Assessment of the anaerobic biodegradability of macropollutants

Irini Angelidaki1;* & Wendy Sanders21Environment & Recourses DTU, Building 115, 2800 Lyngby, Technical University of Denmark; 2WageningenAgricultural 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. Thispaper 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 com-pounds. Hydrolysis of complex substrates is also discussed. Furthermore, factors important for anaerobicbiodegradation are shortly discussed.

1. Introduction

Anaerobic degradation or digestion can bedened as a biological conversion process withoutexternal electron acceptor such as oxygen as inaerobic processes or nitrate/sulphate as in anoxicprocesses. In the anaerobic process organic carbonis converted by subsequent oxidations and reduc-tions to its most oxidized state (CO2), and its mostreduced state (CH4). A wide range of microor-ganisms catalyze the process in the absence ofoxygen (McInerney et al. 1980). The main prod-ucts of the process are carbon dioxide and meth-ane, but minor quantities (usually less than 1% ofthe total gas volume) of other products such asnitrogen, nitrogen oxides, hydrogen, ammonia,hydrogen sulphide and other volatile compoundsare also generated (McInerney et al. 1980). Themixture of gaseous products is termed biogas andthe anaerobic degradation process is often alsotermed the biogas process. As the result of theremoval of carbon, organic bound non-carboncompounds are released to their soluble inorganicform.

With the increasing application of the anaero-bic digestion process there is an urgent need toreview methods for estimation of the biodegrad-ability and methane potential of wastes used in foranaerobic digestion.

A substance or a compound is biodegradableif it can be decomposed by the action of micro-organisms. Microorganisms can use this com-pound as energy source and as carbonsource. A compound can be mineralized i.e. con-verted besides to new microbial biomass to theend carbon products i.e. to carbon dioxideand methane. In some cases complete mineraliza-tion is not seen and the compound can beinvolved in microbial metabolism with only trans-formation (also called primary biodegradation)of the compound to intermediates, but with-out total conversion to end products. Anorganic compound can be processed dur-ing anaerobic degradation through metabo-lism (the compound is supplying energy andcarbon source for the micro-organisms) or by co-metabolism (the compound is converted only atthe presence of another, usually easily degrad-able organic compound such as glucose, ethanoletc.), that is supplying micro-organisms withenergy and carbon for their cell mass built up).

In this paper biodegradability with referenceto macro-pollutants only, will be treated,since micro-pollutants do not generateenough biogas in order to determinebiodegradation through biogas productionand there are also uncertainties related toadsorption.

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

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2. Some general considerations concerning/inuencing anaerobic biodegradation

There are several physical, chemical and physio-logical factors in the environment that aect bio-degradation of organic compounds, such asavailability of the compounds, the availability ofelectron donors and acceptors, oxygen concentra-tion, temperature, pH, moisture, salinity, sorptionof chemicals to particulate material, concentrationof the chemicals. Dierent factors might havedierent inuence according the specic charac-teristics of the compound.

2.1. Redox conditions

One of the major factors governing biodegrada-tion is the nature and availability of electron ac-ceptors. From a thermodynamic point of view,oxygen is the most favourable electron acceptor.Under anoxic conditions, the biodegradation willoften depend on the availability of electron ac-ceptors, such as nitrate, iron, sulphate or carbondioxide. In truly anaerobic conditions there is ab-sence of inorganic electron acceptors other thanCO2, and small amount of energy is gained byconsecutive oxidations reductions of the organicmatter, or CO2 is used as electron acceptor. Theenergy released in a redox process as a result ofelectron transfer from one compound to another,is used for the maintenance and growth of themicrobial population in the environment.

2.2. Temperature

Temperature aects survival and growth ofmicroorganisms and it also inuences their meta-bolic activities. In general, higher temperaturesthat do not kill microorganisms result in highermetabolic activities. Temperature is the mostimportant variable in controlling the rate ofmicrobial metabolism in anaerobic environments(Westermann et al. 1989).

Anaerobic digestion is applied under threedierent temperature ranges, i.e. the mesophilic(2540 C), the thermophilic (4560 C) and thepsychrophilic (

exist for the release of enzymes and the subsequenthydrolysis of the complex substrate during anaer-obic digestion (Batstone 2000).

