Reviews in Environmental Science and Bio/Technology 3: 117129, 2004. 2004 Kluwer Academic Publishers. Printed in the Netherlands.

117

Assessment of the anaerobic biodegradability of macropollutants

Irini Angelidaki1; * & Wendy Sanders2
1

Environment & Recourses DTU, Building 115, 2800 Lyngby, Technical University of Denmark; 2 Wageningen Agricultural University, Subdepartment of Environmental Technology, P.O. Box 8129, 6700 EV Wageningen (*author for correspondence, phone: +45-45251429; fax: +45-45932850; e-mail: ria@er.dtu.dk)

Key words: anaerobic, biodegradation assays, hydrolysis, macropollutants, methane potential Abstract A variety of test procedures for determination of anaerobic biodegradability have been reported. This paper reviews the methods developed for determination of anaerobic biodegradability of macro-pollutants. Main focus is paid to the nal mineralization of organic compounds and the methane potential of compounds. Hydrolysis of complex substrates is also discussed. Furthermore, factors important for anaerobic biodegradation are shortly discussed.

1. Introduction Anaerobic degradation or digestion can be dened as a biological conversion process without external electron acceptor such as oxygen as in aerobic processes or nitrate/sulphate as in anoxic processes. In the anaerobic process organic carbon is converted by subsequent oxidations and reductions to its most oxidized state (CO2 ), and its most reduced state (CH4 ). A wide range of microorganisms catalyze the process in the absence of oxygen (McInerney et al. 1980). The main products of the process are carbon dioxide and methane, but minor quantities (usually less than 1% of the total gas volume) of other products such as nitrogen, nitrogen oxides, hydrogen, ammonia, hydrogen sulphide and other volatile compounds are also generated (McInerney et al. 1980). The mixture of gaseous products is termed biogas and the anaerobic degradation process is often also termed the biogas process. As the result of the removal of carbon, organic bound non-carbon compounds are released to their soluble inorganic form. With the increasing application of the anaerobic digestion process there is an urgent need to review methods for estimation of the biodegradability and methane potential of wastes used in for anaerobic digestion.

A substance or a compound is biodegradable if it can be decomposed by the action of microorganisms. Microorganisms can use this compound as energy source and as carbon source. A compound can be mineralized i.e. converted besides to new microbial biomass to the end carbon products i.e. to carbon dioxide and methane. In some cases complete mineralization is not seen and the compound can be involved in microbial metabolism with only transformation (also called primary biodegradation) of the compound to intermediates, but without total conversion to end products. An organic compound can be processed during anaerobic degradation through metabolism (the compound is supplying energy and carbon source for the micro-organisms) or by cometabolism (the compound is converted only at the presence of another, usually easily degradable organic compound such as glucose, ethanol etc.), that is supplying micro-organisms with energy and carbon for their cell mass built up). In this paper biodegradability with reference to macro-pollutants only, will be treated, since micro-pollutants do not generate enough biogas in order to determine biodegradation through biogas production and there are also uncertainties related to adsorption.

