MEfiSMt PEOD¥CTIOH BY MABROBIS
PUMMXOI OF P1AR WASTE
l9X£XJCftM AlfHOIT M»1IGST
A THESIS
Bubaitt©4 to
©Bieoi sfsm c®imm
itt partial fulfillment ©f
tli© requlr«aent>s for tk@
clogs?©© of
MSfSR Of SOXEHOB
3mQ 1048
PHOTO I
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leacl of Bepartmeat of /Fopd f ©©Baology
eh&lxwm of SekooX eraduat® ©omiitt®©
f^TiT1gn^liB»i^#a^>M>i*iiiiahw*M»w<wwi>*ii'^'ff wniir'a^w^w»»^WMWII mm II^J^U
Deaa of ovaAuate school
AcmoMMmmw
fh® author is grateful to S* B« Wiegaai* H©ad of t&e
FOcS f©ctoology Dapartmeat^ Oragoa State eollog®, aad
Br» ©* jT* ffertblngfeoKij Aaalstant Pfofea?©? of tM© toopartment ©f Fooa ^ocSmology, Orogosi Stato College, far th©ir
valtmbl© assistance in th© preparation aafi in ©uggestiag
tMs problem* fh® wit©i? tji^i©^ fiiPth©^ to
OS:SH?@SS MSJ
Indabtedndde to- ©3?.* W* B# Bolioa*. Assoeiat© frof^tsor of
the lopartaeat ©f iaot®ri©l©^sr# ©^0goa State Cotlege* for
tk® valmabl© suggostioas
BM
guidaae® in eas?ry.lag out tho
©s^fimoatal wox^ls aai for asaista&eo in intarpretiag thd
data*.
Ho ais©' aoknododgee th® 2ie3$fi& ^aietaaoo of
©r* tt* W, fha^er* SOQdartiti Afiseeiftte^ ^©partaeat of
Bacteriology* Oragea AgplcmXtwal B^pofimoat Station,
aai ®fe©iia0 OasiOFff* Assoeiata Arofaeao* of th® ©opart*
a©at of food foelmoi©^..
TABIE OP COSflSTS
Pag©
GmfTM
I
'fetroductlon............................
1
II
E€)¥£©w of 1,1 tefatttf©.^...****#.,.♦.,..........
S
III
Analytical ::otIiods.
14
IT
Mai^sis ©f Pear W&sfc© ,,,,*,...*•..*»»*,.■«•
16
IT
fjpfcMMnaipjf iBaptrlttesits * * ♦ *♦***♦........
17
?!
F03m©j4tatloia ©f foa^ Waste ©a a
^rg©p lAlieratoi^r Seal©*................
SB
VII
DiscuEClon of Results..............*....
42
fill
Suimiary and Gone 1'asIons.................
SO
BIK.20@l^Fffif*.....................................
§3
LlSf
OF
TmmB
Page
I
Pacifie HorthwestJ eaaned Fear Pacfe..........
$
II
Analysis of Poar I'iaote......................
16
III
Bailf Aeeomxt ©f SO^llt^x4 Femodoter*
Swaiup Bad Ino culusi............................
26
XV
Sally Recount ©f gO*llt©r WGrmnter-*
Sewage Sludge Inoculuia*.....*.,»............
31
¥
Sally Mcoim* of Qwlites? JPewmtLteVf
Swamp Mud Inoeulwa...«....*..............♦♦.
S5
VI
'&\mm%y of FerKontors.,.....................
39
VII
Caloulated Heat ?alti§. of Methane ^redtieed
froHi Dry Poar Uaute.........................
43
Lias
OF
rsmsm
VtWFRB
X
fepeplaeafeal aa^ tealsrfeleal Iquipment used
in th@ Aaa©i?ol)l© F^faentatlon of P^ar
Uacte..................................... *.
23
II
Daily Aftcotuxfe of g@*litw Fevnoste^
Sr/ai^) m&& Inoculua........,.».«.........,..,
S9
III
Daily Aocomfc of so»liter Fe^m©at0j?f
Sewage Sludge laoculum......................
M
II?
Bally Aeeount of 5*lit@f Fei'aonter,
Swamp Hud fcoeulum..........................
07
¥
Hcs»o*>fl0pa Developed in Farmefit©!*;,.
Swaap tfed inoculum..........................
46
?I
M©j?o*flo^a Devolopea In Fermeatey,
Setmg© Sludge Isaoeulum.................«.....
4?
FEHMElfAf 101 OF ?MH MSfB
CBfkPfSR I
Tk© dispeoal. ©f pear ^astd ffe® prooeeaiag plo&ts ia
the faeific fo^thuest jn^eentfi special srofeXeBS to tl©
casmiag iadastx^1 lidoftusQ thB vmt© is relatively hlgOk in
eugor and ba^.a M# bieehenieal, ©^fg©,ia a^Kami (B«0«D«')*
If «lTO$e§ iat© streams tfe© Q&£to6fty6*ate ©Bt ©tter ©Fgaal^
fivdbatanoee oawe-** ^pm &mmkpQ®$xmv vBAoeia^ble-edevs oM
a lepletjioa of tls© din solved, o^g©a of tM stjpsew. feelce
requtoen^ita neeeaaary to support novm,% a<|ttatio life*
Th0 oac^govt def ioioB^ fflsir ©xt©M fai? io^a sts*©^% flo*
poia&i9g upon the asaomfc #f wet©. imttodmoed* Bim m*® flm
©t ®tr©eiss#. tea&etBANBMiit aiaA.ethev fetors*
^ ««® ia»
ataneee oaxmlng ixidusts^ies ar$ ©©jpi^us offead^ps la ad4»
Stag teavy pollatloii loads to atrea&ft*
lam^ etatoBfeave
already^ passed law© reatrioting atrean polltttloo*
Ja
Oregon, tfee State Sanitary Aathority fe©© recently notified
offendera that alleviation'Sietitod a nuat to. established
within the near future (31)*
If the reatrletive lane
already enaeted twre e-sforoed .maiay food iadustriea «rauld
b© coHpelled to close down* The preaent ©scteait of atreaa
pollution will not be peiratjitted iMefinitelgr, and the
ometm©nt of mor© sti?l3ag©nt laws and their enforcement may
to© ©xp©et©& In th© future*.
Wastes as?© prisaarily th© r©^
sponsifeillty of th© industry aad disposal must b© eoa*
slddred as part of overhead operating cost*
Methane has long bean knoun as on© of the products
©f anaoroMc bacterial decoiaposition of organio matter*
fh© studies h©r© reported wr© undertaken to investigate
th© mothan© fermentation and its praetieability a© a
souro© of gas for supplemental fuel in plant operation*
while at th©' Btm® bim® ainiialaing stroaEi pollution*.
In
this Miaerohio foraentatlon of the waste* carbon dioxide
as tsrell as metfean© i& fojmed*
fh© ratio varies from Itl
to It® depending upon th© nature of the traet© and con*
ditlons of th© formentatlon (18]l*
^e carbon dioxld©
could b© recovered and used in th© iaanufaetur© of dry ic©*
It would appear that if the methane formed were used for
fuel this fermentation may b© th© most economical method
for th© treatment of wast© from larg® canning plants.
Eh© following information is presented to ©mphasise
th© tr©m©ndous quantity of wast© material ^uhioh requires
disposal in some manner by th© packer*
fh© wast© ob*
tained from culling, stemming, peeling* and coring of
pear© is approximately SIS to 40 percent of the fruit received for processing (24)*
In th© faclflc Mortterest
the av©rag© yearly canned p©ar pack for 1941*1945 ims
s
SrS95*42S eased (44),
Based ©n the ©atiraat© of §6 to 38
eases per t-oa of froah produet fFoeosaed* aad assuming 40
poroe-at loss ia prooessiag^ the average ^aarly pear msfc®
is 36,000 tons.
Tattle 1
facifie Sor^hcrest caaned l^ear Met <44)
Tot&l oaees paekei
Year
f ons freah produet
4f2©7,9Q3
XU2
|02#OOO
194®
2,020,156
?6|000
1944
3,042,510
82,000
85,000
1948
fofeal
I©»977,XX5
488,000
5,396*423
fa.,QO0
qMmmml«HitmmBtmm*mimm*
Averageper yestr
Various proposajs for utilization of pear mate Ift-*
elude fejraaentatios t© sueh frodmets as aeetle aeid m&
etlj^l aleohol @M feeding t© litrostoek*
Other posai-
feiiltiee tjMeh offer more or lees reegmpenae t© offset
disposal costs are dehydration and utilization of the
dried product for fuel or stoek feed, and the production
©f food feast.