1. The organism secretes enzymes to the bulk li-quid, where they will either adsorb to a particleor react with a soluble substrate (Jain et al.1992).

2. The organism attaches to the particle, secretesenzymes into the vicinity of the particle and nextthe organism will benet from the released dis-solved substrates (Vavilin et al. 1996).

3. The organism has an attached enzyme that mayalso act as a transport receptor to the interior ofthe cell. This method requires the organism toabsorb onto the surface of the particle.

Research on aerobic sludge has indicated thatmost of the extracellular enzymes are associated tothe biomass (Morgenroth et al. 2002). Only a fewstudies have been done on anaerobic sludge butalso in this case the enzymes seem to be cellassociated (Hobson 1987; Philip et al. 1993).Mechanisms 2 and 3 seem therefore more likelythan mechanism 1. This indicates that good con-tact between biomass and substrate is a pre-requisite to the hydrolysis.

2.3.2. Aspects related to the enzymaticdepolymeriationHydrolytic enzymes can be endo-enzymes, whichprefer to cut the bonds towards the middle of themolecule, or exo-enzymes, which prefer to cut thebonds near to the edges of the macromolecule. Theenzyme substrate specic activity is thought tofollow MichaelisMenten kinetics.

The overall eect of the digestion temperatureon the hydrolysis rate originates from the com-bined temperature eect on the enzyme kinetics,bacterial growth and solubility of the substrate.

For instance, the GibbsHelmoltz equationgives the relation between temperature and pKa ofthe enzymes. The change in charge will have con-sequences for the structure of the enzyme resultingin changes of catalytic eciency, amount of activeenzyme and binding of the substrate (Chaplin &Bucke 1990). In general, the rates of reactions varywith temperature in accordance with the Arrheniusequation. Veeken and Hamelers (1999) digestedseveral biowaste components, such as orangepeels, bark, leaf and grass at 20, 30 and 40 C.They found a good Arrhenius relation between the

rst order hydrolysis constant and the digestiontemperature (R2 0.9840.999) with an averagestandard free energy of activation of 4614 kJ/mol.

The eect of the pH on the hydrolysis is com-plicated. The net eect of pH on the hydrolysisrate is specied by the pH optima of the dierentenzymes present in the digester and the eect ofpH on the charge/solubility of the substrate. Thelatter especially applies to the digestion of sub-strates that contain proteins.

The production and activity of enzymes can beinhibited by hydrolysis products. The productionof proteinases by microbes can be inhibited bycomponents, such as amino acids, high inorganicphosphate levels and glucose. With respect to theproduction of cellulases similar ndings were madeas for proteinases (Zeeman 1991). The productionof cellulases is inhibited by high glucose levels but isstimulated by low glucose levels. However, no ef-fects of the concentration of free amino acid on theproduction of cellulases were reported (Glenn1976). Also NH4 can inhibit the hydrolysis of cel-lulose (Zeeman 1991). From the results it washowever unclear if the inhibition was directly fromthe free ammonia or indirectly from the resultinghigh volatile fatty acid levels. El-Mashad (2003)studied the eect of dierent ammonia concentra-tions (range 13.7 g NH4 N/l) on the hydrolysis ofliquid cattle manure at 50 and 60 C in batchreactors. The inoculum used for the experimentswas digested cow manure adapted to an ammoniaconcentration of 1.1 g NH4 N/l. At both temper-atures a negative linear relation between the rstorder hydrolysis constant and both total and freeammonia concentrations was established.

Accumulation of long chain fatty acids at thelipid-water interface causes inhibition of the lipaseactivity by physicalchemical changes of theinterface, e.g. the surface tension (Verger 1980;Angelidaki & Ahring 1992).

2.3.3. Aspects related to the physical stateand structure of the substrateAn important factor for the hydrolysis is thephysical state and structure of the substrate and itsaccessibility for hydrolytic enzymes. It is thereforeobvious that the hydrolysis rate of particulatesubstrates is lower than that of dissolved polymersas with the former only part of the substrate isaccessible to the enzymes. Macro-pollutants in

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waste (water) can be found in dierent physicalstates, in particles, dissolved or emulsied. Parti-cles are the most commonly found, for example 6090% of the total organic load in domestic sewageconsists of particles (Elmitwalli 2000).