118 2. Some general considerations concerning /inuencing anaerobic biodegradation There are several physical, chemical and physiological factors in the environment that aect biodegradation of organic compounds, such as availability of the compounds, the availability of electron donors and acceptors, oxygen concentration, temperature, pH, moisture, salinity, sorption of chemicals to particulate material, concentration of the chemicals. Dierent factors might have dierent inuence according the specic characteristics of the compound. 2.1. Redox conditions One of the major factors governing biodegradation is the nature and availability of electron acceptors. From a thermodynamic point of view, oxygen is the most favourable electron acceptor. Under anoxic conditions, the biodegradation will often depend on the availability of electron acceptors, such as nitrate, iron, sulphate or carbon dioxide. In truly anaerobic conditions there is absence of inorganic electron acceptors other than CO2 , and small amount of energy is gained by consecutive oxidations reductions of the organic matter, or CO2 is used as electron acceptor. The energy released in a redox process as a result of electron transfer from one compound to another, is used for the maintenance and growth of the microbial population in the environment. 2.2. Temperature Temperature aects survival and growth of microorganisms and it also inuences their metabolic activities. In general, higher temperatures that do not kill microorganisms result in higher metabolic activities. Temperature is the most important variable in controlling the rate of microbial metabolism in anaerobic environments (Westermann et al. 1989). Anaerobic digestion is applied under three dierent temperature ranges, i.e. the mesophilic (2540 C), the thermophilic (4560 C) and the psychrophilic (<20 C). In general, the overall process rates double for every 1 C increase in operating temperature up to an optimum temperature 60 C (Harmon et al. 1993). The optimum temperature for growth usually is close to the upper limit of its range. A further increase of the temperature beyond its optimum temperature can result in a sharp decrease of the growth rate (Chen & Hashimoto 1978). The eect of temperature on anaerobic degradation is theoretically only inuencing the degradation rates and not the ultimate biodegradability of a component. However, the biodegradation rates can be so slow that the achievable biogas potential appears to be lower than at optimal conditions. Kaparaju and Rintala (2003) have found methane potential to be 2.4, 12.6 and 15.7%, at 5 C and 10 and 15 C respectively, compared to the methane potential achieved at 35 C, after 345-days digestion of manure. The eect of temperature on the achievable methane potential of manure was investigated by Hashimoto et al. (1981). It was found that temperatures in the range between 30 and 60 C (at 5 C intervals) did not aect the achievable methane potential of manure. At higher temperatures (65 C) the experimentally determined methane potential was lower. The lower potential was attributed to unstable fermentation rather than decreased substrate availability at that temperature. 2.3. Hydrolysis An important step of the anaerobic biodegradation process is the hydrolysis of the complex organic matter. During the anaerobic digestion of complex organic matter the hydrolysis is the rst and often the rate-limiting step. The hydrolysis can be dened as the breakdown of organic substrate into smaller products that can subsequently be taken up and degraded by bacteria (Morgenroth et al. 2002). Substrate for hydrolysis can be directly present in the waste or can be formed by microbial activity such as internal storage products, or bacterial biomass. During hydrolysis of macro-pollutants polymers such as lipids, protein, carbohydrates are depolymerized to glycerol and long chain fatty acids, to amino acids and to oligo- and monosaccharides for lipids, proteins and carbohydrates respectively. 2.3.1. Hydrolysis mechanism Hydrolysis takes place extracellular by enzymes excreted by the biomass. Three main mechanisms

119 exist for the release of enzymes and the subsequent hydrolysis of the complex substrate during anaerobic digestion (Batstone 2000). 1. The organism secretes enzymes to the bulk liquid, where they will either adsorb to a particle or react with a soluble substrate (Jain et al. 1992). 2. The organism attaches to the particle, secretes enzymes into the vicinity of the particle and next the organism will benet from the released dissolved substrates (Vavilin et al. 1996). 3. The organism has an attached enzyme that may also act as a transport receptor to the interior of the cell. This method requires the organism to absorb onto the surface of the particle. Research on aerobic sludge has indicated that most of the extracellular enzymes are associated to the biomass (Morgenroth et al. 2002). Only a few studies have been done on anaerobic sludge but also in this case the enzymes seem to be cell associated (Hobson 1987; Philip et al. 1993). Mechanisms 2 and 3 seem therefore more likely than mechanism 1. This indicates that good contact between biomass and substrate is a prerequisite to the hydrolysis. 2.3.2. Aspects related to the enzymatic depolymeriation Hydrolytic enzymes can be endo-enzymes, which prefer to cut the bonds towards the middle of the molecule, or exo-enzymes, which prefer to cut the bonds near to the edges of the macromolecule. The enzyme substrate specic activity is thought to follow MichaelisMenten kinetics. The overall eect of the digestion temperature on the hydrolysis rate originates from the combined temperature eect on the enzyme kinetics, bacterial growth and solubility of the substrate. For instance, the GibbsHelmoltz equation gives the relation between temperature and pKa of the enzymes. The change in charge will have consequences for the structure of the enzyme resulting in changes of catalytic eciency, amount of active enzyme and binding of the substrate (Chaplin & Bucke 1990). In general, the rates of reactions vary with temperature in accordance with the Arrhenius equation. Veeken and Hamelers (1999) digested several biowaste components, such as orange peels, bark, leaf and grass at 20, 30 and 40 C. They found a good Arrhenius relation between the rst order hydrolysis constant and the digestion temperature (R2 0.9840.999) with an average standard free energy of activation of 4614 kJ/ mol. The eect of the pH on the hydrolysis is complicated. The net eect of pH on the hydrolysis rate is specied by the pH optima of the dierent enzymes present in the digester and the eect of pH on the charge/solubility of the substrate. The latter especially applies to the digestion of substrates that contain proteins. The production and activity of enzymes can be inhibited by hydrolysis products. The production of proteinases by microbes can be inhibited by components, such as amino acids, high inorganic phosphate levels and glucose. With respect to the production of cellulases similar ndings were made as for proteinases (Zeeman 1991). The production of cellulases is inhibited by high glucose levels but is stimulated by low glucose levels. However, no effects of the concentration of free amino acid on the production of cellulases were reported (Glenn 1976). Also NH 4 can inhibit the hydrolysis of cellulose (Zeeman 1991). From the results it was however unclear if the inhibition was directly from the free ammonia or indirectly from the resulting high volatile fatty acid levels. El-Mashad (2003) studied the eect of dierent ammonia concentrations (range 13.7 g NH 4 N/l) on the hydrolysis of liquid cattle manure at 50 and 60 C in batch reactors. The inoculum used for the experiments was digested cow manure adapted to an ammonia concentration of 1.1 g NH 4 N/l. At both temperatures a negative linear relation between the rst order hydrolysis constant and both total and free ammonia concentrations was established. Accumulation of long chain fatty acids at the lipid-water interface causes inhibition of the lipase activity by physicalchemical changes of the interface, e.g. the surface tension (Verger 1980; Angelidaki & Ahring 1992). 2.3.3. Aspects related to the physical state and structure of the substrate An important factor for the hydrolysis is the physical state and structure of the substrate and its accessibility for hydrolytic enzymes. It is therefore obvious that the hydrolysis rate of particulate substrates is lower than that of dissolved polymers as with the former only part of the substrate is accessible to the enzymes. Macro-pollutants in