At preseBt,. kowroir, majaj of the casmeyies mv®%'&
gptoiil th© iBa&erift& aad wash it domi the s©w©r with oater^
or dlBobarge it into son© coavenieat str^asi with ep
without paratXlmboary lagoojai&g*
CHAPflH II
Metban© M.® long fe©©a ImoMt as smsrsla gas aM fi?©
damp»
It aecura In nattite 'Gfe$ro'9'®xl, ca?gaiaie .isjattej? ^Mter^
goes anaerobic aoeompoalfclon, as in raarshos, mines,
manwr© pil^s* wterXogg^d aolla, ©nd a©s>fci© tanks* Aa
©stensiTO r©^i©w of tfet© lltoratm*® was preaented T&y
Buvcrell (It)* fhayor (40)t esM Barker <0#S) ©n th© Garly
invettlgatlon® -of fete ia@fc!a&n# fermontafeiom and th© or*
ganlsmi ol>s©rved* fSaoir retrieua ioan toe smnmarlged as
f ollevTS •
fh© first r©00rd0i observation that methane Is ft
forffl©ntatioa product is that of folta (VPfi) who eon*
eltadoi it ©rlgiiaatod. froa rottteg organic amttor* Among
fch© oarlf in^ostigator© to rocogni^© tfo© mioroblal
natmro of th© feraantation tiras Hopp©«»Soyl©r (1S8@), who
obtained si©than® and carbon dioxid© frcaa ©aloltim aootato
placed in rlvor mad* laa© <l©Oi) observed a sarelna
daveloplng in a eultw© of &©mj$.ng laavoa containing
ac©tat©5 lator (1913) h© found methan© produced # p©rhaj>®
seeondaril^t froa aeoton©*
OjaolianeM {^904*1916) -studied th© mothan© f©rs©nta<'
tion of eollulos© and ©thanolj acetic and butyric aoida
t7©r© first formed in th© collulos© fenasntation*
l©
6
described a @3*e2Jlael(, bacterium tpGn fch© fei^aeatatioB of
alcofeol.
Two mefch&fte orgenisma* a rod^shaped bacterium aad a
sa^cisaa^ ^©3pe described by Sotegea (1.906) Ao fouad them
to ^uaatitatively deeoapos© vaFloue fatt^ acid ©alts to
atetha&e and carboa dioxide*
S© also ©howed that the
bacteria coiald form metbais® % the reduofcien ot carbon
dioxide tTith molecular hgrdrogeft*
$his action is control
ireraial, certaiii imvestigatore coafirming it ^Jhile
other© eoatend that the aetieja is iadireot in that the
methane arise* from acetic acid .produced spiergistieally
V <S3Lo*ftpWuiitt ftflfitittQ» (40) *
there is also evideric©
tisat the iiethase feaeteria caa reduce carboti dioxide to
©ethane using bjrdrogeii sulf ide as the' reducing agent (40) •
more receatly Schnellen (34) has described lethai^esarcina
bar^erii tjhich «an reduce carbon monoside with water to
give aethaae*
It mierececcus tevnantixig aeth^l^ et^l and butfi
alcohols and acetone to methane was described by
Oreengreg* (1#20)* fh®raoj>hilic rod-stoped methane
bacteria active on certain fatty acids wez® described by
Coolhaas (19S8)#
An extensive study of the wtbane produc^ig bacteria
«ae carried out by Barker (a)*
frier to these investiga*
tions the information available in literature
TMS
eoitaparatttr®!^ lifctlL©*
Th© ©fganiaas possess cultup© char*
acteristtes tshieh amis© sttt% fegr orainaz^- labofafeoyy ps?©*
oeOttres ©«tr©m©J,y diffieult*
fh© iifflculty, aoooMi^g
t» farfe©» (31^ rosulfe® ffom th.® faet that th© organisa®
ard nt^let aaa^rofeoe ^Mok a© not form &pep**t feav» a low
^at© of deirelepaeiit ana ar© ©xta?©®©i^ ©easltlir© t© ©v®»
tfaees of fr®©- osygoa.
isa ai^ltloa to a l^fifogen demtow*
th@y require earlnm diexide a«
G
tbe&r <i©!V®l©p»@at la es sulture*
fey^rogea aeeepter for
Barker ^•'Qeloped mefhois
tm tfbtatalng hlghlgr purified etilturee ©f f©«r types off
methane frMueisig orgasissjs., obieh tta^r be identified oa
the: baeie of their (3i©tinet siopphelogical ant ph^sialogi*
eal eharaet©ristic$# as fo&laa*$
i»
Methamoaare.ltoa m©thaMe&.#
Lar@# sphorieal eella
eharaeteristleall^ grouped to form oiahical
sarcsim paeicefcs.
spore forni&g*
PermanentXy iwiotile &M noa*
Fera^ats aoetie aael butyric
aei&s with the produetiosi of netfesne*. hut a©t
etbjrl. or hutyl aleohol*
2.
iethahoooocms saago*
©ram variable.
Snail apherloal cells i^hieh
oeoiir slnglsr is email groupa* or in large, ir*
ropilar and sonewhat
BXSMJ
aggregatea.
motile aM lionspore forsaimg.
Ito*
Ferments aoetic
aad but^rie acids with the production of aethane,
but aot etSb^l or butyl ale ©hois*
©ram variable.
s
xient^r iamotll©* noaspore f^paiag redd* Site rod®
a*€> eharaetaristieail^ jola©d Into i©Bg thread®
t?Meh lie porallol to one anothoj? so as to fosm
feimdles. Fetfaseata ae©tIo and bmtyyie aoida isifch
ttm pFOdaetioB of a©tMit®, hut not ©tJsgrl aleolxol.
©ram aogativ®.
4«
Metlaaaofeaet©rluai omeliaaslvii. 'fhlB.f fff©^©^-^
bent, inaotil©, noaspor© formlag rods*
F©j^a©nts
©thyl alcohol t© acetic acid and teutyl alcohol
to Ijutyrlc acid vXth m®thm® foraiation* ^hQ saa©
©pgaai^m pjpo'bat&ly also feraeats butyric acid to
acetic acid*
Acatic acid is act jtoymenbed*
F^x^ ydard aft0i» Ba^kej**^ (6) fipat pmblieatloa etaar*
actess'Izing the aoi»phological aad p%siolcgical oharaet©*?Istics of highly purified cultiiupoa of M@tlaaa© produciag
bacteria h© iescsi'Ibod th© Isolatioa of a pur© culture of
o
Hetfaaaobactegium pmeliangteii by f©llo^teg rigid proc©*»
dur©®*
la light of tfoese pm^culturo stmdiea thd rao^pl^*
logical descriptioa ua$ r.0:i?is©d»
to b© motil© aad spor« fonoiagt
the orgaaisa na@ foussd
Flhil© the spor-®© have^
relatively losr heat resistance they withstaad proloaged
exposure to air> although vegetative cell© are highly
sensitive to free oxygea..
Inasiaueh as the bacteriua pro-
duces spc^es the generic name should be revised*
Methaaobacillus botog appropriate.*
Jn addition to feeing £©X9»®<I direetSy fsww carbon -di*
oxide and carbon aonoxide, methan© has boon shoun to arise
from action of mothano bacteria upon various other organic
compottM©*
fheso includ© methyl alcohol (2), othyl alco*
jheOs (2*19), butyl alcohol (2,19)^ acetone (2)# ani wl«
ous fatty &0%m t2tl9,l&,$7§Z&}*
Buswoll (X9) observod that various fatty acids could
quantitatively be convertod to methan© and carhon dloxid©,
thus confirming previous reports of their utilisation*
From a sorles of experiment e with, fermentation of noriaal
fatty acids t&m fomlQ to valeric, inclusive, and of
oleic acid, he concluded that the proportion of methane
increased with length of the carbon chain, the carbon di*
oxid#tmthan© ratio beiag approxiaaatei^r Is® for the longer
chain acid©*
Buawell and co*tsr©rl£er3 (If) studying the
significance of the methan© fermentation in sewage disposal eoneluded that almost every type of organic material,
such as fatty acids, proteins and carbohydrates may be
evontually deeomposM to give rise to methane and carbon
dioxide.
Barker (2) coneluded from the numerous investigations
on the methan© fermentation that regardless of what or*
ganio compound was fermented th© only hydrocarbon or re*
duetlon product formed is methane*
fh© investigations of
fhB.j®T (39) furfcker support this view*
M Isypothesis that
th© feyaentatlOB of acetat@s might b© a 4©eaa?feox3rlatioii^
and that hi^.©*' f^tty aelds ©hmild give rise to th© oorl»©spoa<liBg3ty high©** hydroeapbon, was not aubstantigtoS •
fh© only p©i?missibX© eoaoiusioa was that amerobi© ioeoa*
position of saturated fatty acids by methane bacteria
leads to th© pro^tictioa of oothano a© tha only foydpoeai^
bon*
Althou^i Buswll sad loave {%®$&?} po a tula tod that
tha methan© fe3?aj©jataitioa of so^isgo involws ©xidatioa**
roduotioii of pspotoias and e&^feofeydr&tos by vj&tort BapJcoi*
(2) poiht©d otit ft fttndaxaoiital dofieioBoy of th© th®©ji»y Xu
that %M ^ooa not explain v;liy Eotfc.anG is tho on® and only
yoductiOtt product of tho substraLto*
Barfeo^ '(3^5) oxproased tho boliof that th© deeoajp©*
sitioa ift oipude f#a?ii©atations is th© vosttfA df tho action
of a mixed flora rathor thaa of methaae baoteria alone ♦
fho*»e is ia genoral no ee^taiaty that asthan© avisos by
diroet ooaveysicm of all of theso organie substrates*
fh© complex cojapouhds sueh as proteins and carbohydrates
are in all likelihood first attaekod by saprophytio
aoaorobos which produe© the lo^or fatty acids aad alcohols
as their ultlmat© dissimilation products*
^h@se sub-
stances then secondarily undergo th© isothan© forsaentation.
