General method for determining anaerobic biodegradation potential.

A simple, generalized method was refined and validated to test whether an organic chemical was susceptible to anaerobic degradation to CH4 + CO2. The method used digested sewage sludge diluted to 10% and incubated anaerobically in 160-ml serum bottles with 50 micrograms of C per ml of test chemical. Biodegradation was determined by the net increase in gas pressure in bottles with test chemicals over the pressure in nonamended sludge bottles. Gas production was measured by gas chromatography and by a pressure transducer. The latter method is recommended because of its speed, accuracy, and low cost. Sewage sludge from municipal digesters with 15- to 30-day retention times was found to be suitable. The sludge could be stored anaerobically at 4 degrees C for up to 4 weeks with satisfactory test results. p-Cresol, phthalic acid, and ethanol are suggested as reference chemicals to confirm sludge activity and method reliability. A revised anaerobic salts medium was developed which minimizes problems of a biological gas production (CO2), avoids precipitation, and meets the requirements of the anaerobic microbiota. When greater than 75% of the theoretical gas production was observed, the chemical was judged to be degradable, and when 30 to 75% of the expected gas was produced, it was termed partially degradable. This method has been tested on more than 100 chemicals of various physical properties and found to reproducibly determine anaerobic biodegradation potential. Of the chemicals tested, 46 were found to be anaerobically degraded. Sludges from nine different municipal treatment plants were surveyed for their ability to degrade nine chemicals which differed in susceptibility to degradation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolic biomarkers for monitoring anaerobic naphthalene biodegradation in situ

During the anaerobic biodegradation of naphthalene and methylnaphthalene, unique metabolites are formed by specific microbial carboxylation and ring-reduction reactions. Groundwater samples from an anoxic, shallow aquifer contaminated with gasoline were examined for the presence of these metabolites by extraction, derivatization and gas chromatography coupled to mass spectroscopy. Several metabolites [2-naphthoic acid (2-NA), tetrahydro-2-naphthoic acid (TH-2-NA), hexahydro-2-naphthoic acid (HH-2-NA) and methylnaphthoic acid (MNA)] were found to be present in the groundwater samples. The concentration of 2-NA at each monitoring well was quantified and correlated to the zones of contamination. The presence of the other metabolites in the same wells as 2-NA was used as confirmation that the anaerobic pathway was indeed active. The distribution of metabolites at this site shows that they can be used as biomarkers for demonstrating in situ biodegradation.     

Sulfate-reducing anaerobic ammonium oxidation as a potential treatment method for high nitrogen-content wastewater

After sulfate-reducing ammonium oxidation (SRAO) was first assumed in 2001, several works have been published describing this process in laboratory-scale bioreactors or occurring in the nature. In this paper, the SRAO process was performed using reject water as a substrate for microorganisms and a source of NH(4) (+), with SO(4) (2-) being added as an electron acceptor. At a moderate temperature of 20°C in a moving bed biofilm reactor (MBBR) sulfate reduction along with ammonium oxidation were established. In an upflow anaerobic sludge blanket reactor (UASBR) the SRAO process took place at 36°C. Average volumetric TN removal rates of 0.03?kg-N/m3/day in the MBBR and 0.04?kg-N/m3/day in the UASBR were achieved, with long-term moderate average removal efficiencies, respectively. Uncultured bacteria clone P4 and uncultured planctomycete clone Amx-PAn30 were detected from the biofilm of the MBBR, from sludge of the UASBR uncultured Verrucomicrobiales bacterium clone De2102 and Uncultured bacterium clone ATB-KS-1929 were found also. The stoichiometrical ratio of NH(4) (+) removal was significantly higher than could be expected from the extent of SO(4) (2-) reduction. This phenomenon can primarily be attributed to complex interactions between nitrogen and sulfur compounds and organic matter present in the wastewater. The high NH(4) (+) removal ratio can be attributed to sulfur-utilizing denitrification/denitritation providing the evidence that SRAO is occurring independently and is not a result of sulfate reduction and anammox. HCO(3) (-) concentrations exceeding 1,000?mg/l were found to have an inhibiting effect on the SRAO process. Small amounts of hydrazine were naturally present in the reaction medium, indicating occurrence of the anammox process. Injections of anammox intermediates, hydrazine and hydroxylamine, had a positive effect on SRAO process performance, particularly in the case of the UASBR.     