Chyi and Dague (1994), Hills and Nagano(1984) and Hobson (1987) indicated that thehydrolysis of complex substrates is a surface re-lated process and a formation of a biolm on theparticle surfaces is necessary for the completeanaerobic digestion of organic matter (Song 2003).The rate of microbial attachment to the substratedepends on the type of micro-organisms (Song2003). At full microbial colonization the hydrolysisrate of particulate substrates is constant on a perunit surface area basis, although the actual valueseems to depend on the type of inoculum (Song2003). In Table 1 values for the surface hydrolysisrate are presented.

The accessibility of a substrate can also be al-tered by formation of complexes with other com-pounds. For example, cellulose itself is relativelyeasily degradable, but once it is incorporated in alignocellulosic complex, the biodegradability ofthe cellulose is distinctly lower (Tong et al. 1990).

3. Anaerobic biodegradability assays

Anaerobic biodegradability assays are used toestablish anaerobic biodegradability, for determi-nation of the ultimate methane potential of wastes,but are also used for determination of the rate ofthe biodegradation in general.

3.1. Methods for determination of anaerobicbiodegradation

Biodegradability assays are based on the measure-ment of either formation of one or more productsinvolved in the biological reaction under investiga-tion or measurement for substrate depletion.

Methods based on product formation aremonitoring either the end product (biogas) orintermediates production such as volatile fattyacids. Most methods are based on monitoringbiogas production. Biogas production is measuredeither as volume increase under constant pressure(volumetric methods), or measurement of pressureincrease in constant volume (manometric meth-ods), or measurement of methane formation bygas chromatography (Rozzi & Remigi, 2001). Gaschromatography is used to measure content ofmethane and carbon dioxide of the biogas thatends up in headspace of closed vials (Dolng &Bloemen, 1985). Soto et al. (1993) compared liquiddisplacement systems to gas chromatographymethods and concluded that the latter are moreaccurate for low methane productions.

Gas chromatographic methods can be dividedin two groups:

1. Using a GC with thermal conductivity detection(TCD) where both methane and carbon dioxideare measured. By using a reference gas e.g.nitrogen in the headspace and regular samplingthe volume of biogas can be estimated based onthe molar fractions of CH4, CO2 (Soto et al.1993).

2. Using a GC with ame ionization detection(FID), where only methane is measured (An-gelidaki and Ahring 1993). The measurement iscompared with methane standard with knownmethane content. This method is simple andfast; one methane measurement takes less thanhalf a minute. Thus, many samples can be tes-ted with relatively low time consumption.

Methods based on substrate depletion, requireusually more complex analysis. Substrate deple-tion can be determined either as lumped parameter(volatile solids (VS), chemical oxygen demand(COD), dissolved organic carbon (DOC), etc.) ordirectly analysis of the compound that is beingused as substrate (Rozzi & Remigi, 2001).

Table 1. Surface related hydrolysis rate assessed for dierent substrates

Substrate Hydrolysis rate

(mg COD/cm2/day)

Inoculum Temperature (C) Reactor Reference

Starch 1.0 Granular sludge from potato factory 35 Batch Sanders et al. (2000)

Rice 1.1 Not indicated 35 Batch Palmowski et al. (2001)

Hay 0.01 Not indicated 35 Batch Palmowski et al. (2001)

Cellulose 0.33 MSW leachate 38 CSTR Song (2003)

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In Figure 1 the principles of biodegradationassays are summarized. When the biodegradationassay is used for determination of biodegradationrates, primary depletion may be only representa-tive of hydrolysis or acidogenesis step, whenmethanogenesis is the limiting step, while CH4production is reecting overall biodegradationonly when methanogenesis is non-limiting.

3.2. Experimental set-up for biodegradability tests

When evaluating literature it is clear that com-monly two dierent experimental set-ups are usedto establish the biodegradability and hydrolysisrate of particulate substrates, i.e. batch (Veeken &Hamelers 1999) or continuous (Miron et al. 2000)experiments. In the batch approach, the selectedsubstrate (waste) is incubated in closed vials orasks at a specic temperature with an amount ofmethanogenic inoculum. After incubation the de-gree of degradation of the substrate is assessed atpre-set time intervals to determine the rate andultimate biodegradation or hydrolysis. Controlswith only inoculum added are included in order toaccount for the biogas produced from organicmatter contained in the inoculum.

