Laboratory experiments were conducted to study the effect of temperature, sample preparation, reducing agents, light intensity and pH of the media, on bio-gas and methane production from the microbial anaerobic decomposition of alligator weeds (Alternanthera philoxeroides. Efforts were also made for the isolation and characterization of the methanogenic bacteria

Latif, A.

English

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https://ntrs.nasa.gov/search.jsp?R=19770012785 2020-03-22T11:33:58+00:00Z
S1,NT kNNUAL REPORT
i
NASA GRANT NSG. 8036
BIO-GAS PRODUCTION FROM ALLIGATOR INA:DS
Tnitiation date: June 1, 1976
Principal Investigator: Dr. Abdul Iatif
Research Assistard" :
Mr. Daniel Malone
Mr. Mohammed Rizvi
Techincal Advisor:
Mr, William Wolverton
NASA N.S.T.L.
Bay St. Louis, Mississippi
Participating Tnstitution:
Department of Biological Sciences
Alcorn State University
Lerman, Mississippi
.{NASA-Ch-1496C9) BIG-GAS FROLUCTION IRCM	 N77-19729
ALLIGATOR WEELS Semiannual Report (Alccrn
State Univ., ICTMan, Miss.)	 11 p NC A02/MF
A01	 CSCL 06C	 Unc7,as
G3/51 219tb7
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Aio-gas Production From Alligatur Weeds
T.aburatory cxperirnerts were conducted to study the effect of temperature,
swnple t:reparati on, :•< ,',uc i ng agents, 1 1 ght intensity and nl of the medi ,i^ on
bic--gas and methane production from the microbial anaerobic (1-compositiun of
311 igator weeds (Al t.e rnranthera pni loxcroides (Mart. ] Gri.esb.) . Efforts ..,_re
also made for the isolation and characterization of the met.hanogenic bacteria,
responsible for methane production.n. Briefly, methodology aJrd results are pre-
sented as follows:
Materials and Methods
Alligator weeds were collected from a water	 pond located at Cros-
by Chemical Company, Picayune, 14ississippi, Harvesting was done rr,anually by
rakes and the plants were tranL3ported to the Alcorn State University Crunpus
in pl ra:,t,i c bags. Several fermentation units were constructed to conduct xp-
eriments to evaluate the effect of temperature, sample preparation, light in-
tensity, reducing agents, and p1i of the media, on the prodrircti on of bio-gas
and methane from the rni cr('bial anaerobic decomposition of alligator weeds.
A fermentation unit was constructed as follows: A large Coke bottle (,)'jo
MI) was sealed to the atmosphere with two hole rubber stopper. One outlet
was fitted with a rubber septtun for gas chromatographic sampling, and the ut-•
her outlet was connected with plastic tubing to a sealed container i'ill<_d with
water acidified with sulfuric acid. The di:;placement of water in the second
container by bio-gas produced in the fermentation bottle pr •uv i ded a conveni-
ent method of measuring the volume of bio-Las produced. Samples for gas
chr• ornatogr •aphi.c analysis were taken through the rubber septtun. Pt,rkins }?lrr r
81.1 Cas Chromatographs was used to estimate the methane content of the bio-
gas.	 P
.1-
Alligator weeds were chomped into approximately one inch long pieces and
placed in fer •,r,e ntation Mottles. Fach fcr•rnentation bottle had 250 grarn(w(t
weight) chopped slants, 20 ml of fresh r •urnen content from the cow's stomach
and 250 ml of water. The purpose of adding inoculiun from the rrunen conternt
was to initiate an aerobic decomposition-process and supply methanogenic bac-
t(_^ri al populations. :)i nce absolute anaerobic conditions are necessary for
the growth of methanogenle bacteria, all units, once sealed, were not di.stur•-
l)ed. A : ealant was used to insure air-tightness around the tubes and rubber
stopper. There were five treatments. Fach treatment was replicated four times.
The detail of \,ario:rs treatments is given below.
Treatment No. 1:_ T^ rr. .: ature
Four fF.rment.atlon units were placed in an incubator maintained at 35°C
and four other units were incubated at room tcrnperatur • e(21, c C ± 3`C).
Treatment No. 2: Sample Pr!=parat.S on
Tn one experiment plants wur •e chopped into approximately one-Inch long
pieces. In the second, plants were blended into a slurry form and in the third
exper• i n ent, the plants were first  boi lei in water for one hour and then blen-
ded into a slurry form.
Treatment No. 3. Tjy.ht Intqns;. ty
Tn one experiment, four fermentation bottles wore placed under bright
light produced by fluorescent tubes(1 4 00 W). Tn the second, f.-r•mentation
bottles ;. .-e covered by al iuni num 1'oil to prevent exposure to light. In the
third experi-ment., the fermentation bottles wur • e left in the laboratory under
ordinary 1 1 1,1it .
Treatment No. /4. ;R(duci np._ Aunt s
f
-2-
I
Cy.iteinE sulfide and cystei ne hydrochloride were used as reducing agents.
Cysteine sulfide was added to four fermentation bottles at the rate of 2 ml
1xrr 100 ml media whereas cystein e hydrochloride wLs added to the other four
bottles at 'he same rate. The concentration of reducing agents was 2.5%. A
control without the addition of reducing agents was also maintaliv-d.
Treatment No. _5- r;ffeet of _I)li
Tl:,: effect of pli was determi ned by ad justi ng the i ni ti al pH of 1 i qui d
content in each fermentation bottle to 7, R, and 9 respectively by the addi-
tion of sodium hydroxide. A control .,:as mai ntained which had a pH of 6.6 .
All the experiments were conducted at room tenperature(24'C +- 3°C) with
the exception of experiments of treatment number 1, in which the incubation
tomper •ahir • e fur one experiment was 35*C. fn all the , xperrI.m(,nt.s llant.s were
chopped into approximately une-inch long pieces with the exception of treat-
ment niunber 2, where plants wore blended into a slur ry form with and without
boiling the plants. Tach fermentation bottle had 250 gvwn(wet weight) alli-
gator weeds, 20 ml of fresh rumen content and 250 rril water.
Tsolatior. and Characterization of Methanopenic Bacteria
"'ifliples for bacterial i sol at.i on and characteri zat i on were taken frorn those
fermentation units which had produced the highest amount of hio-gas arid me-
thane during anaerobic decomposition of alligator weeds. These units were these
which were incubated at 3; 'C
 
