Study on biogas production by anaerobic digestion of garden-waste

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    wastes containing higher percentage of organic matter by anaero-bic treatment is the best possible option. This process results in therecovery of useful energy in the form of biogas, thus reducing fossilfuel and green house gas sources [3].

    Mixed microbial populations endowed with characteristic andspecic metabolic capabilities occur widely in niches of naturalhabitats. Their coexistence is sustained by metabolic interactions

    hydrogen and carbon dioxide for maximumproduction of methane.This is brought about by hydrogen producing acetogenic bacteriawhich grow only in syntrophic association with hydrogen scaveng-ers such as sulphate reducing or methanogenic bacteria [5].

    The present study explores garden-waste as substrate for anaer-obic digestion. Cow dung, paddy eld soil, mine water, and ter-mites were used as inocula.

    Typically, the garden refuse stream consists of a wide range ofmaterial including leaves, grass-clippings, perennial and annualplants material, tree and shrub branches and other woody waste,etc.

    Corresponding author. Tel.: +91 970 9158133/326 2396433.

    Fuel 95 (2012) 495498

    Contents lists available at


    .eE-mail address: (P. Gupta).The extraction of biogas out of solid waste is considered as an is-sue that has only comeup in the recent past. Sandec, theDepartmentofWater and Sanitation inDevelopingCountries at the Swiss FederalInstitute for Aquatic Science and Technology (Eawag), conducted aresearch on biomethanation of biodegradable solid waste for lowand middle income countries.

    Citizens can be relieved from the nuisance of organic waste byscientic treatment through a cost-effective, quick, and non-polluting method or by recycling. Biological conversion of solid

    up into four phases: hydrolysis, acidogenesis, acetogenesis/dehy-drogenation, and methanation. Hydrolytic bacteria bring about ini-tial degradation of complex biopolymers such as cellulose,hemicellulose, proteins and lipids into dicarboxylic acids, volatilefatty acids (VFAs), ammonia, carbon dioxide, and hydrogen. Metha-nogenic bacteria which play a key role in the terminal step of anaer-obic digestion use only a few compounds like acetate, methanol,methylamine, hydrogen and carbon dioxide. VFA and dicarboxylicacids are thus needed to be converted asmuch as possible to acetate,1. Introduction

    Population and pollution are inpopulation there is increase in wasteing countries the most serious enviroare related with inadequate solid wmainly organic waste, dumped in opronmental pollution to soil, groundw0016-2361/$ - see front matter 2011 Elsevier Ltd. Adoi:10.1016/j.fuel.2011.11.006ted, due to increasingation. In many develop-al and health problemsanagement. The waste,ces, causes heavy envi-nd surface waters [1,2].

    that allow the ow of carbon, energy and other intermediatestoward their mutual benet. Methanogenic archeae, a diversegroup of obligate anaerobes, occurring in most anaerobic habitats,form the terminal electron sink during anaerobic digestion of or-ganic matter to methane. Methanogens derive the energy forgrowth only by methanogenesis and are the only organisms knownto produce methane as a catabolic end product [4].

    Methane fermentation is acomplexprocess,whichcanbedividedBio-gasicationStudy on biogas production by anaerobic

    Priyanka Gupta a,, Raj Shekhar Singh a, Ashish SachaaCentral Institute of Mining & Fuel Research (CSIR), Dhanbad, Jharkhand 826 015, IndiabDepartment of Biotechnology, Birla Institute of Technology, Mesra, Ranchi, Jharkhand,

    a r t i c l e i n f o

    Article history:Received 17 August 2011Received in revised form 31 October 2011Accepted 2 November 2011Available online 15 November 2011

    Keywords:Anaerobic digestionInoculum

    a b s t r a c t

    As petroleum and good quenergy is the demand of thin India is also a serious pindigenous natural sourcesfour natural sources i.e. cowis inferred that microbes pof four natural sources.


    journal homepage: wwwll rights reserved.igestion of garden-wasteb, Ambarish S. Vidyarthi b, Asha Gupta a

    coal reserves in India are depleting, hence alternative renewable source ofe. At the same time management of huge quantities of garbage produced

    em. Therefore, anaerobic digestion of garden-waste was tried using somend potential microbes for the gasication of garden-waste. For this studyung, paddy eld soil, mine water and termites were used. From the study itnt in paddy eld soil when enriched on garden-waste gave best results out