120
Table 1. Surface related hydrolysis rate assessed for dierent substrates Substrate Hydrolysis rate (mg COD/cm2/day) 1.0 1.1 0.01 0.33 Inoculum Temperature (C) Reactor Reference

Starch Rice Hay Cellulose

Granular sludge from potato factory Not indicated Not indicated MSW leachate

35 35 35 38

Batch Batch Batch CSTR

Sanders et al. (2000) Palmowski et al. (2001) Palmowski et al. (2001) Song (2003)

waste (water) can be found in dierent physical states, in particles, dissolved or emulsied. Particles are the most commonly found, for example 60 90% of the total organic load in domestic sewage consists of particles (Elmitwalli 2000). Chyi and Dague (1994), Hills and Nagano (1984) and Hobson (1987) indicated that the hydrolysis of complex substrates is a surface related process and a formation of a biolm on the particle surfaces is necessary for the complete anaerobic digestion of organic matter (Song 2003). The rate of microbial attachment to the substrate depends on the type of micro-organisms (Song 2003). At full microbial colonization the hydrolysis rate of particulate substrates is constant on a per unit surface area basis, although the actual value seems to depend on the type of inoculum (Song 2003). In Table 1 values for the surface hydrolysis rate are presented. The accessibility of a substrate can also be altered by formation of complexes with other compounds. For example, cellulose itself is relatively easily degradable, but once it is incorporated in a lignocellulosic complex, the biodegradability of the cellulose is distinctly lower (Tong et al. 1990).

Methods based on product formation are monitoring either the end product (biogas) or intermediates production such as volatile fatty acids. Most methods are based on monitoring biogas production. Biogas production is measured either as volume increase under constant pressure (volumetric methods), or measurement of pressure increase in constant volume (manometric methods), or measurement of methane formation by gas chromatography (Rozzi & Remigi, 2001). Gas chromatography is used to measure content of methane and carbon dioxide of the biogas that ends up in headspace of closed vials (Dolng & Bloemen, 1985). Soto et al. (1993) compared liquid displacement systems to gas chromatography methods and concluded that the latter are more accurate for low methane productions. Gas chromatographic methods can be divided in two groups: 1. Using a GC with thermal conductivity detection (TCD) where both methane and carbon dioxide are measured. By using a reference gas e.g. nitrogen in the headspace and regular sampling the volume of biogas can be estimated based on the molar fractions of CH4 , CO2 (Soto et al. 1993). 2. Using a GC with ame ionization detection (FID), where only methane is measured (Angelidaki and Ahring 1993). The measurement is compared with methane standard with known methane content. This method is simple and fast; one methane measurement takes less than half a minute. Thus, many samples can be tested with relatively low time consumption. Methods based on substrate depletion, require usually more complex analysis. Substrate depletion can be determined either as lumped parameter (volatile solids (VS), chemical oxygen demand (COD), dissolved organic carbon (DOC), etc.) or directly analysis of the compound that is being used as substrate (Rozzi & Remigi, 2001).

3. Anaerobic biodegradability assays Anaerobic biodegradability assays are used to establish anaerobic biodegradability, for determination of the ultimate methane potential of wastes, but are also used for determination of the rate of the biodegradation in general. 3.1. Methods for determination of anaerobic biodegradation Biodegradability assays are based on the measurement of either formation of one or more products involved in the biological reaction under investigation or measurement for substrate depletion.

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Figure 1. Principles for biodegradation assays.

