Bioresource Technology xxx (2008) xxx–xxx

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Bioresource Technology

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Short Communication

Simple and rapid methods to evaluate methane potential and biomass yield fora range of mixed solid wastes

P. Shanmugam *, N.J. HoranPublic Health and Environmental Engineering, School of Civil Engineering, University of Leeds, Leeds, LS2 9JT, UK

a r t i c l e i n f o a b s t r a c t

Article history:Received 13 December 2007Received in revised form 30 May 2008Accepted 5 June 2008Available online xxxx

Keywords:Empirical formulaBiochemical/stoichiometric methanepotentialC/N ratioAdenosine tri-phosphateBiomass yield

0960-8524/$ - see front matter � 2008 Elsevier Ltd. Adoi:10.1016/j.biortech.2008.06.027

* Corresponding author. Address: EnvironmentalResearch Institute (CSIR), Adyar, Chennai 600 020, Ind

E-mail addresses: pashanmugam@yahoo.com (Pleeds.ac.uk (N.J. Horan).

Please cite this article in press as: Shanmufor ..., Bioresour. Technol. (2008), doi:10.1

This paper describes rapid techniques to evaluate the methane potential and biomass yield of solidwastes. A number of solid wastes were mixed to provide a range of C:N ratios. Empirical formulae werecalculated for each waste based on the results of chemical analysis and these formulae were used to esti-mate the COD equivalent and stoichiometric methane potential (SMP). The actual COD and biochemicalmethane potential (BMP) were determined experimentally for each waste and for both parameters therewas a good agreement between the empirical and experimental values. The potential of adenosine tri-phosphate (ATP) to act as an indicator of biomass yield (mg VSS mg�1 COD removed) was determinedduring the anaerobic digestion process. The biomass yield determined from ATP analysis was in the range0.01–0.25 mg VSS mg�1 COD removed which corroborated well with previously reported studies. Empir-ical formula based SMP together with ATP measurement were shown to provide rapid methods to replaceor augment the traditional BMP and VSS measurements and are useful for evaluating the bioenergy andbiomass potential of solid wastes for anaerobic digestion.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

The techniques used in UK for disposing of solid wastes arechanging rapidly and largely as a result of the Landfill Directive,biodegradable waste is now being diverted away from landfill. ThisDirective requires that by July 2010 biodegradable municipalwaste (BMW) going to landfill shall be reduced to 75% of its1995 tonnage, rising to 35% of this by 2020 (Defra, 2007). Diversionis also being aided by increases in land fill tax, currently around£22/T. Other drivers that contribute to this change include climatechange and the potential fiscal rewards from the Renewable Obli-gation Certificates (ROC’s). As a result a large tonnage of biodegrad-able waste stream previously sent to landfill now requires dealingwith by other means. The opportunity to convert this waste to en-ergy is obviously an attractive one and current technologies forachieving energy from waste focus on biological and thermal op-tions. The biological route is essentially the application of anaero-bic digestion (AD) which is able to reduce the amount of volatilesolids in a waste feed by up to 70%, whilst at the same time gener-ating a source of renewable energy as methane. The Department ofEnvironment, Food and Rural affairs (Defra, 2007), has estimatedthat the potential annual market for anaerobic digestion is £400

ll rights reserved.

Engineering, Central Leatheria. Tel.: +91 44 24911386.. Shanmugam), n.j.horan@

gam, P., Horan, N.J., Simple a016/j.biortech.2008.06.027

million. In addition AD currently attracts 2 ROC’s with a value of£30/MWh.

