EE Final

0
How can we convertorganicfoodwaste intomethane gasthroughanaerobicdigestiontopowerhouses
in developing nations?
Adhitya Jayasinghe
CHEMISTRY KILLING TWO BIRDS WITH ONE STONE

ABSTRACT
The paper seeks to investigate the proposed conversion of organic food waste into
sustainable energy, CH4, methane to power houses in developing nations. The pressing issue of
energy scarcity has filled the minds of politicians, engineers, and scientists across the world.
There have been numerous proposed solutions from tidal power to geothermal, but most of
these alternatives require a rather large initial fee, making it difficult for developing nations to
adopt. Therefore, the researcher looked to find an alternative source of energy that developing
countries would be able to adopt; biogas. Biogas and biofuels have been used for thousands of
years, but the researcher chose to approach the idea of biogases from a different perspective.
How can we kill two birds with one stone and solve the issue of energy scarcity and waste
pollution?
To answer this question, the researcher used an age-old method of using cow manure
as an initial fuel source. The researcher tested different ratios of cow manure and food waste in
order to find the optimal ratio of methane production, because the structure of pure food
waste would be far too rigid for the anaerobic methanogens to digest. The solution of Basal
Carbonate Yeast Trypticase and different ratios of food waste and cow manure went through
the process of Hydrolysis, Acidogenesis, Acetogenesis, and Methanogenesis. The researcher
withdrew gas from each sample daily and put it through a gas chromatographer and recorded
the yield of Hydrogen H2, Methane CH4, and Carbon Dioxide CO2. In the preliminary stages of
the experiment, the researcher saw the highest yield of methane gas coming from the solution
of pure cow manure. After three to four days, the researcher recorded an exponential yield
increase from the solution that contained 75% food waste and 25% cow manure.

Tables of Contents
Introduction……………………………………………………………………………………......
Research Question………………………………………………………………………………...3
Background Information…………………………………………………………………………..3
3.1 Hydrolysis……..……………………………………………………………...……….3
3.2 Acidogenesis………..………………………………………………...…………….…4
3.3 Acetogensis…...…………………………………......………………...………………5
3.4 Methanogenesis………………………..……………………………...……………….5
3.5 Obligate Anaerobe ……………………...…………………...…….………………...6
Method…………………………………………………………………………………………….7
4.1 Synthesizing Basal Carbonate Yeast Trypticase………….………………...……........7
4.2 Dilution of Substrates…………………………..…………………………………......8
4.3 Food Waste and Cow Manure Substrate Inoculation……..………………………......9
4.4 Gas Chromatography……………………...…………………..……………………..10
Results……………………………………………………………………………………………11
Interpretations of Results…...........................................................................................................13
Conclusion……………………………………………………………………………………….15
Bibliography..........……………………………………………………………………………....16
Appendix I ………………………………………………………………………………………19

1
INTRODUCTION
Indiaisone of the fastestdevelopingnationsinthe worldtodayatan average growthof 7.02%
GDP overthe pastfour years.1
Today,Indiaisdelvingthroughastage of industrialization,ahighlyenergy
intensivestage of growthforany country.CountriessuchasUnitedKingdom, the UnitedStates,Japan,
Germany,and France wentthroughthisstage of developmentdecadesago.A commonsocial stigma
createdby these countriesis thatthere is a positive correlationbetweendevelopmentandpollution,
and the drop of standardof livinginexpectancyof arapidincrease of standardof livinginthe near
future.Currentexamplesof unsustainable industrializationcanbe observedinChinaandSaudi Arabiain
the past twodecades.Waste pollution,increasingsocial-incomegaps,andincreasingfrequency of
terminal diseaseare justdropsof waterin a seaof side-effectsof unsustainablegrowth.2
The question
has beenthrownaroundnumeroustimes: how candevelopingnationsprogresswithoutresultingina
depressionof standardof living?There are multiple alternativesoutthere;solarpower,nuclearpower,
windpower,hydroelectricpower,ethanolbiofuels,andthe listgoeson.All these sourceshave the
abilitytoyieldenormousamountsof energy,buttheyall come withone draw-back;price.For
developednations,these are logical investments,butfordevelopingnationssuchasIndiaor Rwanda,it
isimprobable thatindividualswillhave the moneytoinvestintothesetypesof technologies.
Overthe past decade there hasbeena surge inresearchintoalternative energysourcesthatare
feasible atthe micro-scale. One of these manyalternativesisbiogas. Driedcow manure hasbeenused
as a source of fuel since 3200-2700 BCE, and isstill beingusedtodaytopowercooking,water-heating,
and fertilizingfields3
.In1995, manure accountedfor almost21 percentof netenergyexpenditure in
rural India.4
The Ministryof NewandRenewable Energyinitiatedthe National BiogasandManure
1 “GDP Growth (annual %)” See India for GDP statistics.
2 Effects of unsustainablegrowth in India.See Pandey and Khanjan 113-115.
3 The uses of cow manure in history.See Miller 71-73.
4 Rural India’s Energy Expenditures. See Sampat 1.