In Figure 1 the principles of biodegradation assays are summarized. When the biodegradation assay is used for determination of biodegradation rates, primary depletion may be only representative of hydrolysis or acidogenesis step, when methanogenesis is the limiting step, while CH4 production is reecting overall biodegradation only when methanogenesis is non-limiting. 3.2. Experimental set-up for biodegradability tests When evaluating literature it is clear that commonly two dierent experimental set-ups are used to establish the biodegradability and hydrolysis rate of particulate substrates, i.e. batch (Veeken & Hamelers 1999) or continuous (Miron et al. 2000) experiments. In the batch approach, the selected substrate (waste) is incubated in closed vials or asks at a specic temperature with an amount of methanogenic inoculum. After incubation the degree of degradation of the substrate is assessed at pre-set time intervals to determine the rate and ultimate biodegradation or hydrolysis. Controls with only inoculum added are included in order to account for the biogas produced from organic matter contained in the inoculum. The continuous set-up uses completely stirred tank reactors (CSTR) operated at a specic temperature and at dierent hydraulic retention times (HRT). Analyses are made from the euent once steady state has been established. The continuous set-up is much more laborious than the batch set-up. Batch experiments can be performed in a single-ask batch reactor or a multiple-ask batch reactor (Sanders 2002; Rozzi & Remigi, this jour-

nal). The latter reactor is actually a system of several small batch reactors of equal contents that allows more thorough homogenization than one large batch reactor. This type of batch reactor is mainly used for assessment of the biodegradability and hydrolysis rate of low homogeneity wastes such as lipid containing wastes. 3.3. Inoculum The anaerobic digestion process is a complex process requiring the presence of several dierent microorganisms. It is of great importance to nd appropriate inoculum containing the necessary microorganisms for the degradation process to proceed. Digested sludge is often the used inoculum (Owen et al. 1979). However, in some cases, microorganisms adapted to specic conditions such as high ammonia concentrations are needed (Angelidaki & Ahring 1993). Another important factor is the amount of inoculum added. Low amount of inoculum is often wished as inoculum also contributes to product formation (biogas) and thus can blur the results if biogas production from inoculum is relatively high compared to the compound (or waste) under investigation. On the other hand a relatively small amount of inoculum can lead to overload of the process with acidication as a result. If the ammonia concentration in the medium is high, or substrate contains high concentration of proteins (releasing ammonia during degradation), accumulation of VFA will not lead to acidication due to the buer capacity supplied by ammonia (Angelidaki & Ahring 1993, 1994; Sanders et al.

122 2002a). The degree of primary biodegradation must of course in this case be assessed via substrate depletion instead of methane production, or as sum of product formation (VFA and methane). Acidogenic conditions can be prevented by estimation of the maximum VFA production during the assay, so that sucient inoculum can be added to provide enough methanogenic activity to remove the VFA at all times. As the hydrolysis depends on the concentration of the substrate that is to be hydrolyzed, the highest production of VFA is to be expected in the rst day after incubation. Based on this the minimal amount of inoculum that has to be added can therefore be calculated as follows: Xss Vww kh Vinoculum VSSinoculum Ainoculum Vreactor Vreactor Vinoculum Xss Vww kh VSSinoculum Ainoculum As the hydrolysis constant is unknown applying Equations 1 and 2 requires an estimated value for the hydrolysis constant. However, as the minimum amount of inoculum to be used in the experiment is wanted, it is preferable to use an overestimated value for the kh than an underestimated value. 3.4. Medium An important factor associated with the inoculum is the medium, which can be added to the inoculum during degradation experiments. Medium consists of several minerals and does not contain any signicant amount of organic carbon. Synthetic medium can be added as a source of nutrients of micronutrients, growth factors vitamins and trace metals necessary for growth of the microorganisms. Synthetic media are used in cases that lack of important for growth components might be limiting microbial growth. Important components needed for growth, are shown in Table 2. An example of a synthetic basic medium used in anaerobic tests [modied from previously described basic medium (Angelidaki et al. 1990) is shown in Table 3. 3.5. pH pH plays a major part in anaerobic biodegradation. pH inuences the activity of the hydrolytic enzymes and the microorganisms which are active

1 2

where Xss is the concentration of hydrolyzable substrate in waste (water) (g l1 ), Vww , the volume of waste (water) in batch reactor (l), kh , the rst order hydrolysis constant (day1 ), Vreactor , the volume of the batch reactor (l), Vinoculum , the volume of the methanogenic inoculum (l), VSSinoculum the volatile solids suspended content of the methanogenic inoculum (g VSS l1 ) and Ainoculum is the methanogenic activity of the inoculum (g g1 VSS day1 ).