However, AD is a capital intensive project and in order to under-take a basic process economic feasibility study, it is important tohave an indication of the likely methane yield available from thedigester feedstock. A number of techniques are available to providethis information including the biochemical methane potential(BMP), dynamic respiration rate (DR4), and the COD test (Owenet al., 1979; Environment Agency, 2005). In addition, a number ofindicative ratios can be used such as the soluble COD to volatile or-ganic solids ratio. Of these, the BMP test is the most popular but, aswith any simple batch test that is intended to provide informationon the likely performance of a full-scale continuously operatedprocess, the results of the BMP require interpretation with caution.In particular, the BMP test is conducted over a period of 28 days,whereas conventional AD rarely operates at retention times above15 days. Many researchers have searched for feasible alternativesto the BMP test, but with limited success and consequently ratherthan attempting to search a replacement, it is perhaps more appro-priate to develop additional protocols to complement the resultsfrom the BMP and aid in its interpretation (Shelton and Tiedje,1984; Mendez and Lema, 1993). This paper intends to evaluatetwo such complementary test procedures. The first of these in-volves determination of the empirical formula of biomass thatcan be used for stoichiometric product estimation (Erickson,1978, 1980). The empirical formula developed for activated sludgefrom aerobic biological treatment was C5H7NO2 (Rittmann and

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McCarty, 2001; Tchobanoglous et al., 2005) and routinely used asbasic design rule to calculate the oxygen requirement for BOD re-moval. In a similar way Rittmann and McCarty (2001) and Hansen(2005) have proposed that stoichiometric methane potential (SMP)determined from the assessment of empirical formula based or-ganic solid wastes will provide information on the energy balancefor fermentation processes.

Even with the SMP test, information is not available as to thebehaviour of bacteria during the AD process and those conditionswhich provide an optimum environment for their growth. The di-rect measurement of volatile suspended solids (VSS) cannot be ap-plied in solid wastes digesters as it cannot distinguish betweenbacteria and suspended organic solid wastes particles and so thereis a need for an alternative method of VSS measurement for solidwaste digesters to predict biokinetic design constants. The quanti-fication of adenosine triphosphate (ATP) has been used by a num-ber of researchers as an anaerobic cell viability assay method(Chung and Neetheling, 1988; Yu et al., 2002; Kim et al., 2007).The ratio of ATP to VSS (Hwang and Hansen, 1998) has also beenused to assess both cell viability and biomass yield, thus providingimportant information on the behaviour of the organisms duringthe AD process. Hence, this present study investigates the SMPand ATP analysis as complementary tests to the BMP and VSS forevaluating the suitability of a range of organic solid wastes as sub-strates for AD.

2. Methods

2.1. Characterisation of solid wastes

A number of solid waste sources were used in this study. Muni-cipal solids waste (MSW) was collected in bulk and autoclaved at130 �C to permit storage without deterioration. Leather fleshing(LF) and primary chemically treated sludge (LS) were collectedfrom the tannery effluent treatment plant at Holmes Hall tanneryin Hull. The leather fleshing was minced and homogenised witha commercial blender to 6 mm diameter before feeding to the di-gester. Wastewater treatment sludge included primary sedimenta-tion tank sludge (PST), mixed liquor from a sequencing batchreactor (SBR), and surplus activated sludge (ASP) were collectedfrom the Knostrop wastewater treatment plant at Leeds. The char-acterisation of total solids (TS), and volatile solids (VS) were carriedout using Standard Methods (APHA, 1998). Elemental analysis ofcarbon, hydrogen, nitrogen and sulphur was undertaken usingCHNS analysers Model Thermo Flash EA 1112 series. Samples wereoven dried at 103 �C and then combusted at 1800 �C in the CHNSanalyser in a steam of helium with measured amount of oxygen.This produces N2, CO2, H2O, and SO2 which are then separatedand quantified by gas chromatography using a 5 mm diametersteel (length of 2 m) packed column, helium carrier gas with a flowrate of 40 mL/min, detected with a Propack model TCD.

2.2. Development of empirical formula for stoichiometric methanepotential (SMP)

From the calculated values of C, H, N, and O an empirical formulawas computed for each solid waste following the procedure devel-oped by Rittmann and McCarty (2001) and Hansen (2005). This for-mula was used to determine the COD equivalent of which permittedthe calculation of the stoichiometric methane potential (SMP).

