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.     

Assessment of the influence of thermal pre-treatment time on the macromolecular composition and anaerobic biodegradability of sewage sludge

Laboratory and pilot-scale experiments were carried out in order to evaluate the influence of thermal pre-treatment time on waste-activated sludge properties and anaerobic biodegradability. Six experimental conditions were analyzed from 0 to 30 min of hydrolysis time. Solubilization of macromolecular compounds, changes in the main sludge properties and anaerobic biodegradability of the sewage sludge were evaluated. A similar carbohydrate solubilization degree was achieved, from 53% to 70% and 59% to 75% for lab- and pilot-scale experiments, respectively. In the case of proteins, the values of solubilization were lower in the pilot-scale experiment than in the laboratory, with 31-45% and 47-70%, respectively. Ammonia and volatile fatty acid did not undergo important changes; however the sludge dewaterability enhanced at increased pre-treatment times. All the pre-treatment conditions had a positive effect with regard to anaerobic biodegradability and by fitting experimental data with a simplified mathematical model, it was concluded that the maximum biogas production rate is more influenced by the pre-treatment time than the total biogas production.     

Equipment used for testing anaerobic biodegradability and activity

Recently there has been a growing demand for information on the biodegradability and microbial toxicity of xenobiotic compounds. The environmental fate and effect of these chemicals in aerobic and anaerobic conditions has been extensively studied using a variety of different methods. This paper reviews the different protocols, equipment and instruments used in anaerobic biodegradation and activity tests, including the more sophisticated automated techniques recently developed. The different systems for detecting biogas production are given particular attention. As well as gasometric methods, the different instrumental techniques used to follow the fate of compounds under anaerobic conditions, via analysis of substrate consumption and product formation are evaluated.     

Anaerobic Biodegradation of Indole to Methane

Methane gas was produced from a laboratory, granular activated carbon, anaerobic filter treating a synthetically prepared mixture of polycyclic N-aromatic compounds. The biodegradability of the individual polycyclic N-aromatic compound present in the mixture was investigated. Experimental results obtained from test bottles containing methanogenic enrichment cultures suggested that indole was degraded to methane and carbon dioxide under strict anaerobic conditions.     

The influence of structural components of alkyl esters on their anaerobic biodegradation in marine sediment

Ester-based organic compounds are one type of synthetic base fluid added to drilling mud used during off-shore oil-drilling operations in the Gulf of Mexico. Concern over the environmental impact of synthetic base fluid (SBF) contaminated rock cuttings discharged into the Gulf of Mexico has prompted the promulgation of EPA regulations requiring that all SBF be tested for biodegradability in marine sediment prior to their use in the Gulf. In order to allow the design or selection of suitably biodegradable esters, the anaerobic biodegradability of a variety of ester compounds was tested using a marine sediment inoculum to reveal the effect of: (a) increasing the chain length of the acid moiety, (b) increasing the chain length of the alcohol moiety; (c) alternating the relative size of the alcohol and acid moieties, (d) branching in the alcohol moiety, and (e) the presence of an unsaturated bond in the acidic moiety. The chemical structure of esters was found to affect the completeness and rate of anaerobic biodegradation, and would affect their ability to be certified for use as an SBF in the Gulf of Mexico. Recommendations for ester usage include using esters that have a total carbon number of between 12 and 18 and avoiding the use of branched alcohols (or acids by inference). The presence of an unsaturated bond in the acid (or alcohol by inference) increased biodegradability of the ester.     

Influence of alternative electron acceptors on the anaerobic biodegradability of chlorinated phenols and benzoic acids.

Nitrate, sulfate, and carbonate were used as electron acceptors to examine the anaerobic biodegradability of chlorinated aromatic compounds in estuarine and freshwater sediments. The respective denitrifying, sulfidogenic, and methanogenic enrichment cultures were established on each of the monochlorinated phenol and monochlorinated benzoic acid isomers, using sediment from the upper (freshwater) and lower (estuarine) Hudson River and the East River (estuarine) as source materials. Utilization of each chlorophenol and chlorobenzoate isomer was observed under at least one reducing condition; however, no single reducing condition permitted the metabolism of all six compounds tested. The anaerobic biodegradation of the chlorophenols and chlorobenzoates depended on the electron acceptor available and on the position of the chlorine substituent. In general, similar activities were observed under the different reducing conditions in both the freshwater and estuarine sediments. Under denitrifying conditions, degradation of 3- and 4-chlorobenzoate was accompanied by nitrate loss corresponding reasonably to the stoichiometric values expected for complete oxidation of the chlorobenzoate to CO2. Under sulfidogenic conditions, 3- and 4-chlorobenzoate, but not 2-chlorobenzoate, and all three monochlorophenol isomers were utilized, while under methanogenic conditions all compounds except 4-chlorobenzoate were metabolized. Given that the pattern of activity appears different for these chlorinated compounds under each reducing condition, their biodegradability appears to be more a function of the presence of competent microbial populations than one of inherent molecular structure.     

