtain values of the biokinetic parameters pertinent to the aerobic, autotrophic biodegradation of thiocyanate. Nonlinear regression techniques were employed using both initial thiocyanate utilization rate data and single thiocyanate depletion curves. Both types of data were necessary to overcome the problems arising from the linear nature of the substrate depletion curves and the high correlation of the biokinetic model parameters inherent in nonlinear regression analysis. The aerobic biodegradation of thiocyanate followed a substrate inhibition pattern that was successfully described by the Haldane-Andrews model. Although regression analysis did not yield unique biokinetic parameter estimates, the following parameter value ranges were obtained: maximum specific substrate utilization rate (k), 0.26 to 0.44 mg SCN-/mg biomass h; half-saturation coefficient (Ks), 2.3 to 7.1 mg SCN-/L; and inhibition coefficient (Ki), 28 to 109 mg SCN-/L. Based on the estimated biokinetic parameter values, a design and operation diagram was constructed that depicts the steady-state thiocyanate concentration as a function of solids retention time for a completely mixed, continuous-flow reactor.

Aerobic biodegradation of selected monoterpenes.

Batch experiments were conducted to assess the biotransformation potential of four hydrocarbon monoterpenes (d-limonene, alpha-pinene, gamma-terpinene, and terpinolene) and four alcohols (arbanol, linalool, plinol, and alpha-terpineol) under aerobic conditions at 23 degrees C. Both forest-soil extract and enriched cultures were used as inocula for the biodegradation experiments conducted first without, then with prior microbial acclimation to the monoterpenes tested. All four hydrocarbons and two alcohols were readily degraded. The increase in biomass and headspace CO2 concentrations paralleled the depletion of monoterpenes, thus confirming that terpene disappearance was the result of biodegradation accompanied by microbial growth and mineralization. Plinol resisted degradation in assays using inocula from diverse sources, while arbanol degraded very slowly. A significant fraction of d-limonene-derived carbon was accounted for as non-extractable, dissolved organic carbon, whereas terpineol exhibited a much higher degree of utilization. The rate and extent of monoterpene biodegradation were not significantly affected by the presence of dissolved natural organic matter.

Transformation of trichloroethylene by sulfate-reducing cultures enriched from a contaminated subsurface soil.

Trichloroethylene (TCE) was reductively dechlorinated to cis-1,2-dichloroethylene (cDCE) by sulfate-reducing cultures enriched from a contaminated subsurface soil. The highest observed transformation rate of TCE was 213 mumol l-1 per day at 35 degrees C. The predominant biotransformation product was cDCE. However, further dechlorination of cDCE was not observed in most of the cultures. Methane production was insignificant and active sulfate reduction was achieved by maintaining excess sulfate. A comparison of sodium sulfide and sodium dithionite for their effect on the transformation of TCE revealed that the latter is a better reducing agent. The extent of TCE transformation in 25 days was ca. 20% higher in the dithionite-amended cultures. A decrease in the rate and extent of TCE transformation was observed with an increase in the concentration of bromoethanesulfonate up to 50 mM.

Fermentation of Insoluble Cellulose by Continuous Cultures of Ruminococcus albus.

The hydrolysis and fermentation of insoluble cellulose (Avicel) by continuous cultures of Ruminococcus albus 7 was studied. An anaerobic continuous culture apparatus was designed which permitted gas collection, continuous feeding, and wasting at different retention times. The operation of the apparatus was controlled by a personal computer. Cellulose destruction ranged from ca. 30 to 70% for hydraulic retention times of 0.5 to 2.0 days. Carbon recovery in products was 92 to 97%, and the oxidation-reduction ratios ranged from 0.91 to 1.15. The total product yield (biomass not included) per gram of cellulose (expressed as glucose) was 0.83 g g, and the ethanol yield was 0.41 g g. The product yield was constant, indicating that product formation was growth linked.

Kinetics of Insoluble Cellulose Fermentation by Continuous Cultures of Ruminococcus albus.

Data from analyses of continuous culture fermentation of insoluble cellulose by Ruminococcus albus 7 were used to derive constants for the rate of cellulose hydrolysis and fermentation, growth yield, and maintenance. Cellulose concentration was 1% in the nutrient reservoir, and hydraulic retention times of 0.5, 1.0, 1.5, 1.75, and 2.0 days were used. Concentrations of reducing sugars in the cultures were negligible (less than 1%) compared with the amount of hydrolyzed cellulose, indicating that cellulose hydrolysis was the rate-limiting step of the fermentation. The rate of utilization of cellulose depended on the steady-state concentration of cellulose and was first order with a rate constant (k) of 1.18 day. The true microbial growth yield (Y) was 0.11 g g, the maintenance coefficient (m) was 0.10 g g h, and the maximum Y(ATP) was 7.7 g of biomass (dry weight) mol of ATP.

A kinetic model for anaerobic digestion of biological sludge.

The principal objective of this study was the development and evaluation of a comprehensive kinetic model capable of predicting digester performance when fed biological sludge, preliminary conversion mechanisms such as cell death, lysis, and hydrolysis responsible for rendering viable biological sludge organisms to available substrate were studied in depth. The results of this study indicate that hydrolysis of the dead, particulate biomass-primarily consisting of protein-is the slowest step, and therefore kinetically controls the overall process of anaerobic digestion of biological sludge. A kinetic model was developed which could accurately describe digester performance and predict effluent quality.

Alkaline treatment of wheat straw for increasing anaerobic biodegradability.

Wheat straw was treated with NaOH and anaerobically digested for methane production. Alkaline treatment resulted in a greater than 100% increase in biodegradability of wheat straw. The potential of a process flow scheme employing high alkali concentration at ambient temperature with solids separation and recycle of filtrate containing residual alkali was explored. The effect of NaOH on the solubilization of cell wall constituents and potential problems of toxicity are discussed. A solubilization model was developed which is used to predict biodegradability of whole samples based on solids and filtrate biodegradabilities. Energy requirements and chemical costs are also addressed.