All invostisations prior to Barker's (2) classical
u
vjotfte ©a th© feioeheaisfcrj of the ©©than© £©?ffi©»fcat&oB tj©p©
bas©^ <m th© fefiHsxitstiGn &£ ©rgaad© aaterial with mix&&
sii@s»©*|,l©fa fresi mud os* acti^© ©©^©g© ©olid©* tysiag hlgfe*
If piMPlfiei eulfcw©© ©f Mfe» <M»3Uan,a3)iiiiL,< Barls©r (S)- show©.*!
tfe© rdaetion proesei© ©s follows foi? ©tfc^l ©M bot^l
alcohol©•
(1)
8O^eBgdB«^0«C&8
aCgliyOOOB-i-CS^^ HgO
tQiild fehl© orgamlism eaxmot ^©Ss^fi^ogonat© ©©©ti© aei<3#
other
KBthan©
bacteria attack It in the follo\7ing samiorr
Barkor (2) ©©selM©© tfoat uetimte ©©tually arls©© bj a
r©<Su©ti©& ©f ©art>©si diojcl'd© %n at laasst th© special ©as©
of tli© ferEsantation of ©th^l m& btat^l aleoSiola, ama probably ala©
1B
fermentation of aeotl© amsl butyrle aclia*
fhe g©n©ral ©quation for tij© aetiiaMi© fermajatatioxi i© tli©r©^
for©,
48g&+Ge2—* 4&^GI^Slg@
in tshich IgA r©'P?©8©nt'S $.nf GQmp&im&, ©rgaul© ©r Imorgaia*
to, t7hi©"h ©©n b© aetl"yat©d by tk© ii©than© bacteria a©
that it ©asa aot as liy^r©g©ia doaator for th© r©du©tl©si of
carbom aiozifi©.
5h© m©than® f@raentatloa as a proo©ss ©f ©arboa
%2
dloxMd ra^uctiom thus appeals closely amiogaqis to th©
wall knmftt iwoeossea of nitrate smd sulfate reductions
where SBg* SHg* and CBU are the ultimate reduction
products•
la recent years esperiment© in the practical application of the production of potjer ®&® fuel m® *&$ ©merohie
is©than© fermentation have been successful,
thee© studies
included a variety of industrial and agricultural wastes*
fhe fermentation* howeverf has not heen placed on a com*
aerciai hasis for the sole production of fuel gas*
Eudolfs
and leukelekian {33) showed that the- theriaophilic digestion of semage solids resulted in higher field of gas per
gram of "volatile11 matter added than did noraml temperature digestion,
fh© gas was 70 percent eombuisMt»l©•
fhfey
reported that it was possible to feed a thermophilie sei?*
age fermenter at a rate of 77 pounds of volatile isatter
per day per 1000 cuhic feet.
Buswell {21) studied the
fermentation of milk wastes and obtained from 3*34 to 12*4
cubic feet of gas per pound of dry waste.
Boruff (1©)
investigated the anaerobic fermentation of beer-slop, containing S to 4 percent solids and found that It could be
X3
formeated tk©raiophil£eallf to produce £a®% g@s« Froa aa
average dally
YOIUEI©
of X»500^000 gallons of mis waste
StfgQOftOQQ ouMe f©«t of gaa oottld ^e pro&uead* eoatatoins
gg to 68 pepc^ttt Bfttbasio*
B(»uft (M) nude © ftetfo©^
^v&f of feh© £®^E0i3Lt@ti©2& ©f eatti© and hog panaaM n&gR&es
ana pftoldas house seyoeaimga* m taoaA tibab it
TWWB
pos*
eiblo t© feed -t© a fes*me)atee &t a rat© ©f 4»@ to 5«$ graass
dry w©i^it, per day pes? liter ©f tanK oap^^ity*
FaefelJiS
kome®. seve^ningei pfodmeei 40& eo gaa p©^ gg'em oap 6*4 ©mfeio
jpeet poi? pm®&*
i*o© kog i>a«^©k maat#e tMf obtajUEied ©S3,-
e:i ©f gas pm graa ©y 9*0 ©uM& foot per pounds oeHatsaift*
ins 88 aaQ 78 ^©s^ooat a©fcte®@ ^apeotiveSy'* BttmiwtX aad
Bowaff (88) atudied l^iattr «6* posai^ility of fexvietitias
paato eelluloae aattaffiala ta-'ioetfeagie*
ffey a^ooeaafall^.
©Bijloyei ^Jais ptpoeeaa on straw^ '0ovfi0tft}3E0#. a&i stafela .
iaaimf© ©a a pilot plant fe&eia*
^hey reeoveved 4*0 to 2*9
en^i© feet of ga^ p©:2? poimdt d$y ^«i^t#! of aaterial fed*
14
frequent «naj,y:sip of the sluig© was neeessagy i®P
Itttita, tkms |3©BialttiiBg elos© ©ontfoi* flat rate of fo©d-»
■lag was #@gtt3.afc©i i» ace©xH3'®n@© tii^ the seisa ia irolafeiie
acids, ga0 analysis * ^©iTaa© of g60# an^ pE* ffeea© faetors
iimst "b© r®galat©dl ^© aiaiataim ooMi'feioms favorafel© #03?
ai!aa@!»©&i© €@eosiposifelon ©f tfe© pear msfc©*
fla^ #©ii^©#itioii of th.® gas waa ^©t©!r®3ii0d laritlt an
0x»sat ©ppayalsm^,. Biaa«faotwp®# by Pi&he? Seientlfi^
C^apaay*
flie procedtar© as giv©^ in feMe fiafe,@l? gas Mteiy*-
sis mam«al mas ae0<a foip 4©t;©rffiiaisig -various coraponoat©
(ISO)*
Ift e€msist©4 c>f tafeiiag ia0asur#<3 voiuaie 0f gaa
©^l>i©#, a^moviag various ©oapoaejatj-t
©BO-
by on® with afpfo*
priato absorbents and raeasuring th® deereaa© 1m volume
eaused fey ©aefe roi^'ml* Th® reauXts && wportei a© per*
e&nt &y volum©.
earboa dioxide was dgtexwined fey ab*
#©rpti©a ia a aoiutim of aoSium t^rftresid^ ia aa absorb
tlm pipette* lefeMa© oM Je^rdrogeH wre deteratoed ai*
aulfcaaeousiy by a^osr ©embustioa ia ooEygexi ia th© px»eseao©
of a gl©t?iag platinum coll.
U&© voium© of carbon dioxida
thus foxwed is ©fuivaient to the a»©uiit of me than©,. $Jhii©
twioo this treXwRBL is th© eontraotiOB due to th© eombustim
15
of ®©than©»
fhla eoatraction subtracted from the total
ooBtractloa leave® the ooatpaction due to the eoabustioa
of hydrogem.
tho
VOIUMO
ftio-wthlrds of this contraetloxi is efual t©
of h^^iKigetk*
fh© residu© left after reaovlag
coiabustlble ccaaponGnts was assumed to be nitrogen•*
The proeedur© ae glveii in Stsiadard Methods for Bx«*
amijaation of Water azid Sewage ms used for detef^inatio^
of volatile acids {!)*
f© i© oe #asipl® S.S oc of con*
eentrated swlfartc acid was added and the volm© xaad© np
to 200 o©#
The solution was then distilled and the dls-
tillat© titrated with o#l norraal aodiua h^dr©sid©t using
phenolphthallen as indicator*
Volatile acida are ex*
pressed in p.p.ra. as acetic acid*
leaeiupementQ of pS ^ere made eleetJPOBetrioall^ using
a Colosmn pH electrometer and glass electrode.
%ata for carbon dioxide in fables III, I?, and V iaeltided
AX^T %S present, a® it van absorbed ^Ith the carbon di»
0x140.* Althou^ HgS was not separately deterBiined, odor
and blaefe appearance of the sludge indicated that some
w&s produced•
16
CHikPTEE I?
Malyals of f©ar Waat©
Boltydrated p©ar tjast© ©specially prepared for this
stuay by th© Takisia Valloy Itraporating Company waa ased
for eonv©al©m©0 and to aak© possible carrying on tb.® ex*.
perimtntal ^ork regardless of th© season of th© year.
Composition of th© material is shorn by th© following
analysis as laad© by th© !»grl0&ltmrai Chemistry Bepartmentj,
Oregon Agricultural ii£p©rim©nt Station.
fable it
Analysis of Peas? Wast©
(Dry trt. basis)
I.loisture........................ 2.4
protoin.......................... $ .9
Fibsr.. * • • •.. •.»• •«• • *..«. •••*•♦«**• <&% .3
Fat............................. 1.0
Ash............................. 2.4
Carbohydrnte....................65.0
^h© moistur© ©ontent of th© dehydrated wast© as us©d
in th© following ezpsriments tsras found to b© higher, 8.S
percent.
Total carbon as detorminod by combustion analy-
sis was found t© b© 42*5 percent.*
# Determined by Mr. G©orge Harnik, Bacteriology Dept. lab*
fhe apifa^afcus m& proee^^pe tis®<i iB the prelim&ii&x,y
stoAiee to iaitlat© ©a aetlv© aetnaa© f«RB9Atati«tt ^sr©
s«fg©0t©i' Iff W* x&at* A. fha^p.^ 8«ire*&& of his prn^Ii*
cations m tmtkm® g©»©3l© w©y# found iavaluablo (39^40) •
tyi® eaqpeaPtaon^al f^menfcars cosst^tei ^f a msabov Qt SO©oe• and one-liter bottles eoataining an empIchaQnt modium
aad ^ayious aaoiajats of »«nuqp m& or ao'ttv^ s|i,ml@# ffom
Conp A^alr sewage disposal syateia as sorn'oea of inoctilwa.