Grey water biodegradability

Knowing the biodegradability characteristics of grey water constituents is imperative for a proper design and operation of a biological treatment system of grey water. This study characterizes the different COD fractions of dormitory grey water and investigates the effect of applying different conditions in the biodegradation test. The maximum aerobic and anaerobic biodegradability and conversion rate for the different COD fractions is determined. The results show that, on average, dormitory grey water COD fractions are 28% suspended, 32% colloidal and 40% dissolved. The studied factors incubation time, inoculum addition and temperature are influencing the determined biodegradability. The maximum biodegradability and biodegradation rate differ between different COD fractions, viz. COD_ss, COD_col and COD_diss. The dissolved COD fraction is characterised by the lowest degradation rate, both for anaerobic and aerobic conditions. The maximum biodegradability for aerobic and anaerobic conditions is 86 and 70% respectively, whereas the first order conversion rate constant, k ₂₀, is 0.119 and 0.005 day⁻¹, respectively. The anaerobic and aerobic conversion rates in relation to temperature can be described by the Arrhenius relation, with temperature coefficients of 1.069 and 1.099, respectively.     

Effect of low power ultrasonic radiation on anaerobic biodegradability of sewage sludge

The effect of low power ultrasonic radiation on anaerobic biodegradability of sewage sludge was investigated. For this purpose, soluble substances and variation of microbial system of sewage sludge subjected to low power ultrasonic radiation were tested. The well known hydromechanical shear forces and heating effect of low frequency ultrasound plays a major role in the sludge pre treatment process. More, the increase of soluble substance may partly result from the destruction of microbial cell by excess ultrasonic pretreatment, which will inhibit the anaerobic process. By orthogonal tests, the optimal parameters were found to be an exposure time of 15 min, ultrasonic intensity of 0.35 W/cm² and ultrasonic power density of 0.25 W/ml. Under the optimal condition, anaerobic biodegradability of sewage sludge (R_(vss/ss) %) was increased by 67.6%. Consequently, it can be concluded that low power ultrasonic pretreatment is a valid method for improving anaerobic biodegradability of sewage sludge.     

Feasibility of anaerobic co-digestion as a treatment option of meat industry wastes

The anaerobic biodegradability of meat industry wastes was investigated in mesophilic batch reactors and combined with a mathematical model for describing their biodegradable fractions. The characteristics and methane yield achieved when digesting waste sludge, suggested the use of this as co-substrate for enhancing the biodegradability of the other wastes. The co-digestion experiments showed that it would be feasible to co-digest cow manure or ruminal waste with waste sludge, but biodegradability of pig/cow slurries was not improved, being strongly influenced by the ammonium concentration of co-digestion mixture. By applying the mathematical model, it was observed that when increasing the amount of waste sludge in the co-digestion mixtures, the amount of inert and slowly biodegradable fractions decreased leading to an increase in readily biodegradable fractions, volatile solid removal efficiencies and methane yields. These results suggest that using readily biodegradable wastes as co-substrate, the anaerobic biodegradability of complex organic wastes can be improved.     

Survey of the Anaerobic Biodegradation Potential of Organic Chemicals in Digesting Sludge

The degradation potential of 77 organic chemicals under methanogenic conditions was examined with an anaerobic digesting sludge from the United Kingdom. Degradation was assessed in terms of net total gas (CH₄ plus CO₂) produced, expressed as a percentage of the theoretical production (ThGP). The compounds tested were selected from various chemical groups and included substituted phenols and benzoates, pesticides, phthalic acid esters, homocyclic and heterocyclic ring compounds, glycols, and monosubstituted benzenes. The results obtained were in good agreement with published surveys of biodegradability in U.S. digesting sludges and other methanogenic environments. In general, the presence of chloro or nitro groups inhibited anaerobic gas production, while carboxyl and hydroxyl groups facilitated biodegradation. The relationship between substituent position and susceptibility to methanogenic degradation was compound dependent. The following chemicals were completely degraded (≥80% ThGP) at a concentration of 50 mg of carbon per liter: phenol, 2-aminophenol, 4-cresol, catechol, sodium benzoate, 4-aminobenzoic acid, 3-chlorobenzoic acid, phthalic acid, ethylene glycol, diethylene glycol, triethylene glycol, sodium stearate, and quinoline. 3-Cresol, 4-chlorobenzoic acid, dimethyl phthalate, and pyridine were partially degraded. Although the remaining chemicals tested were either persistent or toxic, their behavior may differ at more environmentally realistic chemical-to-biomass ratios. Our findings suggest that biodegradability assessments made with sludge from one source can be extrapolated to sludge from another source with a reasonable degree of confidence and should help in predicting the fate of an organic chemical during the anaerobic digestion of sewage sludge.     