2.3. Biochemical methane potential (BMP) assessment

The BMP was determined in anaerobic batch reactors of 500 mlcapacity Duran bottles with hermetically sealed stoppers and con-

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trolled gas opening valves. The effective volume maintained was400 ml and the gas phase was 100 mL. Nutrient medium was pre-pared using a modification to the method described by Owen et al.(1979) and added at 1 mL per litre of reactor volume. An internaltemperature of 35 �C was maintained by incubating the reactorsin a temperature controlled mechanical shaker. Samples weremixed at 140 rpm for a 15 minutes period followed by 15 minuteswith no shaking. The quantity of biogas produced was measured byconnecting the gas opening valve on the reactor to the inlet tube ofa hermetically sealed, water displacement aspirator bottles filledwith 5% NaOH to scrub CO2. A measuring cylinder at outlet ofthe aspirator bottles collected displaced water which measuresCH4 at atmospheric temperatures and pressure (Nm L CH4. day�1).The initial and final characterisation of 500 mL bottles was takenfor mass balance analysis and the serum bottle samples were usedfor anaerobic process evaluation analysis. The BMP of seed sludgewas simultaneously carried out in control reactors, and subtractedfrom the VS and gas yield with solid waste and the BMP yield ofsolid waste was calculated as Nm L gm�1 VS removed. The con-tents of the serum bottle were withdrawn periodically and centri-fuged at 6000 rpm for 1 h and the supernatant was collected forproduct analysis of total alkalinity (T.Alk), volatile fatty acids(VFA), and ammoniacal nitrogen (NH3–N).

2.4. ATP–cell viability and biomass yield

Analysis of ATP was based on quantification of luminescence re-leased from the reaction of luciferase with ATP (Chung and Neeth-ling, 1989). Samples were diluted with 20 mM Tris-EDTA at pH7.75, boiled for 30 min and equilibrated to room temperature.Supernatant ATP was recovered by centrifugation at 6000 rpmfor 5 minutes. The luminescence reaction of sample ATP and lucif-erase was measured as a Relative Luminescence Unit (RLU) in aPromega GloMaxTM 20/20 model luminometer, following additionof back titre luciferase supplied by Promega. A calibration graphwas prepared from the RLU at varying ATP concentrations withluciferase to quantify the sample ATP. The COD and VSS contentover time was calculated using 1.4 mg COD mg�1 VS and 250 mgVSS mg�1 ATP (Hwang and Hansen, 1998). This was then used tocalculate biomass yield as mg VSS mg�1 COD removed.

3. Results and discussion

3.1. Characterisation of a range of raw solid wastes

The important parameters for characterising the suitability of awaste for AD are carbon to nitrogen ratio (C/N) and VS content, andthe microbiological performance parameters such as BMP and bio-mass yield are necessary for reactor design estimations. A range ofsolid waste types with differing characteristic were selected forthis study (1) with the volatile organic solids ranging from 60 to75% and the C:N 3.2 to 21.6. The LS had the highest organic content,whereas the LF had the highest nitrogen content and thus a lowerC/N ratio. Primary sludge had the highest ash content and this islikely to be a result of the presence of silt, clay and sand particles.

3.2. Empirical formulae, stoichiometric COD and methane potential(SMP)

Based on the chemical analysis provided in Table 1, the empir-ical formula of each waste was calculated (Table 2). The calculatedempirical formula was then used to determine the theoretical COD(CODe) of the waste. When expressed in terms of the measured vol-atile solids this gave an average value of 1.4 g CODe g�1 VS, a valuethat is typical of such wastes, for instance Han et al. (2005) re-

nd rapid methods to evaluate methane potential and biomass yield

Table 1Characterisation of range of solid wastes

Parameters Leather fleshings ASP sludge SBR sludge Leather sludge PST sludge MSW

1. Total dry solids (%) 21.9 0.9 0.4 21.5 3.8 41.72. Volatile solids (% of TS) 81.3 70.3 62.0 81.5 60.3 63.03. Carbon (% of TS) 35.5 37.6 28.3 39.9 28.7 27.54. Hydrogen (% of TS) 9.4 5.7 5.1 6.4 4.6 3.85. Nitrogen (% of TS) 11.2 7.0 4.2 3.5 2.4 1.36. Oxygen (% of TS) 25.3 20.1 24.5 31.6 24.7 30.57. Sulphur (% of TS) 0.6 0.5 0.8 1.3 0.5 0.48. Ash content (% of TS) 18.7 29.7 38.0 18.5 39.7 37.0