Alternative methods for determining anaerobic biodegradability: A review

The characterization of solid wastes is a necessary step before they can be used in anaerobic digestion. The quantities of different compounds (carbohydrates, proteins, lipids and fibers) and anaerobic biodegradability (capacity to produce methane) are important information required to characterize waste. The Biochemical Methane Potential (BMP) test is one of the most relevant tests for assessing the biodegradability of waste materials. The BMP test is run under anaerobic conditions, using bacteria populations, which makes it very time consuming, i.e., about 30 days. This paper presents alternative methods for determining the anaerobic biodegradability of solid waste. First, we describe the already existing tests for characterizing organic matter. Then we correlate an aerobic test with an anaerobic test in order to estimate anaerobic biodegradability and biogas production. This shortens the analysis time to 5 days. Models using physico-chemical characteristics as input data (total carbohydrate, total nitrogen, fiber, etc.) can predict the amount of methane produced by correlation. Pyrolysis is a very fast analytical test that can be used to characterize solid waste. Lastly, spectroscopy techniques seem to be useful for determining biodegradability, in particular by taking into account the interaction between different molecules in the organic matter.     

Kinetics of anaerobic biodegradation of glycerol by sulfate-reducing bacteria

A kinetic model has been developed and kinetic parameters of anaerobic degradation of glycerol, an abundant by-product of biofuel manufacturing, by a consortium of sulfate reducing bacteria (SRB) in a closed system have been determined. The following main species of SRB has been identified in the consortium: Desulfovibrio baarsii, Desulfomicrobium sp., and Desufatomaculum sp. The proposed model included processes of glycerol degradation, sulfate reduction, and inhibition by metabolic products, as well as effects of pH and temperature. The suggested equation for the anaerobic glycerol degradation was based on Edward and Andrew’s equation. The following kinetic parameters of the anaerobic glycerol degradation were obtained for the initial glycerol concentration from 0.15 to 4 ml/l and sulfate concentration of 2760 mg/l at 22°C: maximum specific growth rate of SRB μ_max = 0.56 day⁻¹, economic coefficient of ashless biomass from glycerol of 0.08 mol SRB/mol COC, and yield of ashless biomass from sulfate of 0.020 mol SRB/mol SO₄. It was shown that the optimum molar ratio of $ {{C_{Gl} } \mathord{\left/ {\vphantom {{C_{Gl} } {C_{SO_4 } }}} \right. \kern-\nulldelimiterspace} {C_{SO_4 } }} $ {{C_{Gl} } \mathord{\left/ {\vphantom {{C_{Gl} } {C_{SO_4 } }}} \right. \kern-\nulldelimiterspace} {C_{SO_4 } }} for SRB growth was 0.8. Initial boundary concentration of inhibition by undissociated hydrogen sulfide was 70 mg/l. Dependence of the specific growth rate of bacteria on the temperature was approximated by the Arrhenius equation in the temperature range of 20–30°C with the goodness of fit R² = 0.99.     

Sulphur fate and anaerobic biodegradation potential during co-digestion of seaweed biomass (Ulva sp.) with pig slurry

Seaweed (Ulva sp.) stranded on beaches were utilized as co-substrate for anaerobic digestion of pig slurry in three-month co-digestion tests in pilot scale anaerobic digesters in the laboratory. The methanogenic potential of Ulva sp. was low compared to that of other potential co-substrates available for use by farmers: 148 N m3CH4/t of volatile solids or 19 N m3CH4/t of crude product. When used as a co-substrate with pig manure (48%/52% w/w), Ulva sp. seaweed did not notably disrupt the process of digestion; however, after pilot stabilisation, biogas produced contained 3.5% H2S, making it unsuitable for energy recovery without treatment. Sequentially addition of the sulphate reduction inhibitor, potassium molybdate, to a final concentration of 3mM, temporarily reduced H2S emissions, but was unable to sustain this reduction over the three-month period. According to these pilot tests, the use of seaweed stranded on beaches as co-substrate in farm-based biogas plants shows some limitations.     