A comprehensive review of the screening methodology for anaerobic biodegradability of surfactants

A biodegradability assay should mimic in situ conditions as closely as possible. If this is not entirely possible, the assayshould at least include inoculum from the site. This review attemptsto condense current literature on anaerobic biodegradability assayand propose a clear assessment methodology to determine the fatesurfactants in anaerobic environments. It has been well documentedthat surfactant concentrations toxic to the microflora can lead tounwarranted failure of biodegradability assays. Thus, an important recommendation is to first perform a toxicity evaluation with relevant controls. Based on the results of this evaluation, a Tier 1biodegradability assay that assesses the rate of formation of reducedendproducts or the consumption of a particular terminal electron acceptor is recommended and supported by current literature. Balancedchemical equations for the complete mineralization of the substrateare then used to compare the amount of transformation that actuallyoccurred with that theoretically expected. When required, resultsshould be confirmed by Tier 2 testing, which includes monitoring ofsubstrate disappearance over time using a variety of analytical tools.These recommended procedures are scientifically defensible and havethe potential of providing environmentally relevant information on thefate of surfactant materials in the environment.     

Comparison of experimental methods for determination of toxicity and biodegradability of xenobiotic compounds

Different methods for determining the toxicity and biodegradability of hazardous compounds evaluating their susceptibility to biological treatment were studied. Several compounds including chlorophenols and herbicides have been evaluated. Toxicity was analyzed in terms of EC₅₀ and by a simple respirometric procedure based on the OECD Method 209 and by the Microtox^® bioassay. The values of EC₅₀ obtained from respirometry were in all the cases higher than those from the Microtox^® test. The respirometric inhibition values of chlorophenols were related well with the number of chlorine atoms and their position in the aromatic ring. In general, herbicides showed lower inhibition, being alachlor the less toxic from this criterion. For determination of biodegradability an easier and faster alternative to the OECD Method 301, with a higher biomass to substrate ratio is proposed. When this test was negative, the Zahn-Wellens one was performed in order to evaluate the inherent biodegradability. In the fast test of biodegradability, 4-chlorocatechol and 4-chlorophenol showed a complete biodegradation by an unacclimated sludge upon 48 h. These results together with their low respirometric inhibition, allow concluding that these compounds could be conveniently removed in a WWTP. Alachlor, 2,4-dichlorophenol, 2,4,6-trichlorophenol and MCPA showed a partial biodegradation upon 28 days by the Zahn-Wellens inherent biodegradability test.     

Fate and biodegradability of sulfonated aromatic amines

Ten sulfonated aromatic amines were tested for their aerobic and anaerobic biodegradability and toxicity potential in a variety of environmental inocula. Of all the compounds tested, only two aminobenzenesulfonic acid (ABS) isomers, 2- and 4-ABS, were degraded. The observed degradation occurred only under aerobic conditions with inocula sources that were historically polluted with sulfonated aromatic amines. Bioreactor experiments, with non-sterile synthetic wastewater, confirmed the results from the aerobic batch degradation experiments. Both ABS isomers were degraded in long-term continuous experiment by abioaugmented enrichment culture. The maximum degradation rate in the aerobic bioreactor was 1.6–1.8 gl^–1 d^–1 for 2-ABS and a somewhat lower value for 4-ABS at hydraulic retention times (HRT) of 2.8–3.3h. Evidence for extensive mineralization of 2- and 4-ABS was based on oxygen uptake and carbon dioxide production during the batch experiments and the high levels of chemical oxygen demand (COD) removal in the bioreactor. Furthermore, mineralization of the sulfonate group was demonstrated by high recovery of sulfate. The sulfonated aromatic amines did not show any toxic effects on the aerobic and anaerobic bacterial populations tested. The poor biodegradability of sulfonated aromatic amines indicated under the laboratory conditions of this study suggests that these compounds may not be adequately removed during biological wastewater treatment.     