The continuous set-up uses completely stirredtank reactors (CSTR) operated at a specic tem-perature and at dierent hydraulic retention times(HRT). Analyses are made from the euent oncesteady state has been established. The continuousset-up ismuchmore laborious than the batch set-up.

Batch experiments can be performed in a sin-gle-ask batch reactor or a multiple-ask batchreactor (Sanders 2002; Rozzi & Remigi, this jour-

nal). The latter reactor is actually a system ofseveral small batch reactors of equal contents thatallows more thorough homogenization than onelarge batch reactor. This type of batch reactor ismainly used for assessment of the biodegradabilityand hydrolysis rate of low homogeneity wastessuch as lipid containing wastes.

3.3. Inoculum

The anaerobic digestion process is a complexprocess requiring the presence of several dierentmicroorganisms. It is of great importance to ndappropriate inoculum containing the necessarymicroorganisms for the degradation process toproceed. Digested sludge is often the used inocu-lum (Owen et al. 1979). However, in some cases,microorganisms adapted to specic conditionssuch as high ammonia concentrations are needed(Angelidaki & Ahring 1993).

Another important factor is the amount ofinoculum added. Low amount of inoculum is oftenwished as inoculum also contributes to productformation (biogas) and thus can blur the results ifbiogas production from inoculum is relatively highcompared to the compound (or waste) underinvestigation. On the other hand a relatively smallamount of inoculum can lead to overload of theprocess with acidication as a result. If theammonia concentration in the medium is high, orsubstrate contains high concentration of proteins(releasing ammonia during degradation), accu-mulation of VFA will not lead to acidication dueto the buer capacity supplied by ammonia (An-gelidaki & Ahring 1993, 1994; Sanders et al.

Figure 1. Principles for biodegradation assays.

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2002a). The degree of primary biodegradationmust of course in this case be assessed via substratedepletion instead of methane production, or assum of product formation (VFA and methane).

Acidogenic conditions can be prevented byestimation of the maximum VFA productionduring the assay, so that sucient inoculum can beadded to provide enough methanogenic activityto remove the VFA at all times. As the hydrolysisdepends on the concentration of the substrate thatis to be hydrolyzed, the highest production of VFAis to be expected in the rst day after incubation.Based on this the minimal amount of inoculumthat has to be added can therefore be calculated asfollows:

XssVwwkhVreactor

VinoculumVSSinoculum AinoculumVreactor

1

Vinoculum XssVwwkhVSSinoculumAinoculum

2

where Xss is the concentration of hydrolyzablesubstrate in waste (water) (g l1), Vww, the volumeof waste (water) in batch reactor (l), kh, the rstorder hydrolysis constant (day1), Vreactor, thevolume of the batch reactor (l), Vinoculum, the vol-ume of the methanogenic inoculum (l), VSSinoculumthe volatile solids suspended content of the meth-anogenic inoculum (gVSS l1) and Ainoculum is themethanogenic activity of the inoculum(g g1 VSS day1).

As the hydrolysis constant is unknown apply-ing Equations 1 and 2 requires an estimated valuefor the hydrolysis constant. However, as the min-imum amount of inoculum to be used in theexperiment is wanted, it is preferable to use anoverestimated value for the kh than an underesti-mated value.

3.4. Medium

An important factor associated with the inoculumis the medium, which can be added to the inoculumduring degradation experiments. Medium consistsof several minerals and does not contain any sig-nicant amount of organic carbon. Syntheticmedium can be added as a source of nutrients ofmicronutrients, growth factors vitamins and tracemetals necessary for growth of the microorganisms.Synthetic media are used in cases that lack ofimportant for growth components might be limit-ing microbial growth. Important components nee-ded for growth, are shown in Table 2.

An example of a synthetic basic medium usedin anaerobic tests [modied from previously de-scribed basic medium (Angelidaki et al. 1990) isshown in Table 3.