Table 2. Components needed in synthetic media (modied from Madigan (2000)) Compound Nitrogen Function in the cell Chemical form supplied in synthetic media NH4Cl, (NH4)2SO4, N2, KNO3 KH2PO4, Na2HPO4 Na2SO4, KH2SO4, Na2S2O3, Na2S, cysteine, etc KCl, KH2PO4 MgCl2, MgSO4 NaCl CaCl2 FeCl3, FeSO4, various chelated iron (in complexes with EDTA etc.) Cr, Co, Cu, Mn, Mo, Ni, Se, V, Zn Vitamins, amino acids (essential), purines, pyrimidines

Next most abundant after carbon. Major element in nucleic acids and amino acids Phosphorus (P) For nucleic acids and phospholipids Sulphur (S) In amino acids cysteine and methionine, vitamins such as thiamine, biotin, and lipoic acid, coenzymes A Potassium (K) Used by several dierent enzymes Magnesium (Mg) Stabilizes ribosomes, cell membranes and nucleic acids Sodium (Na) Necessary for many enzymes Calcium (Ca) Helps stabilize the bacterial cell wall and is important for stabilizing endospores Iron (Fe) Present in cytochromes Micronutrients Growth factors These are usually necessary for specic enzymes Required in small amounts

123 within certain, usually narrow pH ranges. The anaerobic digestion process occurs in the pH interval of 6.08.3. Most methanogens have a pH optimum between 7 and 8 while the acid forming bacteria often have a lower optimum (Angelidaki & Ahring 1994). If the pH of the waste to be tested is outside the optimal range, and if enough buer capacity is not present, the anaerobic process will be inhibited. This will lead underestimation of the methane potential. tion vary signicantly among the published methods (Owen et al. 1978; Eleazer et al. 1997; Owens & Chynoweth 1993; Adani et al. 2001; Harries et al. 2001; Heerenklage et al. 2002; Hansen et al. 2003). Some of these dierences originate from the purpose of measuring the methane potential and from the type of waste samples measured. According to the DTU protocol for determination of the methane potential the sample is inoculated with 6090% w/w (depending on the organic load of the waste) digested manure from a reactor operating at thermophilic temperature (55 C) (Hansen et al. 2003). Large inoculation volumes are ensuring high microbial activity, low risk for overloading and low risk of inhibition. Thermophilic temperature would ensure fast reaction rates. Furthermore, digested manure has been shown to be superior to digested sludge as it is rich to many nutrients, and has a high buer capacity (Angelidaki & Ahring 1994). Inoculum is degassed by incubation at 55 C before use for a few days to ensure that methane production from inoculum is as low as possible. The sample is diluted with water at dierent dilutions (from undiluted waste to 90% w/w dilution) to ensure that possible toxicity of the sample is not overseen. The dilutions used are depending on the type of waste and the expected toxicity or overloading to the anaerobic process. Serum vials are thoroughly gassed with N2 : CO2 (80 : 20) before addition of inoculum and waste, sealed with butyl stoppers and closed with aluminium crimps. The vials are incubated at 55 C. The test is run in triplicates. The accumulated methane production in the headspace of the vials is

4. Determination of methane potential Methane potential (also called biochemical methane potential) of wastes is dened as the ultimate specic methane production, for indenite degradation time. In practice the degradation time is denite and the methane potential is estimated by extrapolation of a methane time degradation curve. Methane potential can be expressed specifically per amount of waste (l CH4 /kg-waste), volume of waste (l CH4 /l-waste), per mass volatile solids added (l CH4 /kg-VS) or COD added (l CH4 / kg-COD). The volume is usually expressed in standard pressure (1 atm) and temperature (0 C) conditions (STP conditions). Other variations for expressing methane potential are also used. By the same way biogas potential can be dened as the ultimate biogas production. Several methods exist for measuring methane potentials of waste. However, the technical approaches in terms of pretreatment of the sample, inoculum, gas measurement technique and incubaTable 3. Basic anaerobic medium Description of anaerobic basic medium