Molecular weight distribution of Pinus radiata kraft mill wastewater treated by anaerobic digestion

Kraft mill is responsible for massive discharge of highly polluted effluents. The main characteristics of this effluent are high toxicity and low biodegradability due to tannin, lignin and chlorophenol compounds. The composition may vary dramatically depending, for instance, on the utilised feedstock and process. The purpose of this work was to investigate the molecular weight distribution of Pinus radiata kraft pulping wastewater treated by anaerobic digestion by using two types of anaerobic reactors: fixed bed and sludge blanket. Anaerobic sludge blanket (UASB) and anaerobic filter (AF) were operated. In both reactors, the total alkalinity ranged between 1.0 and 1.5 g CaCO3/l, while the organic load rate (OLR) was increasing during operation from 1.2 to 3.3 gCOD/l d. COD and total phenolic compounds (UV215) removal ranged between 30-50% and 13-20%, respectively, while the BOD5 removal ranged 60-90%. However only a partial biodegradation (10-43%) of tannin and lignin was observed. Results from ultrafiltration analyses indicated that the fraction with a molecular weight (MW) < 1000, COD and colour decreased after anaerobic treatment, but the total phenolic compounds increased. In the 1000 < MW < 10,000 fraction, there was no change in COD, UV215 and colour. In the > 10,000 MW fraction, colour and COD fraction increased by 14% and 5%, respectively, after anaerobic treatment. It can be concluded from this study, that treatment with UASB or AF reactors is not enough, under the conditions tested, for a large COD removal from Pinus radiata wastewater.     

Biodegradability of chlorinated solvents and related chlorinated aliphatic compounds

The biodegradability of chlorinated methanes, chlorinated ethanes, chlorinated ethenes, chlorofluorocarbons (CFCs), chlorinated acetic acids, chlorinated propanoids and chlorinated butadienes was evaluated based on literature data. Evidence for the biodegradation of compounds in all of the compound categories evaluated has been reported. A broad range of chlorinated aliphatic structures are susceptible to biodegradation under a variety of physiological and redox conditions. Microbial biodegradation of a wide variety of chlorinated aliphatic compounds was shown to occur under five physiological conditions. However, any given physiological condition could only act upon a subset of the chlorinated compounds. Firstly, chlorinated compounds are used as an electron donor and carbon source under aerobic conditions. Secondly, chlorinated compounds are cometabolized under aerobic conditions while the microorganisms are growing (or otherwise already have grown) on another primary substrate. Thirdly, chlorinated compounds are also degraded under anaerobic conditions in which they are utilized as an electron donor and carbon source. Fourthly, chlorinated compounds can serve as an electron acceptor to support respiration of anaerobic microorganisms utilizing simple electron donating substrates. Lastly chlorinated compounds are subject to anaerobic cometabolism becoming biotransformed while the microorganisms grow on other primary substrate or electron acceptor. The literature survey demonstrates that, in many cases, chlorinated compounds are completely mineralised to benign end products. Additionally, biodegradation can occur rapidly. Growth rates exceeding 1 d^-1 were observed for many compounds. Most compound categories include chlorinated structures that are used to support microbial growth. Growth can be due to the use of the chlorinated compound as an electron donor or alternatively to the use of the chlorinated compound as an electron acceptor (halorespiration). Biodegradation linked to growth is important, since under such conditions, rates of degradation will increase as the microbial population (biocatalyst) increases. Combinations of redox conditions are favorable for the biodegradation of highly chlorinated structures that are recalcitrant to degradation under aerobic conditions. However, under anaerobic conditions, highly chlorinated structures are partially dehalogenated to lower chlorinated counterparts. The lower chlorinated compounds are subsequently more readily mineralized under aerobic conditions.     

Effects of thermo-chemical pre-treatment on anaerobic biodegradability and hydrolysis of lignocellulosic biomass

The effects of different thermo-chemical pre-treatment methods were determined on the biodegradability and hydrolysis rate of lignocellulosic biomass. Three plant species, hay, straw and bracken were thermo-chemically pre-treated with calcium hydroxide, ammonium carbonate and maleic acid. After pre-treatment, the plant material was anaerobically digested in batch bottles under mesophilic conditions for 40 days. From the pre-treatment and subsequent anaerobic digestion experiments, it was concluded that when the lignin content of the plant material is high, thermo-chemical pre-treatments have a positive effect on the biodegradability of the substrate. Calcium hydroxide pre-treatment improves the biodegradability of lignocellulosic biomass, especially for high lignin content substrates, like bracken. Maleic acid generates the highest percentage of dissolved COD during pre-treatment. Ammonium pre-treatment only showed a clear effect on biodegradability for straw.     