Tba onfcichst&Rfe moditus, to^ which & pefreoaft O'thaaol oi*
eootcnto was added aa ©ubata?atD# t?as that devalopod tyt
Bavk^x* (S) and modified by Souli^loftittA (20) •
ft oon*
talaed th@ foXlov&qg ia@podiesit#s iH^Gl 0*1 jteroont^
K^SP'OA
0*04 pei>«a^t> IgGlg D#.01 p^2»ca^t# yefeAt esctrftot
0.003 per^ont^ and tap T/ator, viUi aa 0M#ag of oaloiussi
oarbonat© to buffoii? fch© solutioia at a pH f *# t© 7*4»
Dlssolwd eaa^rgoa wa^ oliialnatM isy boiling ttM eooling
t© 40^ e« prioi* to filling the bottl©©» fo oaeh 100 co*
of ttoaium 1 cc. of a 1 poreoat lagS.fBg0 aol^tlon was
added to remove tpacos of osjygosa and to malntoln anae^obio
# HtoaS* Chosil'Stipy Dapartmont* Xiovio and Oiajpk Coll«g®#
Portland.* Orogon, and Rosoargh Asuoolato^ Dapart^ent of
Bacteriology* Oregon Agriomltural gjtp^rim.snt Station*
Aagust'-Septembar 194? •
1$
eonditioas fop devoloimeat ©f the mothaa© orgaaisas.' she
Dottles uesp© eloeed witJi a tightly fitting on© hoi® ru^er
at^-)per> ©©smected ^7 3mt>b©^ tuning to calibrafeei bottlos
fop colleetimg gas over a eatwated acidified brime &dlt**
ti©m* and placed into a ^^ 0* ineubatcHP*
Ht first ®tfaj% alooH©! tsras added as a evbetvate f®r
tla© development of the aetliaae ox-gmtms as staggea-te-Q t»sf
Barker (11) l>mt later caloiim aeetat® v/as ^aed feecauae of
the type ef methane o^gaaisjod to fee e&peefced* Halle #iwae
nethano bacteria fermont sthaiiol to aothanQ, carbondloxide end a#etie acid,, other0 ferment oria^t-e acid aalte*
auch. as acetate and butyrates to Methane and carfeon dioxIt ©eemed lifeei^ that under atsplefc anaerohioais car*
hol^rdrate© of the grear t?aste would he firit tmm&t©rfiied
t© acetio and hmtyriq aeld hy such haeterla as Closfcrldiitsa
hatyri^ua (3S) and little er no alcohol traoli he prodmeed*
Henc© it appeared deairahle to use acetate aa enriching
auhatrat®•
Many fallarea t© obtain an aetiye fermentation were
emeomtered in preliainary studies, fhie diffieulty was
a result of net' fully appreeiating the aensitiveae©® of
the orgonisias to atsaospherie 63£ygen# their relatively slew
rat© @f develepiaent, and the iisagjortance ef sediaient»
4 number of the bottles in t?hich only a relatiirely
10
ssaail qaantlt^ of suamp m&d os? aewsge sliais® %!&,& ia@e& pro*
dmeea no gas aft©i* two aentli^' imeuMtioa at 86° C.
M
tfeos® bottles in igfetieh app^osdjaat©!^8 om towctte. of tfe®
t&feai vQlvm® was m&9 gas appeap^di after tte1©© tf©©ks t@
oa© momfeh tnomteti^B* With haa^f lnoonlvm of s©wag©
tiltsfSge* gas appeas?©*! wi^ia two dasrs* fho imp02?tasi0# of
a©aiK©iafe was ©mplm&t&M Ijy Baewell ©mi Weava (19) t«li€»
foosia that
$M
two parallel «©f ies of 80 eultaupe© GBtih*
mlf two failed t© proaiuc© gas fpm m&ti& aa^i proploBle
ftQids xftMfa aetiv© sluago was ^s^ei fm tfei© isjoe'ula.ti©a, Imt
*?? failed to totn gai wkon o&ly tfa& aagtovaAtutLt liquor
from tM.s aludge t?a© u$@.d* ^bdsd .same aut&ox4© also Jp'a*
ported 3?@0talta .«» gtudie© to d©t©fmia© the aiiiimra aaomit
of sludg© n$«98fi'&?3r t©
'IBSUS?©
sm««©;$sftil iEoamlatloat*
s&ots* conelusioa was tlmt lO- to 16 graai of $itids©- aolids
pti? liter ^©*»e stteessax^r*
Bpeden (If) was of th© opiaio©
that the ftmetioia ©f the sedlnent was aot m nmoh the
futfitshlag ©f a heauy iaoc«ltsm as it was the fwmt&wfr of
a paftiemlar kind of pi^sieal wriaMuwmt for the ©pgaaism.
fhaF®J? (41) ^as ©f the optoi<m that the fimotion of Vbs
sediment was not clear; hut that the most prohablo esplaaation of it® influoace is that its interstices provide the eoaplet© amerohie eonditioia® required b^ these
orgairisna.
After feraexitationB uer© well estahliehed the
aedlnant appear©^ feiaekj thi® xmB ©v£d@xio@ ©f feh© pres*
©nee of sulfat© ^aiiclng baetes'ia*
Fusft&er eviiSemee w&s
the p2?osiouBC©d odor of tegrdvogm. sulfide*
Siaifete j?@d'aeiag
©ygajaisms may favcs? tk® methaaiQ baoterla T8f providlag them
witti cai*fe©B dioxide ft A feydrogea ©Gfc©pt©3?# F©S as oatalyst^
and 3SgS aft a i»©diacisig agent«
Also th© sulfat© redaeias ■
oi?^Gi$as pvo&tee a> tiaorou^dly ©saae^obis ©aviroameat
sait&fei© tof tM growth of tsh© HMtlmne ergaalsas* fosieIty may develop if the sratlftds eaneentratioa lb©com@s t©©
ii»®at»
fhaf®? (40) showed tthat duXtuvea to nhlah carboa
dlosida aad Is^dro^a aulpMde ^©3?© addad pi?'odttc^d approsi*
mately sis tia^s a* mmoh aethaa© por gran -of sad &* tho
©ontyoi^
Barlee^ (6) j'aoently aada a. .$t^dy of th© ■effoot-
©f HgS on Mathantifetetepigtt om®'iiffigMi aad eeneludad that
It had a fa^orafeli- iafitisao^ withia e©3?taia tiaits of pS
®Ed Bulflda ©oae<sat3?fttioa.
©ofiaito iahi'bitioa oeatuw la
a poorly haff©3?0d a©di«ait laitlal pB S*8» ^ith 80 t© 90
p»p*ia» &JSk
With a pi of 7*6 to 7.8f 118 p^p«a« of Bg0
^as nat toxiai ppofeahlf l>©cama© of lowej? eoaoantratiaii of
tmdissoeiated IgS at the highar pH#
la eaeaaiala&g aa aotiv© f©3?ja©atati©a it
XJQM
ohso^vod
that aotioa nppay«atty take© plae®$- ia th© aedtoant^ siaO'©
all the gaa hQh'bles soem t© as»i8@ thorefrom*
It ^as
fovthar aoticod that the gas feiafohles £ovm&& ia tfea sedl*
aeat would h© f^eqiieatiy fetappod aad,, as thoy aee^mtalatod*
the taoreagea
VOIUMO
would foroe th.® supernatant li%ui^
omt ttoough the gaB Sisoharge tube.
Slight agitation Of
tho %ottl© tromM fsp©© mogt of tha ©ntrappod gas asai the
ll^uii level woald fall. Without ^®y»iodie agitation, on»
t^api^d gas often caused sodim^nt to vis® an<3 float on
%h& Gurfaee,
Sino© t&oaa preliisinarf ©zpericieats ty©2»® |>2*iraa'3Pil|,
fop the puvpose of eatablidhiag ©on^itlons n@e0s$ay^ foj?
activ© fs^sentationo onl|r tualitativ© tdfits for aethane
38
GMPf St VI
fitted with, tnhm toT feeding wast© woA wlth&wmilnQ It^a&v
and Fesidtie so as to avoid exposture to the aiy.
fit® gas
was diselmrgea tttrougla another eoimeetim ani eollected
over saturated aei&ifled brine in oaXibratedi "bottlesj it
img analysed fro^pidatly for earfcen dioxide and laetMne*
latdf la the experiaent* gaeosasters were afctae&ed to tke
gae digeharge tubeft* fke amfeiDer tiab© msed to eenneot the
gasometer v?as long enough to permit agitatleia of the
ferHjenter.
4 general viex? of tM afparatms ia elieon in
Figare !♦
fo the S*liter oarfeoy SO© ^e of ©waap axd ^as added*
For tfe® ^*2,iter fermenter., B liters of Mad ujere' msedf 9
liters of Gwip Adair sewage eliadge were added to eaob of
teo others,
the fermenters were then filled with modified
Barker's (26) aedium tmffered tjlth excess ealei^® earbomate and MagS»9lg0 t?as added as previously deserihed.
33a
order to sialce ©ertaln that mf methane ??Mch wm& prodtseed
was derived frosa the fermentatien of the eubetrate and not
frc«a the organi© sewage ®©lids# one of the 2©*l£ter hot*
ties \ylth setjage slmdge served aa a oontrol.