2
ManagementProgramme in1981 in orderto provide cleanenergytovillagesthathave beenstruggling
withenergydeficits.In March 2014, the MNRE revisitedthe programme and,withthe helpof State
Nodal Agencies,KhadiandVillageIndustriesCommission,BiogasDevelopmentandTrainingcenters,
and the IndianInsitute of Technology,hasbolsteredthe programme,recordingabout82,700 villager-
initiatedbiogasplants.5
However,thisisnota sustainable solutionforthe comingyears,andfailsto
meetIndia’svastenergydemands. Inaddition,itfailstoaddressthe problemof waste pollutioninIndia,
one of the largest sourcesof disease andgreenhouse gasemissions.6
Accordingto the 2010 MNRE annual report,Indiaproducesapproximately55milliontonsof
municipal solidwaste eachyear,andthatamountisincreasingata rate of 1% annually.7
The diverse
cultural boundariesof India’spopulace make itdifficultforgovernmenttotake authoritative actionon
the micro scale,thusrelyingonstate governmentsto assume the responsibility of cleaningthe streets
and rivers.The lackof importance placedonwaste-pollutionhasledIndiatobe visiblyone of the most
pollutedcountriesintermsof surface waste.The vastdepositof surface municipal solidwaste isthe
root of a wide varietyof diseases.8
A pile of waste isanideal habitatformosquitoestobreed,which
leadstothe spreadof Chikungunya,Malaria, andDengue fever.9
Waste pollutionisalsoabreeding
habitatfor flies,whichare carriersof deadlydiseasessuchastyphoid,tuberculosis,leprosy,and
cholera.10
Insteadof scramblingtocreate a cure fora new strandof these diseases,we mustfocuson
tacklingthe source of the disease andpreventthe birthof new pathogens.Bylookingatbothproblems,
the energycrisisandpollutioncrisis,fromthe perspective of sustainabledevelopment,we seethat
we’re able tokill twobirdswithone stone.
5 Ministry of New and Renewable Energy biogas progamme. See 67.
6 Waste pollution in Indiaand the repercussions of this topic.See Sachs.
7 Measurements of waste pollution in India and projected growth of waste. See 5.38.
8 The diseases caused by waste pollution in India.See6.
9 The variety of diseases thatmosquitoes carry.See links for depth.
10 The various diseases flies carry becauseof solid wastepollution.See 35.

3
RESEARCH QUESTION
How can we convertorganicfoodwaste and cow manure intosustainable energyinordertopower
rural village houseswhile alsosolvingthe issue of waste pollutioninIndia?
BACKGROUNDINFORMATION
Biogasis a mixture of carbondioxide (CO2),hydrogen(H2),andmethane (CH4).Itisa clean
source of energythatisproducedthroughthe breakdownof biodegradable materialsby obligate
anaerobesinthe absence of oxygen. Thisprocessisknownasanaerobicdigestion. Foodwaste andcow
manure,whichare composedof cellulose(large polymers),starch(carbohydrates),casein(proteins),
and triglycerides(fats),are the productsusedforthe firststage of the anaerobicdigestionprocess.
3.1 Hydrolysis
The firststage of this process iscalledhydrolysis.Hydrolysis,insimple terms,isthe stage in
whichfoodwaste andcow manure isbrokendownintoliquefiedmonomersandpolymers.Inthisstage
a molecule issplitintotwopartsbyaddinga watermolecule.The cationof the parentmolecule gains
the hydroxyl group(OH-
) while the anionof the parentmolecule gainsthe hydrogenion(H+
). Cellulose
are convertedintoglucose withthe presence of cellulases,anenzyme thatcatalysesthe hydrolysisof
cellulose,andwater(H2O).Caseinsare convertedinto aminoacids,specificallyLysine andHistidine,in
the presence of proteases,anenzyme thatcatalysesthe hydrolysisof caseins,andwater(H2O).
Triglyceridesare brokendownintofattyacidsinthe presence of lipases,anenzyme thatactsas a
catalystfor the hydrolysisof triglycerides.11
Forcarbohydratesthe equationis:
ExampleHydrolysisof Carbohydrates: C6H10O4 + 2H2O <--> C6H12O6 + 2H2
11 Presentation on biogas with breakdown of cellulose,starch,casein,and triglycerides.See 10.