    2011 Elsevier Ltd. All rights reserved.

    SciVerse ScienceDirect


    l sevier .com/locate / fuel

  • 2.1. Collection of sample for substrate

    CI s nfo

    2. le

    r sgr P swas done as per Indian Standard 1350 (Part I) 1984 and reaf-

    and carbon dioxide contents by Haldane Gas Analysis Apparatus

    2 months [8]. Enrichment of each culturewas done by sub-culturing

    contentswere of volatilematter (66.01%) and xed carbon (16.14%).

    tence of methylotrophs in the paddy eld soil [9,10]. Mine water

    el 95rmed in 2001 [6]. All analyses were done in triplicate samples.

    Moisture: Moisture content was determined by drying 10 gsample in a pre weighed dish at 108 2 C in an electric oven(Micro-thermal, Kolkata, India) for 1 h, cooled the dish in a des-iccator, and weighed, repeated the process till constant weight.Volatile Matter: Volatile matter was determined by heating 1 gsample in a pre weighed silica VM crucible covered with lid at900 10 C for a period of 7 min in the mufe furnace.Ash: One gram sample was heated in pre weighed silica cruciblewithout covering with lid (i.e. in presence of air) at 815 C for1 h and repeated the process till constant weight.Fixed Carbon: Fixed carbon was determined by calculation[100 (Moisture + volatilematter + ash)].

    2.3. Collection of natural sources of microbes for inoculum

    Four materials collected to use as source of microbes for thebio-gasication of the garden-waste, care was taken to maintainanaerobic conditions:

    (i) Fresh cow dung sample was collected in a poly bag andzipped immediately.

    (ii) Paddy eld soil was collected from the water logged paddyeld in a bottle during paddy season.

    (iii) Mine water sample was collected from the undergroundsump at the depth of more than 1 m from a gassy-mine nearJharia, Dhanbad (Jeetpur coalmine, ISCO, SAIL India).

    (iv) Termites were collected from a tree of botanical garden[CIMFR, Dhanbad, India] just before the inoculation. Ten ter-mites were crushed by glass grinder and ground materialtaken into 5 ml medium for inoculation.

    2.4. Medium

    Barkers enrichment medium mineral salts [7] of the followingcomposition were used for all the experiments.

    g/l in tap water

    Ammonium chloride 1.00Di-potassium hydrogen phosphate


    Magnesium chloride 6H2O 0.10pH adjusted 7.0

    2.5. Experimental setupapfoAfter mixing properly, a part of the sun-dound to ne size for the proximate analysis.The experiments were conducted in specialparatus that contained one reaction vessel ar the collection of gas by water displacemenied samples waroximate analysi2. Proximate analysis of the garden waste sampr 1 week, and stored in airtight container.

    MFR, Dhanbad, India. Collected material wa dried in the su

    Garden-waste was collected from the botanical garden of2. Materials and methods

    496 P. Gupta et al. / Fuly designed glassnd one reservoirt method. 100 mlinoculum gave consistent methane production. Microbes presentin mine water were able to grow on garden-waste but only 8.9 ccmethane gas was produced in 56 days. Microbes present in termitegut produced 10.88 cc methane gas in 42 days and then declined to5.35 cc in 56 days may be due to some methane consumingcontaminants.

    Comparative results of methane production using different nat-ural sources of inoculum on garden-waste are depicted in Fig. 1. Ontaking direct natural sources as inocula, maximum methane pro-duction was found with termite but within few days, the methaneAsh contentwasonly 7.78. Volatilematter andxed carbon togethermakes garden-waste as potential waste for the bio-gasication.

    3.2. Comparative results with different source of bacteria

    Cow dung inoculum experiments were conducted for a couple ofmonths but no methane was produced. This shows that microbespresent in cow dung were not capable of using garden-waste sub-strate for methane production. With paddy eld inoculum maxi-mum methane (mean value of triplicates 3.94 cc) production wasfound on 28th day and then declined. It may be due to the coexis-methodology where garden waste was used as specic energysource.