Detoxification and partial mineralization of the azo dye mordant orange 1 in a continuous upflow anaerobic sludge-blanket reactor

In batch toxicity assays, azo dye compounds were found to be many times more toxic than their cleavage products (aromatic amines) towards methanogenic activity in anaerobic granular sludge. Considering the ability of anaerobic microorganisms to reduce azo groups, detoxification of azo compounds towards methanogens can be expected to occur during anaerobic wastewater treatment. In order to test this hypothesis, the anaerobic degradation of one azo dye compound, Mordant orange 1 (MO1), by granular sludge was investigated in three separate continuous upflow anaerobic sludge-blanket reactors. One reactor, receiving no cosubstrate, failed after 50 days presumably because of a lack of reducing equivalents. However, the two reactors receiving either glucose or a volatile fatty acids (acetate, propionate, butyrate) mixture, could eliminate the dye during operation for 217 days. The azo dye was reductively cleaved to less toxic aromatic amines (1,4-phenylenediamine and 5-aminosalicylic acid) making the treatment of MO1 feasible at influent concentrations that were over 25 times higher than their 50% inhibitory concentrations. In the reactor receiving glucose as cosubstrate, 5-aminosalicylic acid could only be detected at trace levels in the effluent after day 189 of operation. Batch biodegradability assays with the sludge sampled from this reactor confirmed the mineralization of 5-aminosalicylic acid to methane. Received: 11 July 1996 / Received revision: 18 September 1996 / Accepted: 18 September 1996     

Anaerobic digestion of amine-oxide-based surfactants: biodegradation kinetics and inhibitory effects

Recently, anaerobic degradation has become a prevalent alternative for the treatment of wastewater and activated sludge. Consequently, the anaerobic biodegradability of recalcitrant compounds such as some surfactants require a thorough study to avoid their presence in the environment. In this work, the anaerobic biodegradation of amine-oxide-based surfactants, which are toxic to several organisms, was studied by measuring of the biogas production in digested sludge. Three amine-oxide-based surfactants with structural differences in their hydrophobic alkyl chain were tested: Lauramine oxide (AO-R₁₂), Myristamine oxide (AO-R₁₄) and Cocamidopropylamine oxide (AO-cocoamido). Results show that AO-R₁₂ and AO-R₁₄ inhibit biogas production, inhibition percentages were around 90%. AO-cocoamido did not cause inhibition and it was biodegraded until reaching a percentage of 60.8%. Otherwise, we fitted the production of biogas to two kinetic models, to a pseudo first-order model and to a logistic model. Production of biogas during the anaerobic biodegradation of AO-cocoamido was pretty good adjusted to the logistics model. Kinetic parameters were also determined. This modelling is useful to predict their behaviour in wastewater treatment plants and under anaerobic conditions in the environment.     

Anaerobic degradation of monoaromatic hydrocarbons

Over the last two decades significant advances have been made in our understanding of the anaerobic biodegradability of monoaromatic hydrocarbons. It is now known that compounds such as benzene, toluene, ethylbenzene, and all three xylene isomers can be biodegraded in the absence of oxygen by a broad diversity of organisms. These compounds have been shown to serve as carbon and energy sources for bacteria growing phototrophically, or respiratorily with nitrate, manganese, ferric iron, sulfate, or carbon dioxide as the sole electron acceptor. In addition, it has also been recently shown that complete degradation of monoaromatic hydrocarbons can also be coupled to the respiration of oxyanions of chlorine such as perchlorate or chlorate, or to the reduction of the quinone moieties of humic substances. Many pure cultures of hydrocarbon-degrading anaerobes now exist and some novel biochemical and genetic pathways have been identified. In general, a fumarate addition reaction is used as the initial activation step of the catabolic process of the corresponding monoaromatic hydrocarbon compounds. However, other reactions may alternatively be involved depending on the electron acceptor utilized or the compound being degraded. In the case of toluene, fumarate addition to the methyl group mediated by benzylsuccinate synthase appears to be the universal mechanism of activation and is now known to be utilized by anoxygenic phototrophs, nitrate-reducing, Fe(III)-reducing, sulfate-reducing, and methanogenic cultures. Many of these biochemical pathways produce unique extracellular intermediates that can be utilized as biomarkers for the monitoring of hydrocarbon degradation in anaerobic natural environments.     

Applications of pressure transducers in anaerobic systems

Summary Pressure transducer assay methods are widely used to determine the methanogenic activity of anaerobic sludges and to evaluate the anaerobic biodegradability of organic chemicals. The application of this methodology to pure cultures of eubacterial and yeast species was investigated and the results obtained were in good agreement with conventional methods for assaying microbial growth and for determining the specific activity of resting cells. Because of its simplicity and amenability to computer control, the pressure transducer assay method represents a useful adjunct to the variety of methods in routine use for characterisation of anaerobic species.