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The hydrolysistakesplace inthe presence of alpha-amylaseswhichhelps breakdownthe
carbohydrate intosimple sugars,suchasmaltose orglucose.12
The carbohydrate ismade up of a string
of monosaccharides.Whentwoof the monosaccharidescombine,ahydroxyl ionisremovedfromone of
the monosaccharidesanda hydrogenionisremovedfromthe other.Whenthe bondsare broken,they
mustbe replaced. The watermoleculeissplitintotwoandthe monosaccharide whichlostthe hydroxyl
iongainsthe OH-
anionfrom the watermolecule,whilethe monosaccharidewhichlostthe hydrogenion
gainsthe H+
cation,thus,producingglucose (C6H12O6) andhydrogengas(H2).13
Byhydrolyzingthe
carbohydrate,we are breakingitdownintoa simple sugarwhichcan be usedinthe nextphase,
acidogenesis.
3.2 Acidogenesis
Acidogenesis,the fasteststep, isthe processinwhichacidogenicbacteriadigestthe productsof
hydrolysis,creatingketones,alcohols,hydrogen,carbondioxide,andvolatile fatty acids.The most
commonproductsof thisstage are: aceticacid (CH3COOH) butyricacid(CH3CH2CH2COOH),propionic
acid (CH3CH2COOH),lacticacid(C3H6O3),formicacid(HCOOH),ethanol (C2H5OH),andmethanol (CH3OH).
The byproducts hydrogengas, carbon dioxide,andgaseousammoniagostraighttothe last stage,
methanogenesis,tobe digestedbythe methanogenicbacteria. The volatile fattyacids,alcohols,and
ketonesare thenbrokendownfurtherinthe nextstage,acetogenesis.
Example breakdown of glucose into acetic acid: C6H12O6 <--> 3CH3COOH
12 The function of alpha-amylasein hydrolysis.See 1.
13 Hydrolysis of Carbohydrates,Fats,and Proteins.See Carbohydrates.

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3.3 Acetogenesis
Acetogenesis,the intermediate step, isthe stage inwhichthe productsof acetogenesisare
brokendownintohydrogen,carbondioxide andaceticacidbyacetogenicbacteria.14
Hydrogenplaysa
veryimportantrole inthisstage.The reactionwill occurand acetogenesiswillonlyproceedif the partial
pressure is“lowenoughtothermodynamicallyallow the conversionof all the acids.” The partial
pressure isloweredbybacteriathatare seeking hydrogen;therefore,awayto testthe healthof the
acetogenicbacteriaistomeasure itshydrogenconcentration.
Examplebreakdown ofpropionateto acetate: CH3CH2COO-
+3H2O <--> CH3COO-
+ H+
+ HCO3- + 3H2
3.4 Methanogenesis
The last stage ismethanogenesis.Thisisthe stage inwhichthe productsof acetogenesis:
hydrogen,carbondioxide,andaceticacid,are convertedtohydrogengas,methane gas,andcarbon
dioxide bymicro-organisms.The bacteriathatcarry out thisstepare calledmethanogens.Theseare
strict obligate anaerobeswhichwill becometoxicinthe presenceof oxygen. Methanogensare
chemoautotrophs. The stabilizationof waste isreachedwhenmethane gasandcarbondioxide are
produced.15
Examplebreakdown ofCarbonDioxide andHydrogen Gas: CO2 + 4H2 <--> CH4 + 2H2O
14 Description of the acidogenesis and acetogenesis.See 377-378.
15 Equations for all of the mechanisms with explanations of each one in simpleterms. See all.

6
3.5 ObligateAnaerobes
Obligate anaerobesproducesuperoxide dismutaseandcatalase insmall quantitiesinorderto
remove invasivemolecularoxygenthatwill reduce tohydrogenperoxide (H2O2) andsuperoxide(O2
-
)
inside the cell.
Superoxide Dismutase reaction with O2-: 2O2- + 2H+----> O2 +H2O2
16
Catalase reaction with H2O2: 2H2O----> 2H2O + O2
17
Obligate anaerobesbydefinitionare anaerobesthatare notable to grow inaerobicconditions,
but the productionof these twoenzymesare evidence forthe slight the tolerance (0.2% to8%) of
oxygenthatobligate anaerobesare able tohandle.Thisallowsforminimalerrorinthe labwhen
growingthese anaerobes,butwill deterthe resultsasthe anaerobeswillhave beenexposedtotoxic
conditions.18
Afterthe anaerobesproduce carbondioxide,hydrogen,andmethane,the gasesare
harnessedandsealedwithinanair-tightcontainer.Theycanthenbe combustedwithoxygen,releasing
energywhichcanbe usedforheatingwater,oras a source of fuel forcooking.
Methane combustion with Oxygen: CH4 + O2 ----> CO2 + H2O + energy 19
The byproductof thiscombustionisminimal amountsof carbondioxide andhydrogengas,whichis
equivalenttothe amountof carbon dioxide aplantabsorbsduringitsgrowthcycle,makingthe
combustionof biogasclimate-neutral.20
16 Reaction f superoxidedismutasewith an oxygen radical.See 1.
17Catalasereaction with Hydrogen Peroxide. See 1.
18 Explanation of ObligateAnaerobes and their preferred conditions.See 67-68
19 The combustion of methane with oxygen to produce energy. See 1.
20 Byproducts of the combustion of methane and oxygen. See 1.