Toxicity and biodegradation of formaldehyde in anaerobic methanogenic culture

Formaldehyde is present in several industrial wastewaters including petrochemical wastes. In this study, the toxicity and degradability of formaldehyde in anaerobic systems were investigated. Formaldehyde showed severe toxicity to an acetate enrichment methanogenic culture. As low as 10 mg/L (0.33 mM) of formaldehyde in the reactor completely inhibited acetate utilization. Formaldehyde, however, was degraded while acetate utilization was inhibited. Degradation of formaldehyde (Initial concentration /=60 mg/L), formaldehyde degradation was inhibited and partial degradation was possible. The initial formaldehyde to biomass ratio, S(0)/X(0), was useful to predict the degradation potential of high formaldehyde concentrations in batch systems. When S(0)/X(0) /= 0.29, formaldehyde at higher than 60 mg/L was only partially degraded. The inhibition of formaldehyde degradation in batch systems could be avoided by repeated additions of low concentrations of formaldehyde (up to 30 mg/L). Chemostats (14-day retention time) showed degradation of 74 mg/L-d (1110 mg/L) of influent formaldehyde with a removal capacity of 164 mg/g VSS-day. A spike of 30 mg/L (final concentration in the chemostat) formaldehyde to the chemostat caused only a small increase in effluent acetate concentration for 3 days. But a spike of 60 mg/L (final concentration in the chemostat) formaldehyde to the chemostat resulted in a dramatic increase in acetate concentration in the effluent. The results also showed that the acetate enrichment culture was not acclimated to formaldehyde even after 226 days. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 727-736, 1997.     

Monitoring of two-stage anaerobic biodegradation using a BOD biosensor

A previously developed biosensor for fast estimation of short-term biochemical oxygen demand (BOD_st) was used for off-line monitoring of intermediate products from the initial step of an anaerobic process in laboratory scale. Good agreement was generally achieved between the results from the biosensor method and the conventional 5-day test except for samples with high content of organic polymers. During the period of agreement between the measurement principles, good correlation was achieved between the biogas production rate and the organic loading rate. The results from this study demonstrate that BOD_st can be a successful monitoring parameter to achieve a better process control.     

Synthesis and anaerobic biodegradation of indomethacin-conjugated cellulose ethers used for colon-specific drug delivery

Water soluble cellulose ethers, including methylcellulose and two hydroxyethylcelluloses with different molecular weights, were conjugate with indomethacin at room temperature. The chemical structures of the conjugates were characterized by FTIR, ¹H NMR and UV–vis spectroscopy. The results confirmed that different amounts of IND residues were covalently bonded to cellulose ether backbones through ester linkages. Their anaerobic biodegradation in colonic fermentation was investigated by gel permeation chromatography, gas chromatography and UV–vis spectroscopy. These conjugates were found to have different biodegradabilities, depending on the cellulose ether used and the amount of conjugated indomethacin residues. In vitro release experiments showed that hydroxyethylcellulose-based conjugates with low IND residues content could exhibit a sustained drug release behavior in colonic fermentation and were stable in the simulated media of the stomach and small intestine. Therefore, they are promising candidates for future applications in colon-specific drug delivery.     