It contained
the sa»e asetmt of sewage solid© in the saiae iaeditua| no
Figure I
Experimental and Analytical Apparatus Used In
Anaerobic Methane Fermentation of Pear Waste,
to
m
peas? wast© was added. Th© ferraentatioas trer© pla^Qfl la
m lacutoatoi? ooB@i$tiiig of a iarg© iasaiated packing bos*
aad th.0 tmpQTsxtwp® was Eaa±citaiia©d at 30o0>0*
At fii'St tke fefaeatatloais
VGV®
!aaiatai®©4 nitkia a
pH rang© of 6#8 to 7*4 fcy maing calcius <>a3?l>©nat©* but
since it r0qui3?ed mete, large qtaantifcios ior adjustsients,
lim© yaa latap ©mplc^©^*
It ims fotmd that one© th© f©?*
montation was well established further addition of lime
©as not n©c0s©a3pyf providing t&e i»at© ©f feeding t/aa oonts?©lX©d in aeooMane© t?itti tk© c©ne©ntj?ati©n ©f folatil®
acids that aecumulated. fMa is in agreeaoBt with
Bust?© 11 (2S),
$h© rat© ©f feading isaax* tmat© waa ..gfaiually in*
cfaasaa in r©iatl©n to tfe© volatil® acM p^oauction*
Velatil© a©Ida in ©onjunetion with m©than© ©©nt©nt oi th©
gas ser^exS aa an in&ess of tk© fepiasntation. taking plaea*
t?h©n volatil© aoid8« ©spresaad aa aeati© aei6# 3?©$© afeoira
2000
P»P*EK>
m©thana pr©aucti©n dac^aas©^ e©nsi<l©rabl^j
in Bwah iastanoes faeiing ma tampapajpily atoppad*
?h© 20*lit©3P teymantara inoaulat©^ with siTfasip aad
w©s?© initially gixr©n savsral feedings of 10 a©* ®thaa©l#
but little gas wa pfcxaueai*
Afe$ut< a aontH latar <l^y*
drated p©ar waat© was given at Mi© rat© of % g^aa p©r litaf
p©f» ia^, ©2s©©pting ©ecaaional intar^'uptiona.
Aftai* 23
days, foading was diseontinuad baaaua© of the high
25
©©neefttrafcloja ®f volatile seide (oa* gOOO p.p.a*)* ft^reBtf
days l&tep the a©i<Sl% lja<3 Shopped euff£ei«nt33r to vmrrtfftt
fu^thep f0«d£ag»
Six raoi?© SO^gsmBi fteiinga ^©re giirem to
th© B@5Ct 10 Saorst a^Pisig ^iofct tia© t&e rat a of gas- pro*
duoticm oogusldepfifely increased atid the volatile acMs r^mained quite lot?.
It was th@a deoided to add the peas? waste in inereas*
lag amoaats to dete^nine the aa^iaaam oapacity of the
fersiaters, Dmriag th© ©ueoeedlug M days* %2 feedlnge
we*'© ^d© at appro^isiatelf equal latervals* ireights of
the additioma '^eiag m .follttret ^ 4* S,. 5., 3# e, 7f- 7, 7#
i0.i H&JBt and a flnftl additioa of 3^ gl,:am0 per litea? of
tegmmto&t eapaeity* With the eaeeption of the last feediog* volatile aeids i^eaiaia^d low (ea* S0O0 pfp*m.) aod
total gas pxeAvAtitm was taaintaiixed at approxiKatelf 0*3
liters pes? gram of traste added* fh© final addition of iO©
gs?«seai9 gave §4»1 liters of gas ia S4' hours,, hut volatile
acidity iEcreased to over 3000 p#p*a«
fhe sludge volume
had increased to nearlT 10 liters t^jr this time so in vie^
of this and the increase la acidity the experi«eat was
disemtinued* Bata are given in fable ill*
26
Peat'
csas Frodu'o®d,_
flm©
pB
^olatii© wegt® fotai
€:0o;
§17*
■.**.■-■.-.■.. , acid:. ajdet. .MkP« . **
..^
Say© iailJiai aei jus tool p.p«ia»
grams
©© ' p©r- ' p©^-""
o^sit^ ©©nt;#
1
2
3
4
S
6
7
8
f
10
11
12
IS
14
16
16
17
IS
19
20
21
22
23
24
26
26
27
28
29
30
31
32
33
34
36
36
37
39
6.2
6.4
6*6
6*4
7*1
6*6
6*6
6.6
6*9
7.0
7.1
6.4
7.2
649*1
084
684
6*7
6*6
7*6
1378*7
6.6
2230.8
6.6
20©3
6*9
30
14,333
SI.3
66.8
20
8if.97'9
12*$$®
19.X ' 58.S
18.0
75.5
20
2©
4*583
4,591
7.1
6.6
6.6
g0
7.3
20©3
1696.8
a©
20
20
20
20
20
20
20
20
i,so$
82*0
68«8
2©*S
IS*!
70.0
79.2
25.2
71.0
24 *.§
72.#
2©.7
65.6
33*6
53.5
21*6
6S*t
20*1
68.0
15.2
77.0
2,948 15.3
1*839
1,983 12*7
3,111
2,589
1,366
80.0
5*588
3,078
3*664
1*03®
3*889
4f890
8';71S
^867
3*896
3f6S8
4,076
S,ol3
3,483
8#303
2,076
1*615
1#4§0
1:#3®©
1,410
1,166
1,3©6
81.0
27
f&bl® in (Gontinned)
fim©
pH
In,.,.....
Bays toitlai! m$n
40
41
43
43
44
45
46
47
43
49
SO
51
52
53
S4
35
56
57
S8
ao
61
62
m
64
65
6©
67
63
6©
70
71
72
73
74
75
76
77
78
79
e. a
?©iatil© wast©
, acid
. added
p.p.Ea,
grams
ec
20
20
20
5,263
1,856
3,039
5* 336
1510.0
94S.8
7gS.O
7S7.S
20
20
20
40
6*7
Gas ".iPrbduo'oi.
T&t&t' o©,'2
S.T.P.
24.7
63.7
22*1
64.4
S9.6
63 •&
25*6
72*6
§■,406 44*1
60*3
36*4
47*4
62 *S
60.4
36.8
49.0
27*8
73*8
49.0
62*0
34*4
62.6
3@*6
60.6
40.0
67.7
36*7
61.0
43.7
S3.6
43*0
64 *a
47.4
46*6
4*346
4t7-26.
4f182
3,246
S,§74
<5St.
766 0
480 4
6.7
24© 2
87S 6
30
1046 0
0
120
140
806 0
1218 3
14©
140
1046
200
705.0
S20
1074.0
260
6,790
3#963
2*804
2,470
1,901
8,943
26,060
35^140
16,720
31*060
36,760
39#96©
16,610
66,220
64,970
16#170
17,840
63,940
16*600
12,990
8,545
16^680
66,620
18#750
12fS90
28
1?abiB III (Contiamea)
F®ar ' ' Oaa'groQuc®d_..".,
flae
pH
Volatile tsraate foCaX CO
GH
la
aoif , aided &*$*$*... r.
4..
days initial 'ad^ttatefl '9*p*n*
grams
ec
pat*
p©s?»
80
81
83
84
* p©re©at fey volurn®
lifS00
8,; 984
3088
300
@9»S
54*V
Ut680 41.S
54'* ISO 44*7
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t> ^^
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Figure II
rl
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t*i-
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LI TfER ?: -( >R
;:':
Daily Account of 20=1^63? Ferment er.
Swamp Mud Inoculum
1 ■
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: : :
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;
■i
■ j
1
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ur '!•*.' .-:*:. ■iJji
r 'iii;
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:
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5
29
so
The fermeiater inoculated ^ith sewage sludge was
treated in a similar manner as described for the Biud
inotmlated oar'boy, esoept that preliminary addition of
alcohol was not leade.
The rate of gas prodmction varied
as in the vrnd feraenter but reaehed a maximuEi from 20gram feedings at about the same time, i*e., 4 to S liters
at 48 days.
Similarly, inoroased feedings up to SCO
grams gave nearly proportional increases in gas production,
the last feeding yielding approximately 56,0 liters in 24
hours*
fable I? shows data.
The 5*liter carboy, inoculated with suamp mud, was
initially treated with 20 grains of pear waste per liter,
without any preliminary feedings.
Farther additions were
not Made until Z months later when the volatile acidity
dropped to a favorable value.
g?hree feedings of 5 grasis
per liter were then given during the following 9 days;
with these feedings the volatile aeidity regained low and
the gas yields per gram of pear waste were the highest obtained in any of the experiments*
fable ? gives data.
fable IV
Daily Aooount of 20#lit©r Fexnenter# S©uag@ Slu^g© Ifeoemiusi
fisis
pi
Volatile
in
acl^
iai's iaitSal adjusted p.p.ja.
1
2
9
7.0
0#f
®t®
©*7
4
5
0
7
i
9
10
6«6
6»5
6»0
7.2
S42
6.®
7*4
686.
tsraete
.added
@ram@
5e
. .
pei»*
fiS^
0©nt# eesat®
20
20
2©
IMi
3188
11'.-6
22*#
72*4
©2*4
20
2740
S20^
20
20
20
20
274i
4120
1^80
1280
§0*0
«S«4
S1*0
80.©
18*8
84*8
14*4
49*8
18*4
77.8
10.4
77*8
14 .8
70*8
13.1
78*0
17.4
84.8
44.4
48*8
1888
12
1$
14
IS
IS
17
13
19
20
2i
22
23
24
23
20
27
28
29
SO
01
32
S3
34
35
S6
37
SB
7*3
20
1454
0.8
732
1008
1182
822
©27
7*3
1242
1152
1902
1228
24?©
2639
2720
1S@9
1403
1620
2698
139®
153©
7*a
267®#0
e.e
2059,2
7*0
1780*3
1854.2
20
20
20
§221
2878
4420
4189
1618
m
T&felo Ilf (Ooatlaued)
pi,.... ■,...„,...
3©
40
41
42
4a
4®
4©
4*?
48
49
50
51
mate Total
..
. . aold.