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METHOD
4.1 Synthesizing BasalCarbonateYeastTrypticase
The Basal Carbonate YeastTrypticase isthe culture whichwill be combinedwiththe different
batch digestionsinfivedifferentgas-tightbottles.To make the Basal Carbonate YeastTrypticase we
usedthe followingchemicals:NH4Cl,KH2PO4,K2HPO4,CaCl2 ·2H2O,KCl,MgCl · 6H2O, NaCl,C6H12O6,and
yeastextract[See Appendix 1forChemical Measurements].
The firststepwas to take a 1000mL Erlenmeyerflaskandfill itwith600mL of distilledwater. The
nextstepisto boil the 500mL of water,place the flaskona hot plate,andmaintainaninternal
temperature of 105̊C. Combine the chemicalspreviouslylistedandmix themintothe boilingwater. Stir
the flaskuntil youare able to see a consistentmedium-browncolor.Next, recordthe pHof the solution.
The solution shouldhave apH of 6.8 (±0.1). At thispoint,take a volumetricpipette anddrop0.5 mL of
Rosazurinintothe solution. Place the flaskbackonthe hot-plate andletitreachan internal
temperature of 105̊C. Boil for one minute,until youare able tosee bubblesformingonthe surface of
the solution.Next,cool the mediainthe presence of nitrogengas(N2) fortwotothree minutes.After
the mediaiscooled,fill one of the five bottleswith20mLof mediawhile spargingthe mediawith
Nitrogengas(N2). Next,addthe L-Cysteine hydrochloride tothe media,continue sparging.Quickly
remove the tube andplace a rubberstopperinthe mouthof the glass bottle.Place analuminumcap
aroundthe mouthof the bottle andcut outa three centimeterhole ontopinorderto provide accessto
the mediawheninoculatinginthe future.Repeatuntilall five bottlesare filled.Store the bottles
containingthe Basal Carbonate YeastTrypticase inan ovenforthree days at 65̊C.

8
4.2 Dilution of Substrates
While the culture iscooking,prepare yoursubstrateswiththe differentratiosof dilutedfood
waste and dilutedcowmanure.Take five glassunsterilizedglassvialsandplace theminanautoclave.
Sterilize the glassvialsat121̊C for fifteenminutes.Next,removethe glassesandplace theminan oven
at 100̊C fortwentyminutes. Whilethe glassvialsare inthe oven,startto dilute the cow manure andthe
foodwaste. The target Molarityforeach solution is0.5M, and a volume of 250mL of eachdiluted
solution. Forthe cowmanure calculation,we need250mL of dilutedsubstrate inordertofill all five jars,
the equationwouldbe: 0.5M =
𝒎𝒐𝒍
𝟎.𝟐𝟓𝟎 𝑳
= 0.125 mol. For the foodwaste calculation,we need250mLof
dilutedsubstrate inordertofill all five jars,the equationwouldbe: 0.5M =
𝒎𝒐𝒍
𝟎.𝟐𝟓𝟎 𝑳
= 𝟎. 𝟏𝟐𝟓 𝒎𝒐𝒍. Thus,
we require 0.125 molesof cowmanure in orderto dilute eachsample toa constant0.5M, and 0.125
molesof foodwaste inorderto dilute eachsample toa constant0.5M.
Afterdilutingboththe foodwaste andcow manure to 0.5M, separate the twosubstratesinto
the bottlesaccordingto the presetratios.The firstbottle will be filledwith100mLof dilutedcow
manure,shutwitha rubberstopper,sealedwithanaluminumcap,andlabeled B1C100F0. The second
bottle will be filled with90mLof dilutedcow manure and10mL of dilutedfoodwaste,shutwitha
rubberstopper,sealedwithanaluminumcap,andlabeled B2C90F10. Thiscontinuesasshownin Table
1.
B1C100F0 B2C75F25 B3C50F50 B4C25F75 B5C0F100
DilutedCow
Manure(mL)
100 75 50 25 0
DilutedFood
Waste (mL)
0 25 50 75 100
Table 121
21 – Ratios of cow manure (mL) to food waste (mL) in each bottle.