Thermophilic biodegradation of BTEX by two consortia of anaerobic bacteria

Two thermophilic anaerobic bacterial consortia (ALK-1 and LLNL-1), capable of degrading the aromatic fuel hydrocarbons, benzene, toluene, ethylbenzene, and the xylenes (BTEX compounds), were developed at 60 °C from the produced water of ARCO'S Kuparuk oil field at Alaska and the subsurface water at the Lawrence Livermore National Laboratory gasoline-spill site, respectively. Both consortia were found to grow at 45–75 °C on BTEX compounds as their sole carbon and energy sources with 50 °C being the optimal temperature. With 3.5 mg total BTEX added to sealed 50-ml serum bottles, which contained 30 ml mineral salts medium and the consortium, benzene, toluene, ethylbenze, m-xylene, and an unresolved mixture of o- and p-xylenes were biodegraded by 22%, 38%, 42%, 40%, and 38%, respectively, by ALK-1 after 14 days of incubation at 50 °C. Somewhat lower, but significant, percentages of the BTEX compounds also were biodegraded at 60 °C and 70 °C. The extent of biodegradation of these BTEX compounds by LLNL-1 at each of these three temperatures was slightly less than that achieved by ALK-1. Use of [ring-¹⁴C]toluene in the BTEX mixture incubated at 50 °C verified that 41% and 31% of the biodegraded toluene was metabolized within 14 days to water-soluble products by ALK-1 and LLNL-1, respectively. A small fraction of it was mineralized to ¹⁴CO₂. The use of [U-¹⁴C]benzene revealed that 2.6%–4.3% of the biodegraded benzene was metabolized at 50 °C to water-soluble products by the two consortia; however, no mineralization of the degraded [U-¹⁴C]benzene to ¹⁴CO₂ was observed. The biodegradation of BTEX at all three temperatures by both consortia was tightly coupled to sulfate reduction as well as H₂S generation. None was observed when sulfate was omitted from the serum bottles. This suggests that sulfate-reducing bacteria are most likely responsible for the observed thermophilic biodegradation of BTEX in both consortial cultures. Received: 12 July 1996 / Received revision: 31 December 1996 / Accepted: 31 January 1997     

Sequential anaerobic/aerobic biodegradation of chloroethenes--aspects of field application

Because of a range of different industrial activities, sites contaminated with chloroethenes are a world-wide problem. Chloroethenes can be biodegraded by reductive dechlorination under anaerobic conditions as well as by oxidation under aerobic conditions. The tendency of chloroethenes to undergo reductive dechlorination decreases with a decreasing number of chlorine substituents, whereas with less chlorine substituents chloroethenes more easily undergo oxidative degradation. There is currently a growing interest in aerobic metabolic degradation of chloroethenes, which demonstrates advantages compared to cometabolic degradation pathways. Sequential anaerobic/aerobic biodegradation can overcome the disadvantages of reductive dechlorination and leads to complete mineralization of the chlorinated pollutants. This approach shows promise for site remediation in natural settings and in engineered systems.     

Enhanced anaerobic biodegradation of benzene-toluene-ethylbenzene-xylene-ethanol mixtures in bioaugmented aquifer columns

Methanogenic flowthrough aquifer columns were used to investigate the potential of bioaugmentation to enhance anaerobic benzene-toluene-ethylbenzene-xylene (BTEX) degradation in groundwater contaminated with ethanol-blended gasoline. Two different methanogenic consortia (enriched with benzene or toluene and o-xylene) were used as inocula. Toluene was the only hydrocarbon degraded within 3 years in columns that were not bioaugmented, although anaerobic toluene degradation was observed after only 2 years of acclimation. Significant benzene biodegradation (up to 88%) was observed only in a column bioaugmented with the benzene-enriched methanogenic consortium, and this removal efficiency was sustained for 1 year with no significant decrease in permeability due to bioaugmentation. Benzene removal was hindered by the presence of toluene, which is a more labile substrate under anaerobic conditions. Real-time quantitative PCR analysis showed that the highest numbers of bssA gene copies (coding for benzylsuccinate synthase) occurred in aquifer samples exhibiting the highest rate of toluene degradation, which suggests that this gene could be a useful biomarker for environmental forensic analysis of anaerobic toluene bioremediation potential. bssA continued to be detected in the columns 1 year after column feeding ceased, indicating the robustness of the added catabolic potential. Overall, these results suggest that anaerobic bioaugmentation might enhance the natural attenuation of BTEX in groundwater contaminated with ethanol-blended gasoline, although field trials would be needed to demonstrate its feasibility. This approach may be especially attractive for removing benzene, which is the most toxic and commonly the most persistent BTEX compound under anaerobic conditions.     