3.5. pH

pH plays a major part in anaerobic biodegrada-tion. pH inuences the activity of the hydrolyticenzymes and the microorganisms which are active

Table 2. Components needed in synthetic media (modied from Madigan (2000))

Compound Function in the cell Chemical form supplied in synthetic media

Nitrogen Next most abundant after carbon. Major element in nucleic

acids and amino acids

NH4Cl, (NH4)2SO4, N2, KNO3

Phosphorus (P) For nucleic acids and phospholipids KH2PO4, Na2HPO4

Sulphur (S) In amino acids cysteine and methionine, vitamins such as

thiamine, biotin, and lipoic acid, coenzymes A

Na2SO4, KH2SO4, Na2S2O3, Na2S, cysteine, etc

Potassium (K) Used by several dierent enzymes KCl, KH2PO4

Magnesium (Mg) Stabilizes ribosomes, cell membranes and nucleic acids MgCl2, MgSO4

Sodium (Na) Necessary for many enzymes NaCl

Calcium (Ca) Helps stabilize the bacterial cell wall and is important for

stabilizing endospores

CaCl2

Iron (Fe) Present in cytochromes FeCl3, FeSO4, various chelated iron (in com-

plexes with EDTA etc.)

Micronutrients These are usually necessary for specic enzymes Cr, Co, Cu, Mn, Mo, Ni, Se, V, Zn

Growth factors Required in small amounts Vitamins, amino acids (essential), purines,

pyrimidines

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within certain, usually narrow pH ranges. Theanaerobic digestion process occurs in the pHinterval of 6.08.3. Most methanogens have a pHoptimum between 7 and 8 while the acid formingbacteria often have a lower optimum (Angelidaki& Ahring 1994). If the pH of the waste to be testedis outside the optimal range, and if enough buercapacity is not present, the anaerobic process willbe inhibited. This will lead underestimation of themethane potential.

4. Determination of methane potential

Methane potential (also called biochemical meth-ane potential) of wastes is dened as the ultimatespecic methane production, for indenite degra-dation time. In practice the degradation time isdenite and the methane potential is estimated byextrapolation of a methane time degradationcurve. Methane potential can be expressed specif-ically per amount of waste (l CH4/kg-waste), vol-ume of waste (l CH4/l-waste), per mass volatilesolids added (l CH4/kg-VS) or COD added (l CH4/kg-COD). The volume is usually expressed instandard pressure (1 atm) and temperature (0 C)conditions (STP conditions). Other variations forexpressing methane potential are also used. By thesame way biogas potential can be dened as theultimate biogas production.

Several methods exist for measuring methanepotentials of waste. However, the technical ap-proaches in terms of pretreatment of the sample,inoculum, gas measurement technique and incuba-

tion vary signicantly among the published meth-ods (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 pur-pose of measuring the methane potential and fromthe type of waste samples measured.

According to the DTU protocol for determi-nation of the methane potential the sample isinoculated with 6090% w/w (depending on theorganic load of the waste) digested manure from areactor operating at thermophilic temperature(55 C) (Hansen et al. 2003). Large inoculationvolumes are ensuring high microbial activity, lowrisk for overloading and low risk of inhibition.Thermophilic temperature would ensure fast reac-tion rates. Furthermore, digested manure has beenshown to be superior to digested sludge as it is richto many nutrients, and has a high buer capacity(Angelidaki & Ahring 1994). Inoculum is degassedby incubation at 55 C before use for a few days toensure that methane production from inoculum isas low as possible. The sample is diluted with waterat dierent dilutions (from undiluted waste to 90%w/w dilution) to ensure that possible toxicity of thesample is not overseen. The dilutions used aredepending on the type of waste and the expectedtoxicity 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 alu-minium crimps. The vials are incubated at 55 C.The test is run in triplicates. The accumulatedmethane production in the headspace of the vials is

Table 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; concentratedHCl, 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; DLDL-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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followed by sampling gas from headspace of thevial with a pressure-lock syringe, and subsequentanalysis of methane content by GC (FID detec-tion). The methane production from the inoculum(blanks are included where water is added insteadof waste) is subtracted from the methane produc-tion of the waste samples. The methane potential isdetermined from the vials resulting in the highestmethane potential. At least two dilutions shouldresult in the highest methane potential, else higherdilutions of the waste should be tested.