9
By doingthiswe create a wide varietyof substrateswhilekeepingthe control of dilutedcow
manure,andcomparingit to the methane productionof pure dilutedfoodwaste. Aftersplittingupthe
differentsubstratesintothe ratiosprovidedabove,andsealingeachbottle,we place the glassvialsin
the ovenat 65̊C, alongwiththe bottlesof Basal Carbonate YeastTrypticase forthree days.
4.3 Food Wasteand CowManureSubstrateInoculation
After72 hours,remove the six glassvialsfilledwiththe substrate,andthe six glassvialsfilled
withthe Basal Carbonate YeastTrypticase media.The stage of inoculation orthe introductionof micro-
organismsintoa mediumthatwill supportgrowth, will take place inthe BactronIV 900 SHEL LAB, an
anaerobicchamberusedtoprevent the samplesfromthe invasiveoxygenradicals.Place all five of the
substrate filledsamples,all fiveof the Basal Carbonate YeastTrypticase media,andthree 14-gauge 5mL
syringesintothe anaerobicchamberdoor.Toensure thatthe anaerobicchamberiscleanedof gases,
openthe valve onthe nitrogengastank to twentyPascals,lettingthe N2 gasflow intothe anaerobic
chamber.Next,clickthe vacuumbuttonforthirtyseconds,removinganygasesinthe chamber. Next,
release more nitrogengasintothe chamber.Repeatthe lasttwostepsthree timesinorder tofilterout
all of the toxicgases.Whenopeningthe doorstoplace yourhands inside the chamber,pressdownon
the foot-pedal labeledvacuumtomake sure that notoxicgasesare allowedtoenterthe chamber, and
thenpressdownon the foot-pedal labeledgas torerelease the storednitrogengasintothe chamber.
Now,we have achieved anaerobicconditions.Openthe internal doorandreceive the glass-ware and
syringes.Usingone syringe, pierce the rubberstopperwiththe needle,and carefullyextract5mL of
liquidsubstrate fromthe B1C100F0 glassvial.Release the liquidintoone of the Basal Carbonate Yeast
Trypticase media;label thisglassvial B1C100F0-S1.Repeatthese stepsusingacleansyringe eachtime
and transferring5mLof liquidsubstrate fromeachvial containingsubstrate intothe corresponding
mediaandlabelingitaccordingtoits correspondingsubstrate;e.g.B3C50F50-S3. Afterinoculatingall

10
the samples,remove all the samples,turnoff the machine,andclose the valve of the gastank to cease
the release of the nitrogengas. Next,place the tenglassvialsbackintothe ovenat65̊C, ideal conditions
for the anaerobe growth,for24 hours.
4.4 Gas Chromatography
The last stage of thisexperimentistomeasure the concentrationof methanegaswithinthe
biogasproducedbythe varioussamples.Todothis,we must use gas chromatographyandcomputed
integralsinordertomeasure the separationof concentrationsof H2, CH4,andCO2 gases. To start, turn
on the computerlinkedtothe gaschromatographer,inthiscase the ChemitoGasChromatographGC
7610, andopenPeakSimpleV1.47and opena blanktest.Asthe computeris startingup,retrieve your
five inoculatedmediaandthe 5mL thinlayerchromatographysyringe.Openthe valve tostartthe flow
of the carriergas, N2.Nitrogenisthe ideal carriergasbecause ithas a highthermal conductivitywhich
will keepthe tungsten-rheniumfilamentcool and turnon the Main switchandthe Bridge switch and
waittill itthe apparatus reads60̊C. Switchthe machine tomethod8 for the Thermal Conductivity
Detector. Waitfive minutes.Next,withdraw 5mLof gas from yourfirstsample,B1C100F0-S1 and
release itintothe hole atthe top of the ChemitoGasChromatographGC 7610. ClickCtrl+Rto start the
teston the program PeakSimple. The biogasisevaporatedinaninjectorwhichisheatedto200-300̊C.
The evaporatedgasis thenputthroughthe column,PolarpakQ,where itisseparatedintoCH4,H2, and
CO2 as itmovesalongthe N2 carriergas. Overthe course of eightminutesall the data will be recorded
and a graph similartothe one in Figure 1 will be displayedonthe screen. Alongwiththisfigure,the
program will give youthe concentrationsof CO2, CH4, and H2 by computingthe areaunderthe peakusing
integration.The exactequationtofindthe concentrationis: 𝑪𝒐𝒏𝒄𝒆𝒏𝒕𝒓𝒂𝒕𝒊𝒐𝒏 =
𝑨𝒓𝒆𝒂 𝒖𝒏𝒅𝒆𝒓 𝑷𝒆𝒂𝒌
𝑺𝒕𝒅 𝒐𝒇 𝑪𝑯𝟒
𝒙 𝟏𝟎𝟎.
Recordthe data,and repeatthe previousstepsforall five inoculatedmediaeachdayforfive days.

11
RESULTS
Figure 122
Table 2: Separation of the Concentration of gases first 24 hours23
Concentration ofMethane Gas
(%)
Concentration ofHydrogen Gas
(%)
Concentration ofCarbon Dioxide
B1C100F0 12.05 1.28 86.67
B2C75F25 8.13 1.51 90.36
B3C50F50 6.37 2.53 91.1
B4C25F75 5.16 2.66 92.18
B5C0F100 2.47 3.21 94.32
Table 324
(%)
(%)
B1C100F0 16.51 2.21 81.28
B2C75F25 11.37 2.56 86.06
B3C50F50 9.72 2.48 91.14
B4C25F75 6.76 2.13 91.11
B5C0F100 2.50 3.94 93.56
22 Graph that shows the abundanceof each gas relativeto its specific retention time standard for B1C100F0
23 Recordings of the concentrations of methane gas, hydrogen gas,and carbon dioxideof each sampleon day one.
24 Recordings of the concentrations of methane gas, hydrogen gas,and carbon dioxideof each sampleon day two.