    3. Results and discussion

    3.1. Proximate analysis

    In the garden-waste sample,moisture contentwas 10.07%.Major(Meghna, India). The ratio of methane to carbon dioxide deter-mined was in the range of 55:4552:48.

    2.7. Experiments with different source of bacteria

    Four experiments (triplicate of each) using different sources ofmicrobes i.e. cow dung, paddy eld soil, mine-water, and termiteswere carried out using garden-waste as substrate for aboutautoclaved Barkers medium was taken into the reaction vessel,0.325 g of Na2S, 3 ml of 5% solution of Na2CO3, 0.0001 g resazurine,and 10 g UV sterile garden-waste of mesh size below 10 mm werealso added to the reaction vessel and inoculated with 5 ml of inoc-ulum. The reaction vessel was immediately evacuated to removeair and re-lled with inert gas (helium) to establish anaerobic con-ditions. The collection vessel (reservoir) was completely lled withwater. Both the vessels were connected together with black pres-sure tubing. Reaction vessel was also connected with mercurymanometer.

    2.6. Measurements

    Pressure developed due to production of gas was measuredusing mercury manometer everyday and amount of water equiva-lent to the pressure in the vessel drown out of the reservoirthrough stopper at the bottom of the vessel and volume of waterdrained out was measured by a measuring cylinder.

    Gas collected in the reservoir periodically analyzed for methane

    (2012) 495498got declined. Cow dung inoculum did not work on garden-waste,and with paddy eld soil inoculum, production of methane wasvery low. Mine water inoculum gave consistent increasing pattern.

  • Fig. 2. Methane produced from garden-waste using paddy eld soil as inoculum.

    el 953.3. Enrichments of paddy eld soil inoculum

    The culture grown from paddy eld soil on garden-waste wasused as inoculum for rst enrichment. There was a little improve-ment in methane production compared to initial set of experi-ments. Maximum mean value of methane (19.74 cc) wasdetermined on 49th day.

    Similarly in 2nd enrichment experiments the methane produc-

    Fig. 1. Methane production from garden-waste using natural sources as inoculum.

    P. Gupta et al. / Fution was higher than the rst enrichment. Maximum methane(107.90 cc) was determined on 49th day.

    In the similar manner, third enrichment experiments were setup. Maximum mean value of methane (73.91 cc) was determinedon 35th day. Methane detected in 3rd enrichment was less thanthat of second enrichment. Therefore, further enrichments werenot done.

    The results of methane produced by paddy eld soil inoculumand its subsequent enrichments are depicted in Fig. 2.

    3.4. Enrichments of mine-water inoculum

    The culture grown from mine-water inoculum on garden-wastewas used as inoculum for rst enrichment.

    First enrichment of mine water inoculum on garden-waste pro-duced only 12.38 cc methane in 2 months.

    Second enrichment of mine water inoculum produced maxi-mum methane in 42 days (about 30 cc methane) and then slightlydeclined in next 2 weeks.

    Third enrichment ofminewater inoculumproduced only14 cc ofmethane in 28 days and thereafter it declined to 4.66 cc in 2 months.

    No further enrichment was done after 3rd enrichment due todeclined activity of the culture.

    The results of methane produced by mine-water inoculum andits subsequent enrichments on garden-waste are depicted in Fig. 3.

    3.5. Enrichments of termite gut inoculum

    The culture grown from termite gut inoculum on garden-wastewas used as inoculum for rst enrichment.(2012) 495498 497First enrichment of termite inoculum produced 39.98 cc ofmethane in 49 days.

    Second enrichment of termite inoculum produced 45.32 cc ofmethane in 42 days. The methane was declined thereafter.

    Third enrichment of termite gut microbes produced methane inthe same patter as in 2nd enrichment.

    As there was no enhancement in methane production in 3rdenrichment, hence further enrichment was not done.

    The results of methane produced by termite gut inoculum andits subsequent enrichments on garden-waste are depicted in Fig. 4.

    Fig. 3. Methane produced from garden-waste using mine water as inoculum.