Table 2Empirical formula, and anaerobic process evaluation for a range of solid wastes

Solid waste Empirical formula C:N VFA (mg L�1) T.Alk. (mg L�1) NH3–N (mg L�1) VSSe (g g�1 COD eliminated)

Leather fleshing C4H11NO2 3.2 3540 9163 1296 0.09ASP sludge C6H11NO2 5.4 4147 6039 241 0.14SBR sludge C8H17NO5 6.7 4266 7997 466 0.14Chemical sludge C13H26NO8 11.4 2722 7477 999 0.01PST sludge C14H26NO9 14.0 3490 5703 305 0.12MSW C25H41NO21 21.6 4371 6821 414 0.25

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ported values of 1.1 and Rittmann and McCarty (2001) observed1.4. Good correlation was also observed between the CODe deter-mined from the empirical equations and that calculated experi-mentally. Direct measurement of the COD of a solid waste isoften thought to produce erroneous results and thus the volatilesolids are generally used to quantify the organic strength of solidwastes (Han et al. 2005; Lin et al., 1999). However, COD data is re-quired for reactor design to estimate the biokinetic yield. Conse-quently COD estimation based on empirical formula provides anattractive alternative to the experimental COD for design purposes.

3.3. Comparison of SMP and BMP test

The BMP was measured using the same range of solid wastesunder controlled conditions of nutrient addition, heating and stir-ring. The measured BMP ranged from 0.36 Nm L g�1 VS for thechemical sludge to 0.52 Nm L CH4 g�1 VS for the ASP (Table. 2).These values are typical of those observed by others, for instanceLin et al. (1999) reported a BMP of 0.35 Nm L CH4 g�1 of CODe

(equivalent to 0.318 Nm L g�1 VS) for chemically pretreated ASPsludge. A comparison of the SMP and BMP for these solid wastesgave a good linear relationship (Table 2). The SMP of LF, PST,SBR, and LS were 0.58, 0.53, 0.51, and 0.56 Nm L g�1 VS whereas;the BMP was recorded as 0.49, 0.38, 0.38 and 0.36 Nm L g�1 VS,respectively. The observed SMP and BMP of ASP sludge was 0.61and 0.52 Nm L g�1 VS, in line with the findings of Gosset and Belser(1982) and Wook and Hwang (2000) who reported the BMP of ASPsludge as 0.36–0.54. In a similar way, the SMP and BMP of theMSW was 0.40 and 0.36 Nm L CH4 g�1 VS and a number of authorsreport similar findings, for example Davidsson et al. (2007) who re-ported that MSW has a BMP of 0.3–0.4 Nm L g�1 VS. Thus the SMP,which can be undertaken within 24 h, provides a more rapid alter-native to the 28 days BMP test. It has the additional advantage thatit provides information as to the likely COD of the waste under testand such information can be used for further biokinetic evaluation.