12
Table 425
(%)
(%)
B1C100F0 21.88 2.36 75.76
B2C75F25 16.81 2.78 80.41
B3C50F50 10.99 2.13 86.88
B4C25F75 9.45 2.46 88.90
B5C0F100 2.51 2.42 95.07
Table 526
(%)
(%)
B1C100F0 34.39 2.22 63.39
B2C75F25 24.72 2.94 72.34
B3C50F50 18.43 2.57 79.0
B4C25F75 16.07 2.29 81.64
B5C0F100 2.54 2.51 95.07
Table 627
(%)
(%)
B1C100F0 46.03 2.31 51.66
B2C75F25 36.14 2.1 61.76
B3C50F50 27.66 2.58 69.76
B4C25F75 37.25 2.79 59.95
B5C0F100 6.97 2.85 90.18
25 Recordings of the concentrations of methane gas, hydrogen gas,and carbon dioxideof each sampleon day
three.
26 Recordings of the concentrations of methane gas, hydrogen gas,and carbon dioxideof each sampleon day four.
27 Recordings of the concentrations of methane gas, hydrogen gas,and carbon dioxideof each sampleon day five.

13
INTERPRETATION OF RESULS
The data displayedinthe tablesabove show thatthe yieldof methanegasconcentrationgrows
exponentiallyinamatterof days for eachsample exceptforB5C0F100. The reasonis because the
methanogensrefusedtodigestthe pure foodwaste substrate because itwasafar more complex
structure than the easy-to-break-downcow manure.The foodwaste contained complex polymersand
proteinsthattookmuch longertobreakdownthan the carbohydrates,proteins,fats,andlarge
polymersin cowmanure.Thisisevidentbythe ratherlinearincrease of the yieldof methane gas
concentrationforthissample forthe firstthree tofour dayswhichare consideredthe lag-phase inthe
growthof the micro-bacteria.28
Afterthe firstfourdayswe are able tosee the concentrationof methane
gas inthe B5C0F100 sample increase bythree hundredpercent.Ratherthanchoosingtodigestthe
complex polymersinordertogrow at an exponential rate,the methanogenschoose to onlydigestthe
polymerswhenthere isanecessitytosurvive.
On the fourthday of data collection,we see anexponential increase inthe concentrationof
methane gasinalmosteverysample.Thisisbecause of the lag-phase inmicrobial growthexhibited by
methanogens.Insteadof the cellsgrowing,theyspendthe lagphase replicatingvariousproteinsand
DNA in orderto prepare itself forthe nextphase whichisthe logphase.29
Inthe logphase the bacteria
will begintoconsume the productsof acetogenesisandstartto produce methane gas(CH4).Thisisseen
inthe equationstatedearlierCO2 +4H2 <--> CH4 + 2H2O. Inthe logphase of microbial growth,the
bacteriastart to replicate atan exponential rate,dividingandconsumingmore andmore products of
acetogenesis,thusincreasingthe concentrationof methanegas.
28 The lagphaseof the bacteria lasts three days then goes onto the log phase.See 170-171.
29 Explains replication of DNA rather than the growth of Bacteria in the lagphase. See 1.

14
Aside fromthe methane gas,we can see thatthe hydrogengasstays almostconstantfor all of
the results,withafractional jumpor dropfrom dayto day. Thisisbecause the methanogens donot
produce an abundantamountof hydrogengas.Most of the hydrogencationsfromthe firstand second
stage,hydrolysisandacidogenesis,wereattachedtothe carbon atomsto produce methane,andmethyl
whichisconvertedintomethane viaademethylationreactioncarriedoutbythe methanogens.30
Thisis
the reasonwhymethane gasand carbon dioxide appeartobe soabundant.The carbon dioxide startsoff
havingthe highestconcentrationthenslowlymovingtowardszeroasthe concentrationof methane gas
increasesdue tothe digestionof productsfromacetogenesisandexpulsionof methanegas.When
lookingatthe data, it looksalmostasthoughthe carbon dioxide concentrationandthe concentrationof
methane gasare inverselycorrelated,andthiswouldapplyforeverysample.
Whenlookingatthe fourthsample,B4C25F75 we notice a consistenttrendwiththe other
samplesforthe firstfourdays,but thenthere isapproximatelyatwohundredpercentjumpbetween
day fourand five.Thisjumpputsthe substrate whichcontains25% cow manure substrate and75% food
waste substrate exceedsthe B2C75F50 sample aswell asthe B3C50F50 sample.Thiscouldbe the effect
of twothings:erroror the methanogenicbacteriagettingusedtothe largerpolymersandefficiently
breakingdownthe more complex structures,thusyieldinghigherconcentrationsof methanegas.If
there wasto be an errorthenthe most logical eventthatwouldoccurwouldbe a steeprise incarbon
dioxide.Thisisthe mostlogical because otherthananapparatus malfunction,the secondmostprobable
error isfor the aluminumcasingtobreakand have atmospherictoxinspollutethe sample.A verysmall
percentage of the atmosphere’sgaseouscompositionismethane gas.There isamuch higher
percentage of atmosphericcarbondioxide thatcouldhave leakedintothe glassbottle.If thiswastrue
we wouldhave seenadrastic rise inthe carbon dioxide levels,almosttoa pointof one hundredpercent.
Insteadwe see anincrease inmethane gasconcentration,thusprovidingevidence thatthe
30 The demethylation reaction and relation to methanogens. See 2385.