7*0
mm
1012*4
©♦a
6*7
©#
08
06
©s
§4
05
0©
§7
68
©9'
70
71
72
7S
74
75
76
77
78
pea?*
pel?*
29*S
SB#3
2S#ir
m.%
10S1
©S7
§Sl*t
S0*O
67*4
44.6
49*0
©00*6
32.9
61.0
40,1
88*7
88*8
64*8
10.6
67 »a
48.7
53*6
42.7
84*8
41*6
SS.0
40.7
88*4
44.6
62*6
4061
2087
201S
SO
m
20
20
m
40
407*6
m
m
m
W4
SIM
§3
09
6©
©1
eo
ttSSZT
^
6*8
zm
274*©
778*0
1S17
80
80
80
926*6
822*6
840*0
80
100
100
909.4
1029*©
100
140
14©
S92»0
SS7*0
1365*6
1748
1647
269i
2829
3S64
446S"
4466
4646
§198
6810
4866
S449
180
2241
1663
9018
10140
18010
14700
25420
32410
34240
14220
29800
37660
12400
18620
37040
142S©
22210
627$
8139
38660
19060
33
f&fcX© I? (eonttoued)
fla©
pi
¥olatil@ israste Uotai
la
aeid
aade^l S».T*.P,»
clays isilfelial adjusted p.p.m.
graias
ee
70
80
81
S3
8S
84
35
GO^
pea?*
GEA
Z_
p©j?«*
960!
7494
&.$$4
42*4
54*9
X$f9
S0060
40 *t
S$*7
G.O
292.0
1S38*0
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Figure III
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Dally Account of 20'=liter Fermenterj,
Sewage Sludge Inoculum
—
1
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35
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in
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4
5
14
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acid
p.p.ra.
3>f>g»
^c
1030
7*0
30
6.6
xa
e.*
18
6.2
Si
6*2
7.0
3300
sa
3.4
7.0
3300
3.1.
5.5
7.4
7*2
7.8
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38
S©
37
as
m
40
41
42
43
1330
2473
700
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7.0
^rffi
45
46
47
48
4©
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31
32
33
34
35
3@
37
38
par* per*
c©nt« ©0at#
30
6*8
S*g
S.2
addod
grams
'■•■ ^ogfe sy6qtte&__
0*4
7.1
8741
1290
273
438
917
437
1033
733
1.9
88.3
9»8
12.3
83.0
83.3
1337
6.3
G.8
13726
1008
1343
1909
m
Tatolo V (Continuod)
mimttmmtfttmmtm^afM
flsa©
in
pS
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Volatil© was*©
acid
QLM®$
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63
63
64
65
66
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68
69
70
71
72
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74
7§
76
77
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80
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8g
03
84'
05
86
87
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6*6
2226
2445
2207
2441
29*4
75♦9
23.2
57*0
2410
31.0
72*0
2901
3066
S091
24.5
63*4
24*5
6©*4
23.9
67*S
2027
23*S
7#.0
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9©1
21*3• 72«©
1S247
6.*$
6057
2407
27^0
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1582
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S148
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120
25
2060
6*8
171*6
3576
2384
2051
846
5020
2679
1589
1295
5671
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1585
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1502
1321
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25
409
93
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90
97
8*8
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W P®'k»e©a^ 'lay 'Wittme
i9a7905
1006
20
19•§
54*5
20*0
54*7
27*@ .45*5
32*0
69.0
41.7
55.3
34*2
45*0
37.6
54*7
42.0
56*0
37
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Figure IV
Daily Account of 5-liter Permenter,
Swamp Mud Inoculum
....: _;
•
I
;
:
t
:
38
A$ a resnalt of ^nt^appad gas In tli@ sefiiaant, dli'fi•euXfei' was ©nOGunt©r©d wit3^ floating @lmdg©*
fMs imml^
freqmeatlf ping tM rm^b©!? feufeliig aa<3 earn®© th® ina^b©!*
stopper to blou from th© f©^a@Bt®r». roeulting in loss of
gas aa€ liquid* ©ail^ agitatiom was found aeeeflsary f@r
two reasonst first* to releas© ■©atrapped gas to the ©©ii-.
asatf e^i>. soeoM, to ©qmalla© th© pS in th& f©raont@rs«
son^idorafel© variation in pM tms ©Bsor^d Mtwa^n s©&im©nt
and lifaid*
Bass©!! and Boruff (02) from a sorioe of e^*-
ptriffient^ eonelidded that f0r»nt©r& \ihieli w©r© frequently
agitated produced c©nsid@ra'b!^ mor© gas per day ttoan tfeos©
whic& waro not in mf waf di$ttirb©d.
fhe
VOIUIB©
of gas produced, dail^ tsra© rooordod^ along
^itb t£sip@ratw© and barometor Bsasuroments for eorroctiag
tho gas troliaja® to standard eoaditiomg of Oo0# and 760 mi*
loiults of tM three larg© seal© laboratory f@rM©ntatione
ar® Bmm3riLM6& in tafelo fl*
3©
ftftie ¥X
Summarj of fa^meatQrs
Total
Fear
Wast©
F^fl
Pays
^sraa©^
ee
gotal peg
Xiiejpft graia
per*
per*
160.4
9©
9©.^
©00
24.2
65.8
1989.S
84
801.3
403
36.1
62.S
IY
Bludg© 1013.9
SO-litej?
84
607.3
376
29.6
60*4
20-lit©i»
Control
for
# 2V
84
0*1
6.4
76.7
Fer*
aenter
Bioo*
ulxm
fafel©
S^amp
V
mxa
i*iSt©r
Tabl©
XII
20-lltef
fable
n
fflposlti©ii«u
Sewage
# |>i?gr toasis
«m Percent by Volume
«#&J3©€I footiiote on pagd 40,
40
The 20-l!ter fensent-er inoculated with s^ran^ said was
fed a total of 1989.8 grams (d*y weight) of uaat©* added
over a period of 84 days.
Ba this time 801 #3 liters of
gas wex»e reeovoredj averaging* 62.5 pereent aiethan.6 and
S©*! percent carhoia dioxide by voluia©.
T?h® yield per graai
of pear waste was 403 se total gas, of which 2152 cc was
m©tha»©«
fh© SO^liter carboy inoculated with active sewage
siiaagB received a total of 1613.9 grams of waste, dry
weight.
Ihis yielded 607.3 liters of gaa in 84 days.
the average coEipoaitioa ©f the ga© was 60.4 percent
median® and 29 #6 percent carbon dioxide by volna®,
fhis
amcmnt© to 376 e© of gas or 227 cc methane per gram of
tmste.
fh© control feriaenter f«r thi@ run gave 6.1
lit ©re of gas containing 76.7 percent Eaethan© and S.4 percent carboa dioxide,
fhia ii of doubtful aignifioane©.
Buswell (19) states that a control is sublet to error
because of the uncertainty of its representativ© nature
and because the addition of substrate may proaote the
# "Average** values given for coaaposition of the gas are
not true averages. While all ©f the ga@ VB.Q aetered,
all was not collected and sampled because of lack of
gas holders of sufficient size. Appro&iaately ©ne-.
third of the measured volumes in each experiment were
aaapled for analysis. As may be seen from the tables,
distribution of the analyses was adequate to give an
approach to the true average.
41
digestion of organic material In the inoculum*
Highest percentage of methane was obtainod in the
5*11 tor formontor.
weight, wors a<i<Sod#
m 99 days 160*4 grams of tjaate* dry
fhis gave 96*3 liters ©f gas with an
average ©ompo$ltion of 6i,8 porcent rasthgoa© and 24*2 por**
e©nt earbon dioxid© ^ volmme.
fh© total gas p©r gram
was to iras 600 cc# containing 395 cc methane*
Detailed results of individual fsriseintatlons ar©
givon in fables liz,. 2¥# an<a ? and ar© prosentoa graph*
ically in Fignros II, in* ana IV.
42
GBAPTBa ?IX
Digeussion of Results
in ©©efe fQfmentes? therd v&& an initial pepioS of
yeXatitrely low gss produetion and ?$'X&tlvo3gr liigte. acoisBm*
l&tion of volatile aeiaa*
This laot^fi tor ©pproximatoly
SO a&y« tritk oaeh of fcfet© lafgo formos^bors*;
On subsequoat
int©3KE*uptiom of foed.ing# volatil©' ©cMs docreasod ^aS:
m!©tfe©no. p©i?oontag© iaoapeaseS*
ffe©re adiitions of pear
i?ast« wore ro^aaaod* It was. otoeoffvoci that volatile aoMa
feimined low, intioation that in all profeateility a
"balaae©<3 miero^flcra ha<a heeome established.
Following
iaci'oasod *»ato of footing to tak© aivantago of this conditlon^ th© fat© of gas production incroasod smxteedlj as
indicated by the sharply ineroasod slopo of tho total gas
curves*
During the subsequent lifo of th© foipaentation,
although feeding was forced in attompt to determine aafti*
mm capacity, rapid gas evolution continued.
At the same
time volatile aeida remained reasonably low*
nethane in
the gas, although lower than uhen feeding was resuiK©d#
averaged about 0S percent by volum©*
ig^rogen eoncontra-
tion varied irregularly from about 1 to 20 percent by
volume,
fheao variations did not correlate with vari*
ations in other gas constituents.