The basic medium is prepared from the following stock solutions, (chemicals given below are concentrations in g l)1, in distilled water) (A) NH4Cl, 100; NaCl, 10; MgCl26H2O, 10; CaCl22H2O, 5 (B) K2HPO43H2O, 200 (C) Resazurin 0.5 (D) Trace-metal and selenite solution: FeCl24H2O, 2; H3BO3, 0.05; ZnCl2, 0.05; CuCl22H2O, 0.038; MnCl24H2O, 0.05; (NH4)6Mo7O244H2O, 0.05; AlCl3, 0.05; CoCl26H2O, 0.05; NiCl26H2O, 0.092; ethylenediaminetetraacetate, 0.5; concentrated HCl, 1 ml; Na2SeO35H2O, 0.1 (E) Vitamin mixture (componets are given in mg/l): Biotin, 2; folic acid, 2; pyridoxine acid, 10; ridoavin, 5; thiamine hydrochloride, 5; cyanocobalamine, 0.1; nicotinic acid, 5; P-aminobenzoic acid, 5; lipoic acid, 5; D L -pantothenic acid. To 974 ml of distilled water, the following stock solutions were added A, 10 ml; B, 2 ml; C, 1 ml; D, 1 ml and E, 1 ml. The mixture is gassed with 80% N2 20% CO2. Cysteine hydrochloride, 0.5 g and NaHCO3, 2.6 g, are added and the medium is dispensed to serum vials and autoclaved if necessary. Before inoculation the vials are reduced with Na2S9H2O to a nal concentration of 0.025%.

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Figure 2. Experimental set-up for anaerobic biodegradation test used at Denmark Technical University (DTU) for standard biogas potential tests.

followed by sampling gas from headspace of the vial with a pressure-lock syringe, and subsequent analysis of methane content by GC (FID detection). The methane production from the inoculum (blanks are included where water is added instead of waste) is subtracted from the methane production of the waste samples. The methane potential is determined from the vials resulting in the highest methane potential. At least two dilutions should result in the highest methane potential, else higher dilutions of the waste should be tested. 4.1 Biogas/methane potential considerations Anaerobic degradation of organic matter results in mainly, methane and carbon dioxide. Methane is practically non-soluble and ends up for the most in the gas phase. However, more complex conditions are valid for carbon dioxide. Carbon dioxide is more water soluble and at the same time a part of it is 2 mainly ionized to HCO 3 , and a small part to CO3 that might either precipitate or remain as ion. The distribution of inorganic carbon to gas or liquid phase is strongly controlled by pH but also by temperature. Therefore, it is often more reliable to determine methane potential than biogas potential. 4.2. Theoretical aspects for calculation of the biogas potential 4.2.1. COD/VS When considering the biogas process for a specic application the characteristics of the substrate/

waste is naturally of prime interest. Waste and wastewater is often of a complex composition, which is dicult to describe in detail. The most common single parameters used to describe the concentration of waste or wastewater is the chemical oxygen demand (COD) expressed as g-O2 /l, or the volatile solids content (VS) expressed as g-VS/l or %. The COD content describes the amount of oxygen needed to completely oxidize the waste under aerobic conditions, and is determined experimentally by measuring the amount of a chemical oxidizing agent needed to fully oxidize a sample of the waste. The COD is used as a measure of the oxygen equivalent of the organic matter content of a sample that is susceptible to oxidation by a strong chemical oxidant (APHA 1992). During oxidation 9095% of the organic matter is oxidized, depending on the method used. The preferred oxidant used for COD determination is dichromate, due its superior oxidizing ability, applicability to a wide variety of samples and ease of manipulation. The VS content describes the content of organic material in the waste, and is dened as the amount of matter in a dried sample lost after 1 h at a temperature of approximately. 550 C in air (APHA 1992). The method relies on the fact that most organic materials ignite and combust at this temperature, while most inorganic compounds require higher temperatures. Both methods are relatively easy to perform, and give a good rst impression of the strength of

125 a waste. COD is usually used for description of wastewaters, while VS is used for slurries and solid wastes. If the composition of the organic material is known, the relation between COD and VS content can be calculated by setting up the stoichiometry of complete oxidation. As an example the relation is derived below for glucose (Equations (3) and (4)):
C6 H12 O6 M 180 6O2 M 32 ! 6CO2 6H2 O 3

chloride) S, and N will be oxidized to a dierent degree, thus contributing to the nal COD value. Thus a compound having the formula Cn Ha Ob Nc Sd is oxidized with O2 to the following products (Equation. (7)).
Cn Ha Ob Nc Sd xO2 ! y CO2 zH2 O vNH3 wH2 SO4 7