15
methanogenslearnedtoefficientlydigestthe more complex structuresof foodwaste andinturn,yielda
higherconcentrationof methane gas.
Conclusion
The foodwaste and cow manure whichisbrokendownthroughhydrolysis,acidogenesis,
acetogenesisandmethanogenesis,producessignificantamountsof methane gaswhichcanbe usedfor
heatingwater,cooking,orevenheatingthe village housesduringthe night.Aswe have seeninhistory,
cow manure hasbeenusedas fuel ina wide range of forms, fromburningdriedcow manure totoday’s
anaerobicdigestionof cowmanure toproduce methane gaswhichiscombustedwithoxygentocreate
heat. Our goal wasto substitute foodwaste forthe cow manure inorderto solve boththe problemof
waste pollutionaswell asthe energydeficitinIndia. Fromthe results,we seethatinthe primarystages
of methanogenicbacterial digestion,cow manure isthe mostefficientsubstratesource touse interms
of the yieldof methane gas.Butas evidentinsample B4C25F75,whichconsistedof 25% cow manure
substrate and75% foodwaste substrate,once the methanogenslearnedtodigestmore complex
polymers,the digestionof foodwaste yieldedthe highestconcentrationof methanegas.If we were able
to continue the experimentformore days,we couldextrapolatethe dataandsee that the samples
whichcontainedsome amountof a foodwaste substrate wouldyieldthe highestamountsof methane
gas.

16
Bibliography
"Anaerobic Digestion: Microbiology and Biochemistry." 10 Aug. 2014. Web. 22 Aug. 2014.
<http://biogas.ifas.ufl.edu/Internships/2012/files/AD%201.pdf>.
Ananth, Lakshmi. "Hydrolysis in Digestive Process: Basic Step in Chemical Digestion." Web
log post. Suite. 31 Dec. 2010. Web. 23 Aug. 2014. <https://suite.io/lakshmi-
ananth/4re92jw>.
Annual Report. Rep. New Delhi: Ministry of New Renewable Energy, 2009. Web.
<http://mnre.gov.in/file-manager/annual-report/2009-
2010/EN/Chapter%205/chapter%205_1.htm>.
Brooks, George F. "Obligate Anaerobes." Jawetz, Melnick & Adelberg's Medical Microbiology.
New York: McGraw-Hill Medical, 2007. 67-68. Print.
"Combustion of Fossil Fuels." Virtual Chembook. Web. 30 Aug. 2014.
<http://www.elmhurst.edu/~chm/vchembook/511natgascombust.html>.
The Editors of Encyclopædia Britannica. "Amylase." Encyclopedia Britannica Online.
Encyclopedia Britannica, 31 May 2013. Web. 30 Aug. 2014.
<http://www.britannica.com/EBchecked/topic/22046/amylase>.
"GDP Growth (annual %)." Data. Worldbank. Web. 22 Aug. 2014.
"How Does Catalase Break down Hydrogen Peroxide?" Weblog post. UCSB Science Line. Web.
26 Aug. 2014. <http://scienceline.ucsb.edu/getkey.php?key=166>.
Ismail, Nasir, and Baba Abubakar. "ANAEROBIC DIGESTION OF COW DUNG FOR
BIOGAS PRODUCTION." ARPN Journal of Engineering and Applied Sciences 7.2
(2012): 170-71. Questiaschool. Web. 28 Aug. 2014.