4.1 Biogas/methane potential considerations

Anaerobic degradation of organic matter results inmainly, methane and carbon dioxide. Methane ispractically non-soluble and ends up for the most inthe gas phase. However, more complex conditionsare valid for carbondioxide.Carbondioxide ismorewater soluble and at the same time a part of it ismainly ionized to HCO3 , and a small part to CO

23

that might either precipitate or remain as ion. Thedistribution of inorganic carbon to gas or liquidphase is strongly controlled by pH but also bytemperature. Therefore, it is often more reliable todetermine methane potential than biogas potential.

4.2. Theoretical aspects for calculation of thebiogas potential

4.2.1. COD/VSWhen considering the biogas process for a specicapplication the characteristics of the substrate/

waste is naturally of prime interest. Waste andwastewater is often of a complex composition,which is dicult to describe in detail.

The most common single parameters used todescribe the concentration of waste or wastewateris the chemical oxygen demand (COD) expressedas g-O2/l, or the volatile solids content (VS) ex-pressed as g-VS/l or %.

The COD content describes the amount ofoxygen needed to completely oxidize the wasteunder aerobic conditions, and is determinedexperimentally by measuring the amount of achemical oxidizing agent needed to fully oxidize asample of the waste. The COD is used as a mea-sure of the oxygen equivalent of the organic mattercontent of a sample that is susceptible to oxidationby a strong chemical oxidant (APHA 1992).During oxidation 9095% of the organic matter isoxidized, depending on the method used. Thepreferred oxidant used for COD determination isdichromate, due its superior oxidizing ability,applicability to a wide variety of samples and easeof manipulation.

The VS content describes the content of or-ganic material in the waste, and is dened as theamount of matter in a dried sample lost after 1 h ata temperature of approximately. 550 C in air(APHA 1992). The method relies on the fact thatmost organic materials ignite and combust at thistemperature, while most inorganic compoundsrequire higher temperatures.

Both methods are relatively easy to perform,and give a good rst impression of the strength of

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

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a waste. COD is usually used for description ofwastewaters, while VS is used for slurries and solidwastes.

If the composition of the organic material isknown, the relation between COD and VS contentcan be calculated by setting up the stoichiometryof complete oxidation. As an example the relationis derived below for glucose (Equations (3) and(4)):

C6H12O6M 1806O2M 32! 6CO26H2O3

COD=VS 6 32=180 1:067 g COD=g-VS4

For many types of organic waste the oxidationstate of carbon is close to zero (as for glucose) andin these cases the COD/VS ratio will be close tounity.

In a more generalized form, the oxidationreaction for organic material is given in Equation(5).

CnHaOb n a4 b2

O2 ! nCO2 a

2H2O

5and the COD/VS ratio then becomes (Equation 6):

COD=VS na4 b2

32

12n a 16b 6

Organic matter consisting of only C, H, and O istheoretically fully oxidized CO2 and H2O. Whenorganic matter contains also S, and N, it is wishedthat S, and N stay in the reduced form (H2S,NH3). However, depending on the method and thesalt content of the sample (e.g. high content of

chloride) S, and N will be oxidized to a dierentdegree, thus contributing to the nal COD value.Thus a compound having the formulaCnHaObNcSd is oxidized with O2 to the followingproducts (Equation. (7)).

CnHaObNcSd xO2! yCO2 zH2OvNH3wH2SO47

Alternatively HNO3 can be produced instead ofNH3. Therefore one should be aware of the de-nition during such calculations.

4.2.2. Theoretical biogas potentialWhen organic material is degraded anaerobically,the end result is carbon in its most oxidized form(CO2) and its most reduced form (CH4), i.e. anelectron transfer between carbon atoms takesplace. The ratio between CH4 and CO2 depends onthe oxidation state of the carbon present in theorganic material, i.e. the more reduced the organiccarbon content is, the more CH4 will be produced.