Alternatively HNO3 can be produced instead of NH3 . Therefore one should be aware of the denition during such calculations. 4.2.2. Theoretical biogas potential When organic material is degraded anaerobically, the end result is carbon in its most oxidized form (CO2 ) and its most reduced form (CH4 ), i.e. an electron transfer between carbon atoms takes place. The ratio between CH4 and CO2 depends on the oxidation state of the carbon present in the organic material, i.e. the more reduced the organic carbon content is, the more CH4 will be produced. If the composition of the organic material is known and all the material is converted to biogas, the theoretical methane yield potential can be calculated from the following Buswells equation (Buswell and Neave 1930):   a b Cn Ha Ob n H2 O ! 4 2     n a b n a b CH4 CO2 8 2 8 4 2 8 4 This equation is derived by balancing the total conversion of the organic material to CH4 and CO2 with H2 O as the only external source, i.e. under anaerobic conditions. The specic methane yield, usually expressed as (STP l CH4 )/g-VS might then be calculated as:

COD=VS 6 32=180 1:067 g COD=g-VS 4 For many types of organic waste the oxidation state of carbon is close to zero (as for glucose) and in these cases the COD/VS ratio will be close to unity. In a more generalized form, the oxidation reaction for organic material is given in Equation (5).   a b a Cn Ha Ob n O2 ! nCO2 H2 O 4 2 2 5 and the COD/VS ratio then becomes (Equation 6): b na 4 2 32 6 COD=VS 12n a 16b Organic matter consisting of only C, H, and O is theoretically fully oxidized CO2 and H2 O. When organic matter contains also S, and N, it is wished that S, and N stay in the reduced form (H2 S, NH3 ). However, depending on the method and the salt content of the sample (e.g. high content of

Table 4. Theoretical characteristics of typical substrate components Substrate type Carbohydrate Protein* Lipids Ethanol Acetate Propionate (C6H10O5)n C5H7NO2 C57H104O6 C2H6O C2H4O2 C3H6O2 Composition COD/VS g-COD/g-VS 1.19 1.42 2.90 2.09 1.07 1.51 CH4 yield STP l/g-VS 0.415 0.496 1.014 0.730 0.373 0.530 CH4 yield STP l/g-COD 0.35 0.35 0.35 0.35 0.35 0.35 CH4 (%) 50 50 70 75 50 58

*Nitrogen is converted to NH3.

126 Bo;th b a 8 4 22:4 12n a 16b

2

  lCH4 STP g-VS

where 22.4 is the volume of 1 mol of gas at STP conditions. If Buswells equation is combined with the COD/VS relation a similar COD based specic yield becomes: n a b   8 4 22:4 lCH4 2 Bo;th STP 10 b g-COD na 4 2 32 In Table 4 the characteristics of a number of typical organic materials suitable for anaerobic degradation is listed. 4.2.3. Practical biogas potential Although the theoretical biogas potential gives a rough idea of the quality of waste and the potential biogas production, the practical yield obtained in a biogas reactor will always be lower due to a number of factors: A fraction of the substrate is utilized to synthesize bacterial mass, typically 510% of the organic material degraded. At a nite retention time a fraction of the organic material will be lost in the euent, typically 10%. Lignin is not degraded anaerobically. Often a part of the organic material is inaccessible due to binding in particles or structural organic matter. Limitation of other nutrient factors. Under favourable conditions with mainly water-soluble matter, degrees of conversion up to 90 95% can be achieved. If the organic matter is highly particulate or structural such as manures, 3060% conversion is more normal. In order to predict the biogas yield to be expected under practical conditions, it is best to nd experimentally determined biogas yields in the literature or perform small-scale batch fermentations. A prediction of the composition of the gas produced is more complex and depends rst of all on the amount of CH4 and CO2 produced, but also on the pH of the reactor content. The CH4 produced is mainly released to the gas phase, but CO2 is partly dissolved in the liquid phase of the reactor or is converted to bicar-

bonate as a function of the pH. Consequently, the CH4 percentage in the biogas produced will generally be higher than predicted by the stoichiometric ratio.

5. Assessment of the hydrolysis rate First order kinetics are most commonly used to describe the hydrolysis of particulate substrates during anaerobic digestion (Eastman and Ferguson 1981; Pavlostathis and Giraldo-Gomez 1991). dXdegr =dt k h Xdegr 11