  • Future work is needed to demonstrate that paddy-eld soilfrom different locations can be used reliably as an inoculumfor the anaerobic digestion of garden waste.


    Director, Central Institute of Mining & Fuel Research is greatlyacknowledged for providing funds & facilities. Authors are alsograteful to the mine authorities of Jeetpur coalmine, ISCO, SAIL In-dia for the help in collecting mine sump water.


    [1] Christian Mller, Anaerobic digestion of biodegradable solid waste in low- andmiddle-income countries, overview over existing technologies and relevantcase studies, Eawag Aquatic Research, Water and Sanitation in DevelopingCountries (Sandec), Dbendorf; May 2007.

    [2] Veeken AHM, Hamminga P, Mingshu Z. Improving sustainability of municipalsolid waste management in China by source separated collection andbiological treatment of the organic fraction. In: Innovative environmentalmanagement & sustainable development [China]: 2005 ISBN 7802092337,Wageningen UR publication.

    [3] Anhuradha S, Vijayagopal V, Radha P, Ramanujam R. Kinetic studies and

    498 P. Gupta et al. / Fuel 95 (2012) 4954984. Conclusion

    Paddy-eld soil proved to be superior to cow-dung, mine water,or termite guts as a source of microbial inoculums to initiatereactors for the conversion of garden waste to methane.

    Recommended ratio of paddy-eld soil to garden waste 1:100. Repeated sub-culturing (2 or 3) is needed to achieve the fastestrates of methane formation. The addition of paddy-eld soil isonly needed during the startup of the reactor, but not for subse-quent sub-culturing events.

    Sub-culturing should be performed every 40 days to maintain

    Fig. 4. Methane produced from garden-waste using termites as inoculum.maximum rates of methane formation. Residual solids from anaerobic digestion can be used as gardenmulch.anaerobic co-digestion of vegetable market waste and sewage sludge. CLEANSoil Air Water 2007;35(2):1979.

    [4] Rapheal SV, Swaminathan KR, Lalitha K. Metabolic characteristics of an aerobeisolated from a methylotrophic methanogenic enrichment culture. J Biosci2003;28(2):23542.

    [5] Meher KK, Ranade DR. Isolation of propionate degrading bacterium inco-culture with a methanogen from a cattle dung biogas plant. J Biosci1993;18(2):2717.

    [6] BIS Standard IS 1350 (Part I) 1984 (Reafrmed in 2001), Indian StandardMethods of Test for Coal and Coke, Part I Proximate Analysis, Ed 3.1 (199208)BIS 2003, Bureau of Indian Standards, New Delhi-110003.

    [7] Moh RA, Smith MR. The methanogenic bacteria. In: Starr MP, Stolp H, TrperHG, Balows A, Schlegel HG, editors. The prokaryotes (A handbook on habitats,isolation, & identication of bacteria), vol. I. New York: Springer; 1981. p.9545.

    [8] Yadvika, Santosh, Sreekrishnan TR, Kohli S, Rana V. Enhancement of biogasproduction from solid substrates using different techniques a review.Bioresour Technol 2004;95(1):110.

    [9] Reichardt W, Mascarina G, Padre B, Doll J. Microbial communities ofcontinuously cropped, irrigated rice elds. Appl Environ Microbiol 1997;63(1):2338.

    [10] Zheng Yong, Zhang Li-Mei, Zheng Yuan-Ming, Di Hongjie, He Ji-Zheng.Abundance and community composition of methanotrophs in Chinese paddysoil under long-term fertilization practices. J Solids Sediments 2008;8:40614.

    Study on biogas production by anaerobic digestion of garden-waste1 Introduction2 Materials and methods2.1 Collection of sample for substrate2.2 Proximate analysis of the garden waste sample2.3 Collection of natural sources of microbes for inoculum2.4 Medium2.5 Experimental setup2.6 Measurements2.7 Experiments with different source of bacteria

    3 Results and discussion3.1 Proximate analysis3.2 Comparative results with different source of bacteria3.3 Enrichments of paddy field soil inoculum3.4 Enrichments of mine-water inoculum3.5 Enrichments of termite gut inoculum

    4 ConclusionAcknowledgementsReferences


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