However* sine© hydrogen
has high thermal value* Its presence as a potential fuel
48
@a© mlstufe, if not at the expe&se of methatEi©* is ©f ©c©«
nomle elgalficaiiee*
It tsrottld appear deslrahl© t© BO ©©n»
trol the- methane f eraentatloa as to yield as imcfe sael^aiao
as poaslhle* plus aj^ hydrogea at the ©xpens© of non^
eomtoustlbles•
^hla initial lag was prohahly dm© to the tim© r®*
quired for e^tahlishittg the proper aicro*flora, m active
cultare developing aeveral weeks after inoculation*
fh©
initial aecttMilation of volatil© aeids is the result of
prelimltiary activity of such anaerohes a© Jil. butyrlcma^
which attack the carhohydrafc© material prior to the action
of th© ffl®than© organisms,
fhen jaietatoolic prodnets such as
acetic acid accaimilate in exeess of 8000 p.p.m. in an Insufficiently buffered solution* development ©f the raothane
organiQiBis is inhibited,
fhis was well demonstrated in th©
case of the 5*4it®r fermenter, in which a relatively large
amount ©f substrat© was initially added,
fh© resulting
high concentration ©f volatile acids retarded developiuent
of an activ© methane culture*
Th© ©oneeatration of or*
ganic acids that can aeeraaul&tG and still b© transfozroed
to methane depends upon the buffer concentration; only
\?hen the pH does not fall below 6*3 to 6.6 are th© methane
bacteria active*
Addition of small quantities ©f substrat© at fr©qu©nt intervals afforded a mote constant food supply for
44
the arga&isms* and xaalntalned mor© favorafel© conditiosas
for hoglxming of the fesrontatlon*
After th© fermentation
wa.s well ©atahlished^ howevor^ incrsasing amoimts of pear
wasto, up to about 1#S peroent, could b© Introduced without causing volatile acids to iiicipeaso to above 2000 p.p.m,,
uhioh T7a© found to be the inhibit lag concesatratioa ia this
exporiment.
During th® last 30 daya additions averaging
Q,® pe^eont pej? day woapo mad©»
Addition of lima was not
neceissayy after th© foimontatioia booauae aotiv©, pi?ovidii>g
th© rat© ©f feoding tms eontyollod in aoeordanc© with the
eoncontration of volatile Qcids.
ffoQ proportion of m©than© and Carbon dloxldo, as woll
as the total gas produced in th® anaerobic decomposition
of pear wa©t©# dopond upon a nusfoar of factors: ret© of
foodingj eoncentration of subatrato, tiaia of sarapling, and
th© eoneentration of volatilo a#id©#
it waa found that
volatil© acid dotarialnationa give an iraaodiat© plcturo of
th© conditions undor which th© fonpsntation is progrossing*
If th© amomnt of pear waste added to th© foment or tvas
greater than its daily methan© producing capacity* th©
concentration of volatile acids !ncreased|, with a csrres*
ponding increase in the volume of carbon dioxide and decrease in volume of methane.
curves,
This is evident from the
ihen feedings ner© interrupted raethane and th©
volatile acids decreased*
This emphasized the point that
comditlens susfc be imintata.©# In a suitable fealaac© b©*
ttraezi acid foyraatioa and a@i^ formoatatioa to s©tfoa&© for
effieieat fa-rsiantor operatloa*
trafoptwlaat®l|, direefe 8tsu<3y of th® doir®lopffi©at of tii9
miero^flora tms not na-de.
H©woir©y# frequosit saioiposeopio
obs©i?v@tioa0 ^m-T® nad© after tfe® f0s?m©iatatiojii was we.il
©atabllsked* M aB effort to ^fetaiaa iafoOTatioft as to tke
pp©ao!£ttiBat.lsig %jp®® ©f l&aeteipia preBeiit.
piiiototticr©*
grtt^bs* 'Figures ? ama f it shq© titat: the Biiej?o*f l@#a de*
v@lop©a whejs stiaisp' mx&
WLB
used ae inoeulum me S'ifferent
thm 'Aeii sewage sluclg© isas emplefei* ^Jae preioiaiiaati»g
typos of ©rganisms nfeieh appeases uit&. sewage sludge trei*e
roi^sfeape^t
heattt
SOEJO
sia^ly, others in iiaii'fl, sotas sii^ttly
fhe ap|>earaiae© of miraerous Stpeptoeoeeus aM
IliorocooQufl in the femsmtatioia stafte^ with snaraung) Eftad ie
tiotewofthi'.*
!?© speeify whieh 'fflie^oorgaKiisBas
W©J?©
re*
spoiasihl© for the fersaehtatioa is not possible} this phas©
©f the prohlem t?©ul€ require ©xtea^iv© haetsrioiogieai
study.
•To e^pha$|.se the peteutial fuel value of gas ©fetaia**
ahle hf this proeess, th© following eaioulatieiiS hasei ©n
the ©xperteeintal results are presented in fahl© fll.
46
Figure V
Micro-flora Development in the Fermentation of
Pear Waste. Stained with Gentian Violet
(X 1000) Swamp Mud Inoculum.
47
Figure VI
Micro-flora Developed in the Fermentation of
Pear Waste. Stained with Gentian Violet
(X 1000) Sewage Sludge Inoculum,
$abl© ¥12
Galemlated Meat ¥alu© ©f Uettsaa© Produeei
from DJPJ F©ar Hast©
total Gas
„
Forma.mt©r
■""
•"■ ' "'
'pep'
ggam
-"'
^
p@3?
pomad
■—
Tleld
of MetJaano
p©3?
poaad
—'
Gross Calorific
V»l^e»
—■—'—mil;
' p©r
pouEd
il'n1
'I I'liillifniKimUiliil-jlliITi
per
tea
inmMnkiimmm
Stmifsp Mjd
inoculated,
g0-lit@r
40S
6.46
4.04
4*000
8^100*000
376
6*03
S.64
©,600
7*200*000
600
9.64
6.S3
6*300
13*600*000
S©ts?age sludgo
Inooulatod*
20*litOX'
Swamp mud
Inoculated*
S*llt©r
wpmrntrnmimm/mmmmtm
® Based on gross ©slorlfie value of 1016 B.t.u« p©r eu.
ft* of adUhan© uader standard eondlfcioa^.
40
Xa ooasidering th® appllca'bility of th© ^ata to possible industrial purposo* hcref©ves*j» ooptain points should
&© feorn© JUa mind*
fho expopimental roisults her® poported
a?© hasod on the us© of dj?ied poar wasto,
Fx»actical ap-
plication would roquir® the handling of a lax»go iroluM© of
frosh t?aat0.j, eontaining appp^xinatoli' 8S p©3?Q©nt wat©j?»
©hviouslf it would not to© oeonoralcally foaaihl© to dyy
this naato in tho ©potation of a plant on a coaimorcial
basis#
fho profclom is further complicatod in that the
larger voluaies of watory tjasta would introduoo considor*
ahlo amounts of dissolired oxygon into the fenaonfeor*
fhe
experimental rosulta indicated that oxygen had a marked
inhibiting offeet upon the m® thane produeing organisms*
The most foasibi© sis© of formenter for a gltren installa*
tion would ha^o to b© basod on the airorag© ooiiiposition
and daily volume of uast©*
fh©r© would no doubt b© raany
other probloias which would have to b@ inv©stigat©d in the
adaptation of this procoas to th© production of fuel gas*
CBAKKBR ¥11
gugamagy and GQB@jLmsl©ma
mmimri»^m*Jm><.,mJ,gMm
mini mimm
M ^mntMabmm nHkKi*H«m.mwm>*«m
^h© Isiitial. d©^©i©pm©at ©f && a^tlv© aetlfeaii© f-ejmoii*
tati©a requires styisls anaopobie ©oEiitioas .and th® pr©s*»
©me© of siilfat© F-aduoing ©r^miemi app©ar©i t© be ben©*
fleiai in Biaintaialag thes© ©OBidt,ti©&® £m tlielr d0v©lo|>*
mesit,
fh© influ®ne-# of sediment, eueh as awa8© nud ©r
active sowag© sludg©, was ©sseatial In establisMng and
saalntaining mt activ© £®emmtQ&±on*
Is a 2*©®ttlt of ©n«*
trapped gas In tji© eodimentt daily agitatiom was f©tmd
a©0©ssary t© r©3.©as© this gas and to ©q^aliB© the pH ill
tlie fe^manter*
Xt was f ©tmd that ©a©© th© f©3PBi©sit&ti©n was t7©il ©a*
tablished further addition of lime was not neceasary,
providing th© rat© of f®©ding wa« eontrolled in accord^
ane© with th© coneontraticm of trolatil© aeide that aecumi*
iatod.
Shis indicates that a definite Imlane© mist be
itHaintain©d b©tw©©n th© aeid forping ha©t©ria and th©
methan® prodmoing baetoria for th© ©ffi©i©nt operation of
a f©r8i©nt.©r»
The coiaposition of met ton© and carbon dioxide and
total gas produe©d in th© ana©r©bie d©©©mpo^ition of p©ar
past© depends upon a ninaber of faotorss rat© of fooding*
concentration of ©ubatrat©* tira© of gaiflpiing and th©
concentration of volatile aeids that accmmiatoci ♦
If the
amount of poar tmat© added to th© foymenter r/as greater
than its fiaily me than© producing eapaeity* the con«entj?a*
tion of volatile aei&s increased, with a corresponSifig
inerease in the voluae of earfeon <Si0xi^0 and deereaso in
volume of ia©thane* ■ Ucm^vev^ If the addition of p©ar
tff&afc® was 0t©pp®<a temporarily th© volus© of ia®than© in*
er^ased m& the VOIUSB of carhon dioxide and concentration
©f volatile acids decreased correspondingly,
cated that the fermentation
Shis indi*
0
f the waste tools place In
tc?© different- step&f firsts a period-, of h%0x production
of carbon dioxide and lou methane, urith the aocuaulation
of v©.latll® acids and prohahly other csrganie acids*
this
phase nas then followed by a period of low carbon diosx*
id©, with high methan© and corrospondiag decrease in
volatile acids*
m these experiments 37© to 600 cc ef gas per &e&M
of dry weight, er 6*OS to ©*4S cubic feet per pound of
waste, were obtained* containixsg from 60*4 to 65*8 percent
methane by voluMe*
fhla yields a potential fuel vaXite of
3,800 to i.jS00 B#.t+u» per pound of dry wei^at, of wast©
introduced in the fermenter*
The application of the data t© possible induetrial
practice is?ouM require consideration of large volumes cf
fresh waste, containing approximately 8B percent water*
52
Obviottsly i* would not be eeonomloally foasibl© to dry
this wast© In the operation of a plant on a eorasaepclal
ba^la. fiaer© t^oulQ b© m® doabt mangr ©th©i» probieias ishleft
would laav© to bo tovostigated la tjsi© adaptation of thta
process to the tactical production of fuel gas»
5g
X.
toerlcan FUblle Health A©80elatl©ii# StaiiSai?^ mdth**
oia for th© ©xamination of water asid sewage*
9tli M* H««f York, 1946, 21S|>.