Carbon and hydrogen isotopic fractionation during anaerobic biodegradation of benzene

Compound-specific isotope analysis has the potential to distinguish physical from biological attenuation processes in the subsurface. In this study, carbon and hydrogen isotopic fractionation effects during biodegradation of benzene under anaerobic conditions with different terminal-electron-accepting processes are reported for the first time. Different enrichment factors (epsilon ) for carbon (range of -1.9 to -3.6 per thousand ) and hydrogen (range of -29 to -79 per thousand ) fractionation were observed during biodegradation of benzene under nitrate-reducing, sulfate-reducing, and methanogenic conditions. These differences are not related to differences in initial biomass or in rates of biodegradation. Carbon isotopic enrichment factors for anaerobic benzene biodegradation in this study are comparable to those previously published for aerobic benzene biodegradation. In contrast, hydrogen enrichment factors determined for anaerobic benzene biodegradation are significantly larger than those previously published for benzene biodegradation under aerobic conditions. A fundamental difference in the previously proposed initial step of aerobic versus proposed anaerobic biodegradation pathways may account for these differences in hydrogen isotopic fractionation. Potentially, C-H bond breakage in the initial step of the anaerobic benzene biodegradation pathway may account for the large fractionation observed compared to that in aerobic benzene biodegradation. Despite some differences in reported enrichment factors between cultures with different terminal-electron-accepting processes, carbon and hydrogen isotope analysis has the potential to provide direct evidence of anaerobic biodegradation of benzene in the field.     

The anaerobic biodegradation of diethanolamine by a nitrate reducing bacterium

The ability of bacterial cultures to degrade diethanolamine under anoxic conditions with nitrate as an electron acceptor was investigated. A mixed culture capable of anaerobic degradation of diethanolamine was obtained from river sediments by enrichment culture. From this a single bacterial strain was isolated which could use diethanolamine, monoethanolamine, triethanolamine and N-methyl diethanolamine as its sole carbon and energy sources either aerobically or anaerobically. Growth on diethanolamine was faster in the absence of oxygen. The accumulation of possible metabolites in the culture medium was determined as was the ability to grow on certain putative intermediates in the degradation of diethanolamine. A possible pathway for the degradation of ethanolamines by this organism is suggested.     

Photometric method for determining ethanol

A novel enzymatic photometric assay for ethanol determination using alcohol oxidase and peroxidase is described. The sensitivity of the method allows detecting ethanol in biological fluids (saliva and blood serum). Secondary alcohols and other organic compounds do not interfere with the assay. General-purpose spectrophotometers and photoelectric colorimeters can be used in the measurements. Methanol and propanol can also be determined by this technique.     

The anaerobic biodegradation of diethanolamine by a nitrate reducing bacterium

The ability of bacterial cultures to degrade diethanolamine under anoxic conditions with nitrate as an electron acceptor was investigated. A mixed culture capable of anaerobic degradation of diethanolamine was obtained from river sediments by enrichment culture. From this a single bacterial strain was isolated which could use diethanolamine, monoethanolamine, triethanolamine and N-methyl diethanolamine as its sole carbon and energy sources either aerobically or anaerobically. Growth on diethanolamine was faster in the absence of oxygen. The accumulation of possible metabolites in the culture medium was determined as was the ability to grow on certain putative intermediates in the degradation of diethanolamine. A possible pathway for the degradation of ethanolamines by this organism is suggested.     

Ecologically safe method for improved feather wastes biodegradation

Twenty thermophilic actinomycetes were selected that were able to degrade feather wastes in high degree (87–91%). Increased proteolytic and lipolytic activity and exopolysaccharide production of the strains growing on feathers suggested their important role in the complex process of feathers biodegradation. Highest soluble protein content was determined in the fermented broths of the strains 3H, 8H, 4C, M4, and 27A. Based on data obtained, a mixed culture of three selected thermoactinomycete strains – 3H, 8H and M4, has been designed that considerably improved the feathers digestion process. The addition of feathers from 0.7 to 3% and pH values from 7.5 to 8.5 provided highest soluble protein content in the fermented broth of the mixed culture. It was shown that the hydrolysate obtained after 72 h growth of the mixed culture on feathers is rich in soluble proteins and amino acids including essential ones like lysine, threonine, leucine, isoleucine and valine, and rare ones like threonine, proline and serine. Therefore, the obtained value-added protein hydrolysate could be used for preparation of fertilizers or soil amendments, as well as protein source in animal feeding. To our knowledge, this is first report of using mixed culture of thermoactinomycete strains for improved feathers biodegradation. The proposed ecologically safe method is simple and economically viable thus applicable on industrial scale.