If the composition of the organic material isknown and all the material is converted to biogas,the theoretical methane yield potential can becalculated from the following Buswells equation(Buswell and Neave 1930):

CnHaOb n a4 b2

H2O!

n2 a8 b4

CH4 n

2 a8 b4

CO2 8

This equation is derived by balancing the totalconversion of the organic material to CH4 andCO2 with H2O 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:

Table 4. Theoretical characteristics of typical substrate components

Substrate Composition COD/VS CH4 yield CH4 yield CH4

type g-COD/g-VS STP l/g-VS STP l/g-COD (%)

Carbohydrate (C6H10O5)n 1.19 0.415 0.35 50

Protein* C5H7NO2 1.42 0.496 0.35 50

Lipids C57H104O6 2.90 1.014 0.35 70

Ethanol C2H6O 2.09 0.730 0.35 75

Acetate C2H4O2 1.07 0.373 0.35 50

Propionate C3H6O2 1.51 0.530 0.35 58

*Nitrogen is converted to NH3.

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Bo;th n2 a8 b4

22:4

12n a 16b STPlCH4g-VS

9

where 22.4 is the volume of 1mol of gas at STPconditions.

If Buswells equation is combined with theCOD/VS relation a similar COD based specicyield becomes:

Bo;th n2 a8 b4

22:4

n a4 b2

32STP

lCH4g-COD

10

In Table 4 the characteristics of a number of typ-ical organic materials suitable for anaerobic deg-radation is listed.

4.2.3. Practical biogas potentialAlthough the theoretical biogas potential gives arough idea of the quality of waste and the poten-tial biogas production, the practical yield obtainedin a biogas reactor will always be lower due to anumber of factors:

A fraction of the substrate is utilized to syn-thesize bacterial mass, typically 510% of theorganic material degraded.

At a nite retention time a fraction of the or-ganic material will be lost in the euent, typi-cally 10%.

Lignin is not degraded anaerobically. Often a part of the organic material is inac-

cessible due to binding in particles or structuralorganic matter.

Limitation of other nutrient factors.

Under favourable conditions with mainly wa-ter-soluble matter, degrees of conversion up to 9095% can be achieved. If the organic matter ishighly particulate or structural such as manures,3060% conversion is more normal.

In order to predict the biogas yield to be ex-pected under practical conditions, it is best to ndexperimentally determined biogas yields in theliterature or perform small-scale batch fermenta-tions.

A prediction of the composition of the gasproduced is more complex and depends rst ofall on the amount of CH4 and CO2 produced,but also on the pH of the reactor content. TheCH4 produced is mainly released to the gasphase, but CO2 is partly dissolved in the liquidphase of the reactor or is converted to bicar-

bonate as a function of the pH. Consequently,the CH4 percentage in the biogas produced willgenerally be higher than predicted by the stoi-chiometric ratio.

5. Assessment of the hydrolysis rate

First order kinetics are most commonly used todescribe the hydrolysis of particulate substratesduring anaerobic digestion (Eastman and Fergu-son 1981; Pavlostathis and Giraldo-Gomez 1991).

dXdegr=dt kh Xdegr 11with Xdegr is the concentration biodegradablesubstrate (kg/m3), t is the time (days) and, kh isthe rst order hydrolysis constant (day1).

Despite the fact that the rst order kinetics isempirical relation it does reect the major aspectof the hydrolysis of particulate substrates, namelythe fact that the hydrolysis of particles limited bythe amount of surface available. Severalresearchers showed that the hydrolysis mechanismof particulate substrates is surface related (Hills &Nakano 1984; Sanders et al. 2000). In this case theamount of enzymes is present in excess relative tothe available surface area (Hobson 1987) and thehydrolysis rate is determined by the surface areanot by the enzyme activity. Such surface limitedkinetics can be described with a rst order relation(Vavilin et al. 1996; Valentini et al. 1997; Veeken &Hamelers 1999; Sanders 2002a). As it is assumedthat the enzyme activity is associated with thebiomass the rst order constant is not aected bythe biomass concentration.

Although the rst order kinetics were only in-tended to describe the hydrolysis of particles theycan also be used to describe the hydrolysis of dis-solved polymers. Sanders et al. (2002b) showed bystatistical calculations that the production ofmono and dimers from a soluble polymeric sub-strate by a mixture of endo- and exo-enzymes canbe described by rst order kinetics. However, incontrast to the hydrolysis of particles in this casethe hydrolysis rate is aected by the enzymeactivity, thus biomass concentration.