2*
Barker> H* Albert. On tb.© biook^mistry of aethsaa©
f©mation. Arehlv* fur IS£krofe# 7 $404*419,
19S@.
3*
Barls©r# 1* Albert* studies upom th© aetfeano pro*
duclag feaetarla. Areblv* ftw iikrob* 7*4804S8# 1036.
4.
Barker* H* Albert* fh© praduot4<M» ©f 0aprale aini
butyria acids by tke m©feMBt© f©ra©xit&tlon of
©ftopl aleo^ol* Arelilv. tv» Mikrob. 8j41S*421, 1937.
S*
Barker, H* Albert. fh© Eatur© and distribution of
fl^than© producing bacteria. E©pt* froo. 3rd
Batera. Gongr* Hicroblol* 7S8-740J, 1930,
6.
Sarkor, E. Albert* Studies upon the mo than© f©rmm«
tation* I?* Th© isolation and cultur© of
Met^anobaeterluia omelianskii, Aiathoni© Von
i^auwonhook^ ff* Lflkrobiol* Sorol, 6*201*280,
7.
Barker, H. Albert * Hubon, S* and Samer, 1* D* fb©
reduction of radloaetiv© carbon dioxide by
motban© producing bactoria* Proe. Mat. Acad.
Sei* g0j426»43O, 1940.
8.
Barkerj> H* Albert. Studies on the m©tbaae feraoatation* ¥* BlochoEalcal activltios of Jlethano-*
baotcrium osnolianskii * j. Blol* Choa* 1371
iS-3*i67, 1941.
©*
Barker, I. Albert and faba* S* 1. Cloatridium
kluye'orli* in organism concomst in th© fomation of capraic acid from ©tbyl alcohol*
J... Sact* 43$347*»3e3, 194§.
10*
Barker* H. Alb®rt* Studies on th© ©©than© fermentation. VI. fhe iaflmenco of carbon dioxide con*
c^ntration on the rate, of carbon dioxide reduction by aolecular hydrogen. Froc. Hat* Acad.
^©1* g9,tl84«190, 1943.
54
11 •
Barkesv H* Albert*
Personal eommmlcation^ 1947.
12•
Bollen, W» B*
13.
Boyuff, C* 3* asad SUSITOII* A. SI. Fermentation
products of cellulose. Bad, & Sag. Gh©m, 21t
1181^1iaSt 1929.
14.
Borufff* G. S. and Busw©!!, A* 1* garmentatloa
produeta: I^OH coimstalka* lad. & lag* Ch#ia.
22t9Sl»93Sf 1920.
15.
Boruff, 0* S# and Bo*veil, A» fiS* Power and fuel gas
from distillery tmstea* Ind. & Bag. Cli©m. 24.t
SS-S5, 19^2.
16.
Boruff, G* S* Stal>ill»ati©a of patmch manures and
packing hous© screenings, ind. & lag. Chem*
2$1703-708* 1933.
17.
Breden,! 0* R. and lust?© 11^ A* $* ^© use «f ©Nodded
aabestos te methane f^riaentati^ns* j* laet. 261
379*3S3t 1933*
18.
Buswell* A. is* Produetioa of fuel gas tesr anaerobic
fermentation* Ind* & Ing* Gfeem. 22js 1168*1172*
1932.
19.
Bu@tr©li# A» H* and iVeAvo^. S* !.» Laboratory studies
on sludge digestion. 111. State Uator Sus3.
©till. #30, 1930♦
20.
Busweil* A» I. and Boruff* C* &» the relationship
between the chemical composition of or^ini©
aatter and th© quality and quantity of gas
produced during sludge digsstion* Ssmge Wks.
Personal cosimsmlcation, 1947*
J*. 4*434*468, 1932.
21*
Buswell, A* !.> Boruff, C* S. and Wiesraan, G* K.
Anaerobic stabilisation of milk waste. Ind. &
Big. ChSBU 24«1425»1424^ 1932*
22.
Buswell, A. 1* and Boruff, C. S, Mechanical equip*
ment for controlled fermentation of fibrous
materials. Ind. & fug. Qhem. 2Ssl47*149#
1933.
23*
Busweil,, A* M. Important considerations in sludge
digestion* 11. Microbiology and theory of
anaerobic digestion. Sewage Wks* J. 19t28*ȣS*
1947.
§5
24.
Gruess? W* ?• eomaereial fruit aad vegetable ps?oi*
uets. Hew York,, OOoorati^Hlll o©*,> i9Sa* l®lp*
25♦
Everts, W. S« Disposal of wastes from fruit ani
^©getafel© eesmarios* S#.wag© Wks* ^* 108044,.
1©44*
26.
Heukelekian, H. ani ffeiKiGaiann, B» Stud lea on tb©
w&thm® produeing baet^ria* I« Bevelopaent of
a Bi©tho<3 for ©auaeratloa* Sowag© Ws»* S* Hi
426*433, 19S©.
27*
Heu&eXdkim.* H* ®M loinesiam, !♦ Studied ©a th©
saethan© proSueing "bacteria* It* Siuraeratioa
ia iigesting sewage solids. Sewage Iks. £. 11*
4SS-444, 19S9.
28.
Keuiiolekian, H. and Heinonaffii, B. Studios on tk©
motMao produoiag "baateria. III. Mgsstioa of
gowage solids by th© addition of eariete&a
culturoa of iaethaa© ijroducisg orgaaisme.*
Sewage WSB* 3* iljS71*S8&, 19S9*
29.
Heukolekiaa, H. and Hoiaeraarm, B. Studies on th®
mothaao produeiag baoteria* IV. arowth of
metbaa® produoiag orgaaisas ia ©uporaataat
tludg© liquor. Sowag© Wm* «r* llf96§*97:0f 10S9»f
SO,.
Matu©£akt t* M* -Fishor gaa-aaalysis maaual. Be*
•ffisod*. Hew fork, fiafaey Setoatifie QO*f 5*31*
31 •
Oregoaiaa» Buliag mad© oa pollutioa.
Oregon, lev.- 25, 1047* lp.
32«
Porter* ft, ^ofea, Baofcerial oiaemistry and pbyeiol*
ogy* lew York, .foha Wiley & Sons* 194®* 954p.
33*
Hud ©If a, Willem and Hetikelekian, H. TherMophilic
digestion of sewage solids* Bid* & lag* Chem*
2gi90»90f 1930*
34.
Seimellen, Gh* d* f. t. OnderEoekiagea over d©
methonangistiag* Eottordam* Drukkery De
masstad* 1947. 137p.
35.
Stepheasoa* El. and Stricklaada, L. H. Baeterial
formation of methane* Bioehem. J. 27sl617*
1527, 1933.
Portland*
m
36.
Steplionson, 11, and Strickland, L. 1* Th©5 reducfcioa
of su&piiat® to smlpMS©. %• saoloeulax 'S^dx>dgda»
Blo0si«tt» |r» 86t23L&*SS0# 193&.»
ST*
Symoas* 0» £• and Bus^ell^ 4. M*
mentation ©f ©arfeolairtoates*
so©* aSi^oss^gose, i©§3* .
fJi©'iiet;|3aa© fer*
J« iteae^» Cli©a«
§8.
fartfin, 2>* aM Bwrooll* A* s* fh© metlmne f©^a@nte;»
tioa of ©rgmaie aeias and eatfbohydratos•
J* ^laey. Chen* Sde* 56 tl?il*17S§, 1©S4*
30.
1?h@|r03?* &• il* ^aetepiaX genesis of feyirooarbone
from fatt^ aeida* lal# Am, Assoe, Petroleum
Geol. lS?441»4SS.f 19S1.
40.
'SihB.jBff Lm Jk* E©dueti©n of ©aa?l>:0n <3ioKid© to
metbaa© 'by Isydrog^in amlfiie, Louisiana Acad,
Set* 4iSO-SS, 1938,
41*
Thay©P» !»« A*
42.
?an Hiel* e» B. The Mochemisti^r of 'bacteria,
Reir, Bioeiiem. 6*§95*620» 1937.
43*
Warriok,, h* F.,, aeke©#, F* J«* Wirth, H# E* aafl
Sanbornj: i*. H. ES©tho<3e of treating eajmery
waste, lull. 28*$, Sat 1. Ganaef-s Asa©e,#
44,
ftfeatem cann©r and packs?. Statistical Ho v. and
yeayfeook maaber. 39:lSlt 1947,
fepsonal eorasHtmioatlon,
Sept, 1947,
tern*