with Xdegr is the concentration biodegradable substrate (kg/ m3 ), t is the time (days) and, kh is the rst order hydrolysis constant (day1 ). Despite the fact that the rst order kinetics is empirical relation it does reect the major aspect of the hydrolysis of particulate substrates, namely the fact that the hydrolysis of particles limited by the amount of surface available. Several researchers showed that the hydrolysis mechanism of particulate substrates is surface related (Hills & Nakano 1984; Sanders et al. 2000). In this case the amount of enzymes is present in excess relative to the available surface area (Hobson 1987) and the hydrolysis rate is determined by the surface area not by the enzyme activity. Such surface limited kinetics can be described with a rst order relation (Vavilin et al. 1996; Valentini et al. 1997; Veeken & Hamelers 1999; Sanders 2002a). As it is assumed that the enzyme activity is associated with the biomass the rst order constant is not aected by the biomass concentration. Although the rst order kinetics were only intended to describe the hydrolysis of particles they can also be used to describe the hydrolysis of dissolved polymers. Sanders et al. (2002b) showed by statistical calculations that the production of mono and dimers from a soluble polymeric substrate by a mixture of endo- and exo-enzymes can be described by rst order kinetics. However, in contrast to the hydrolysis of particles in this case the hydrolysis rate is aected by the enzyme activity, thus biomass concentration. From Equation (11) the relation between the hydrolysis constant, digestion time and euent concentration for a batch system can be derived (Sanders et al. 2002a).

127 Xss;batch t Xss;added 1 fh fh Xss;added e

kh t

12

6. Potential problems in estimation of methane potential of wastes 6.1. Nitrate, sulphate reducers and methanogens It has been shown that sulphate reducers and denitriers are able to outgrow the methanogens. This is due to the higher energy gained by sulphate or nitrate reduction compared to methanogenesis. Therefore, presence of high concentrations of sulphate or nitrate will result in determination of low methane potentials of the wastes. In cases where the waste (water) contains sulphate and the degree of hydrolysis is measured via methane production the hydrolysis obviously will also be underestimated due to consumption of organic matter for the formation of H2 S. Unless the amount of H2 S produced can be measured direct measurement of the degradation of the individual components for this type of waste (water) is necessary. 6.2. Sorption and bioavailability Sorption is an important mechanism that inuences the fate and eect of organic compounds. When compounds reside in environments with high sorption capacity, they may become unavailable for anaerobic degradation. This can aect determination of their methane potential. 6.3. Problems during COD determination Volatile straight-chain aliphatic compounds are not oxidized to any appreciable degree. aromatic carbohydrates, and some aromatic heterocyclic compounds (e.g. pyridine) are not oxidized. Halogens (Cl , Br J ) can been oxidized. 2 NO 2 exerts a COD of 1.1 mg/mg NO2 N Reduced inorganic compounds such as ferrous iron, sulphide, manganous manganese, etc., are oxidized quantitatively under the species The various COD methods have been developed for water and waste water analyses. However, when samples like manure are analyzed interferences of other additional factors can occur. Furthermore, the samples have to be properly homogenized and diluted, since agricultural and

where Xss , batch(t) is the concentration of total substrate in the batch reactor t days after incubation biodegradable + non-biodegradable part) (g l1 ), Xss;added the concentration of total substrate in the added to batch reactor at start of experiment biodegradable + non-biodegradable part) (g l1 ), fh the biodegradable fraction of substrate, fh 2 [0;1], kh is the rst order hydrolysis constant (day1 ), and t is the digestion time of batch (days). Equation (13) presents the linearized form of Equation (12). From Equation (13) it can be seen that the hydrolysis constant is determined via a linear least square t of the results from the batch digestion, Xss;batch at several moments in time during the batch assay. The biodegradability fh has to be assessed directly from the experimental results, the maximum methane yield of the substrate (Veeken and Hamelers 1999) or the nal euent concentration. ln   Xss;batch t Xss;added 1 fh kh t 13 Xss;added fh

As the concentration of biodegradable substrate is highest at the start of the experiment so will the hydrolysis rate. This means that to obtain enough experimental data for calculation of the hydrolysis constant it is essential that the reactor is sampled at smaller intervals in the beginning of the experiment then at the end. Nevertheless, enough data has to be gathered at the end of the experiment to establish the biodegradability of the substrate. A more direct and accurate method for assessing the hydrolysis constant and biodegradability from batch and continuous experiments is the non-linear least squares t on the assessed euent concentration (Sanders et al. 2002a). This method should be assessed whenever possible. With these calculations the gas production or the COD, protein and carbohydrate content of the blank has to be taken into account. Additionally, it should be emphasised that the biodegradability in Equations (12) and (13) refers to the biodegradability under the applied conditions and may change with the imposed reactor conditions.

128 household wastes contain much higher organic contents that wastewaters normally contain. 6.4. Inhibition of the process The experimentally determined methane potential can be underestimated in cases where waste contains toxicants or the process is overloaded. In such cases dilution of the waste will result in more accurate determination of the methane potential. 7. Conclusions Anaerobic biodegradation is a complex process and the biological approach to determining anaerobic biodegradation or methane potentials leads to substantial uncertainty in the determination. Therefore, the determination procedure should be carefully considered, anaerobic optimal growth conditions secured and results carefully evaluated. References
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