From Equation (11) the relation between thehydrolysis constant, digestion time and euentconcentration for a batch system can be derived(Sanders et al. 2002a).

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Xss;batcht Xss;added1 fh fhXss;addedekht 12

where Xss, batch(t) is the concentration of totalsubstrate in the batch reactor t days after incuba-tion biodegradable + non-biodegradable part)(g l1), Xss;added the concentration of total substratein the added to batch reactor at start of experimentbiodegradable + 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 ofEquation (12). From Equation (13) it can be seenthat the hydrolysis constant is determined via alinear least square t of the results from the batchdigestion, Xss;batch at several moments in timeduring the batch assay. The biodegradability fhhas to be assessed directly from the experimentalresults, the maximum methane yield of the sub-strate (Veeken and Hamelers 1999) or the naleuent concentration.

lnXss;batcht Xss;added1 fh

Xss;addedfh

kh t 13

As the concentration of biodegradable substrate ishighest at the start of the experiment so will thehydrolysis rate. This means that to obtain enoughexperimental data for calculation of the hydrolysisconstant it is essential that the reactor is sampledat smaller intervals in the beginning of the exper-iment then at the end. Nevertheless, enough datahas to be gathered at the end of the experiment toestablish the biodegradability of the substrate.

A more direct and accurate method forassessing the hydrolysis constant and biodegrad-ability from batch and continuous experimentsis the non-linear least squares t on theassessed euent concentration (Sanderset al. 2002a). This method should be assessedwhenever possible. With these calculations thegas production or the COD, protein and carbo-hydrate content of the blank has to be taken intoaccount.

Additionally, it should be emphasised that thebiodegradability in Equations (12) and (13) refersto the biodegradability under the applied condi-tions and may change with the imposed reactorconditions.

6. Potential problems in estimation of methanepotential of wastes

6.1. Nitrate, sulphate reducers and methanogens

It has been shown that sulphate reducersand denitriers are able to outgrow the methano-gens. This is due to the higher energy gained bysulphate or nitrate reduction compared to metha-nogenesis. Therefore, presence of high concentra-tions of sulphate or nitrate will result indetermination of low methane potentials of thewastes.

In cases where the waste (water) contains sul-phate and the degree of hydrolysis is measured viamethane production the hydrolysis obviously willalso be underestimated due to consumption oforganic matter for the formation of H2S. Unlessthe amount of H2S produced can be measureddirect measurement of the degradation of theindividual components for this type of waste(water) is necessary.

6.2. Sorption and bioavailability

Sorption is an important mechanism that inu-ences the fate and eect of organic com-pounds. When compounds reside in environmentswith high sorption capacity, they may becomeunavailable for anaerobic degradation. This canaect determination of their methane potential.

6.3. Problems during COD determination

Volatile straight-chain aliphatic compounds arenot oxidized to any appreciable degree.

aromatic carbohydrates, and some aromaticheterocyclic compounds (e.g. pyridine) are notoxidized.

Halogens (Cl, Br J) can been oxidized. NO22 exerts a COD of 1.1mg/mg NO2 N Reduced inorganic compounds such as ferrous

iron, sulphide, manganous manganese, etc., areoxidized quantitatively under the species

The various COD methods have been developedfor water and waste water analyses. However,when samples like manure are analyzed interfer-ences of other additional factors can occur. Fur-thermore, the samples have to be properlyhomogenized and diluted, since agricultural and

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household wastes contain much higher organiccontents that wastewaters normally contain.

6.4. Inhibition of the process

The experimentally determined methane potentialcan be underestimated in cases where waste con-tains toxicants or the process is overloaded. Insuch cases dilution of the waste will result in moreaccurate determination of the methane potential.

7. Conclusions

Anaerobic biodegradation is a complex processand the biological approach to determininganaerobic biodegradation or methane potentialsleads to substantial uncertainty in the determi-nation. Therefore, the determination procedureshould be carefully considered, anaerobic optimalgrowth conditions secured and results carefullyevaluated.

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