3.4. Evaluation of factors affecting full-scale digester performance

The SMP and CODe offer rapid procedures to estimate the po-tential methane yield from a waste. However, they give no indica-tion as to how the waste might degrade in a full-scale digester.Many other factors influence actual digester performance. For in-

Please cite this article in press as: Shanmugam, P., Horan, N.J., Simple afor ..., Bioresour. Technol. (2008), doi:10.1016/j.biortech.2008.06.027

stance poor VS removal efficiency and low biogas yield are oftenassociated with low C/N, high NH3–N, high VFA and low digesterbuffering capacity (Haandel and Lettinga, 1994; Anderson et al.,2003). In addition, the concentration of ATP is known to indicateboth the cell viability and the metabolic status of a microorganismand it has been used to predict biomass levels in anaerobic diges-tion (Chu et al., 2001; Yu et al., 2002; Kim et al., 2007). In order toestablish the potential of ATP as a simple performance monitor,ATP concentration was measured over the duration of the BMPtest, together with a number of physical parameters that indicatereduced performance. By monitoring a range of solid waste typeswith differing C/N ratios it was hoped that a clear picture wouldemerge as to the status of ATP, as an indicator of reactor stabilityand performance. C/N ratio was selected as the variable parameterbecause it determines both the NH3–N and VFA concentration ob-served in the digester. Increasing NH3–N helps to raise the pH,whereas by contrast an acid pH is governed by VFA’s which neu-tralise HCO�3 , CO2�

3 and acetate ions. However ammonia can betoxic: Callaghan et al. (2000) and Salminen and Rintala (2002) havereported the tolerance level of NH3 in anaerobic digesters acclima-tised to treat high protein wastes as 11.6 g L�1 and 6.0 g L�1,respectively.

Of the six solid wastes evaluated, the cumulative biogas yieldafter 28 days was highest for MSW and the lowest for leather flesh-ing, in line with the C:N ratios for these wastes (Fig. 1 and Table 2).The maximum NH3–N concentration of 1296 mg L�1 was also re-corded for the leather fleshing but this is well below the concentra-tion reported as inhibitory. Alkalinity was in the range of 3800–9700 mg L�1 and the maximum alkalinity was coincident with highNH3–N levels (Table 2). The VFA was observed to peak at around 8days for SBR, ASP and MSW wastes, whereas for LF it was as late as20 days suggesting that a much longer digester retention timewould be required for this waste (Fig. 2). The late decrease inVFA was also coincided with high alkalinity. ATP was measuredat a concentration in the range of 2.01–8.43 mg L�1 and similar val-ues have been reported by others, for instance Hwang and Hansen(1998), Yu et al. (2002), and Chen (2004). The ATP level for LF, ASP,SBR, LS, PST, and MSW were 2.01, 4.21, 3.22, 2.12, 3.22, and 8.43,respectively, and observed as linear with respect to cumulativemethane yield measured during the BMP test (Table 2), thus dem-onstrating the potential value of this parameter. Standard BMP testcan be used to estimate biokinetics design constants. Future work

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0

500

1000

1500

2000

2500

3000

0 10 20 30 40Days Elapsed

Bio

gas

(N m

L) SBRASPPSTChemical SludLeather FlMSW

Fig. 1. Cumulative biogas evolution with elapsed time.

0100020003000400050006000700080009000

0 10 20 30 40Elapsed time (Days)

VFA

(mg/

L)

SBR SludgeASP sludgePST SludgeChemical SludgeMSWLF

Fig. 2. The volatile fatty acids (VFA) levels at elapsed time during the BMP test.

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will therefore focus on correlating the level of ATP attained in theBMP test with the biokinetic constants of a continuously fed sys-tem treating the same waste type.

4. Conclusion

For all the solid waste examined, there was a good corrobora-tion observed between SMP and BMP, CODe and experimentalCOD. The wastes with higher C:N ratio produced methane morerapidly and also provides a guide to the likely operating retentiontime of a continuously fed digester. The ATP measured was indic-ative of both volatile solids removal, amount of CH4 and VSS (bac-teria) produced from the wastes. Thus, the combined application ofempirical formulae, SMP and ATP offers the potential to augmentBMP and VSS tests to estimate both methane yields and biokineticdesign constants for solid wastes anaerobic digesters.

Acknowledgements

The authors would like to acknowledge the help of Mr. NickWaudby, Holme Hall Tannery, Hull (UK) for providing samples ofleather fleshing and chemically treated sludge.

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nd rapid methods to evaluate methane potential and biomass yield