17
Miller, Naomi. The Use of Dung as Fuel: An Ethnographic Example and an Archaeological
Application. Rep. Academia, 10 Feb. 1984. Web. 25 Aug. 2014.
"Ministry of New and Renewable Energy." Ministry of New and Renewable Energy. 2014. Web.
29 Aug. 2014. <http://mnre.gov.in/file-manager/annual-report/2013-
2014/EN/rerp.html>.
"Municipal Solid Waste." National Solid Waste Association of India. Web. 22 Aug. 2014.
<http://www.nswai.com/waste-municipal-solid-waste.php>.
Pandey, Krishnan K., and Khanjan Ajaybhai Kalyani. "Waste to Energy Status in India: A Short
Review." Renewable and Sustainable Energy Reviews 31 (2014): 113-20. Waste to
Energy Status in India: A Short Review. ELSEVIER, 2014. Web. 29 Aug. 2014.
Payal, Sampat. India's Low-Tech Energy Success. Rep. World Watch. Questiaschool. Web. 22
Aug. 2014.
"Producing Power and Heat from Biogas." Biogas an All-rounder. Biogas All-rounder. Web. 27
Aug. 2014. <http://www.german-biogas-industry.com/the-industry/producing-power-
and-heat-from-biogas/>.
Rosenberger, Erich. "What Are the Four Phases of Bacterial Growth." Sciences 360. 6 Dec.
2008. Web. 30 Aug. 2014. <http://www.sciences360.com/index.php/what-are-the-
four-phases-of-bacterial-growth-18035/>.
Sachs, Noah M. "Garbage Everywhere." The Atlantic. Atlantic Media Company, 20 June 2014.
Web. 12 Aug. 2014.
Serna, Emmanuel. "Anaerobic Digestion Process." Anaerobic Digestion Process. WtERT, 25
Nov. 2009. Web. 30 Aug. 2014.
<http://www.wtert.eu/default.asp?Menue=13&ShowDok=12>.

18
"Sludge Treatment." 377-78. Wastewaterhandbook. Web. 25 Aug. 2014.
<http://www.wastewaterhandbook.com/documents/sludge_treatment/831_anaerobic_d
igestion_theory.pdf>.
"Superoxide Dismutase." Worthington Enzyme Manual. Web. 30 Aug. 2014.
<http://www.worthington-biochem.com/sodbe/default.html>.
Thauer, Rudolf K. "Biochemistry of Methanogenesis : A Tribute to Marjory Stephenson." 90.11
(1993): 2358-359. Print.
Thrift, Hanks G. Solid Waste/Disease Relationships. Rep. no. 999UIH6. Print.
United States of America. Centers for Disease Control. Infectious Disease Information.
Washington D.C.: Centers for Disease Control, 2007. Mosquito-Borne Diseases. Web.
24 Aug. 2014.

19
Appendix: BCYT (Basal Carbonate Yeast Trypticase) media
g/L
Dipotassiumhydrogenphosphate 0.3
AmmoniumChloride 1.0
SodiumChloride.
6H2O 0.6
Calcium chloride anhydrous 0.1
Yeast extract 0.08
Trypticase 0.5
Trace elements* 0.5
Trace Vitamins** 1.0mL
Resarzurin 1.0mL
ReducingAgent (CYS-HCl)*** 1.0mL
pH 6.8
Gas Phase N2: CO2 orH2:CO
*Trace Elements
g/100mL
Nitrilotriacetic acid 4.5
Ferric Chloride .
4H2O 0.1
Maganese Chloride.
4H2O 0.1
Calcium Chloride 0.02
Cobalt Chloride.
6H2O 0.17
Zinc Chloride 0.1
Boric Acid 0.019
SodiumMolybdate 0.01
(Nitrolotriacetic acid was dissolved with potassiumhydroxide to pH6.5 and then the othersalts were added).
**Trace Vitamins

20
mg/100mL
Biotin 2.0
Folic Acid 2.0
Vitamin B12 0.1
Pyridoxine HCl 10.0
Thiamine 5.0
Riboflavin 5.0
Nicotinic acid 5.0
DL-Calcium Pantothenate 5.0
P-Amino Benzoic Acid 5.0
Lipoic Acid 5.0
**Trace Vitamins
mg/100mL
Cysteine hydrochloride 2.5
pH 9.0
Sodiumsulphide .
9H2O 1.0
(Added afteradjustingpH)
Preparation of Pre-Reduced Media
The distilled water was taken in a one liter conical flask and boiled till boiling point to displace
the dissolved oxygen. All the chemical ingredients were added to the boiled water and the same was made
up to one liter. One ml each of Resarzurin and trace elements were added to the above solution and mixed
well. The media was precooled under passage of oxygen free nitrogen (to saturate the media with
nitrogen). Gassing was continued till the media temperature dropped to 40°C and then the pH was
adjusted to 6.8 with sodium bicarbonate. The serum bottles of 130 ml were pregassed with Nitrogen to
displace the air and 36 ml of media was dispensed under continuous gassing. Even after pouring the
media, the gassing in the container was continued for a few minutes to avoid post transfer oxygen
contamination. After dispensing the media, the bottles were immediately sealed with butyl rubber stopper
and capped with aluminum crimps. The media was autoclaved at 121°C for 15 minutes. The change of
media colour from royal blue to pink after autoclaving was observed. The reducing agent and vitamin
solution were then added to the media (sodium sulphide in reducing agent and vitamin solution are heat
labile compounds so they are added after sterilization). After half an hour to one hour, the media was
reduced as indicated by decolorization of resarzurin.

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