and those in which the initial pH of the l i.quid
rnedia was adjusted to 9. During the process of isolating pure cultures from
these anaerobi;: inicroorl:anisms, strict anaerobic conditions were maintai.nod
as described by Rr •yant et al. i lie pr•occ._.s of isolation and characterization
is still in progress. Tiic result of this study will be reported I n the anr.-
iial r • :port which will be submitt,c^d in Mlay 1977.
i:-M
Results:
The data from the various experiments which were conducted to study the
	
A
effect of temperature, ::arnplc tu•eparation, reducing agents, light intensity
and pH of the rr,edi a ., on bio--Las and methane production from the microbial
anaerobic decomposition of alligator weeds, rlr•e Ares(-rated in Tables 1, 2,
3, 4, and 5 respectively.
T^ r.a^j-iture
Effect of temperature on the rate and amount of bio--gas production as well
as on the percentage of methane in the bio .-Las was very prominent (Table 1.).
zncrea,A ng incubation temperature of the fermentation units from 24°C t- 30C
to 350C increased not only the rate of bio-gas production but also the methane
percentage in the bio-gas. The methane content of total bio-gas was increased
from 58.1% to 66.cg by increasing the incubation temperature to 35 0C. Further
more hi; her tcmpcu •ature al-c o decreased the l-ag time betw^vn the pr•oductiun of
bio-Las and production of methane gas.
^,. nl e Prer ?r,•^t.fon
Data presented in Table 2, revealed Lhat sample preparation, (alliLator
weeds chopped, blended, and boiled and blend(A) had no effect on the total bio-
gas production. However the methane production from samples which were boiled
and blended, x'2s reduced to a great ^ xtent (47.7%) when compared with chopped
or blended samples (58 .1% and 62.1%).
TJ ght Tntensit
Comparison of the data from experiments in which fermentation units were
exposed to different light intensity, in Table 3 revealed that total bio-gas
produced under bright l i Lht was slightly h i 6her w} en compared with ordinary
;ht or dark conditions. On the other hand, methane content of the bi.o- •gas
was more (06.61) when bio--gas was produced under darknuvs.
Reducip_AAc n s
According to the data presented in Table 1+, addition of cysteine sulfide
and cystei ne. hydrochloride to the fer •morrtati on .)ni is had slightly increased
both, the bio-gas production and percentage of rnethaz.e in the bio•-I;as.
Cysteine sulfide was slightly more effect than cysteine hydrochloride.
Effect of -pH
Pata from the experiments in which pH of the liquid j, edi a in each fLr-
;ientation unit was adjusted before anaerobic microbial decomposition of the
alligator weeds, are presented in Table 5. The data showed that increasing
pH of the media frL'm 6.6 (control) to 'J.0 1 ad a slight decrc, se in bio•-gas pro-
duction as well as in the methane content of the bio-gas produced.
when the pH of the mudia was increased t,o R.0, Lhur • e was a little effect on
bio-gas production but methane content of the bio-t;as produced was increased
up to 61+.0%. By further increase in the pH of the media to 9.0, there was
a sigrni fi cant increase in the bio-gas production as well as in the methane
content of the bio•-gas produced.
.„
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i
Table 1: Bic-gas and Methane Production From t yre Anaerobic Pecotn},osition
of Alligator Weeds at Various Temperatures
Tncubation Temperature
21^ C + _^°C 31°^
Cum.	 Cum. Ci)r.. Cum.
Pao. Days Pic--gas	 Mutl,ane 131o.-gas Methane
—
5 0	 0 20 9
12 45	 0 298
168
20 3>3	 15 1180 613
42 91,0	 503 1495 997
60 1.351	 788 1900 1248
81 2075	 1363 2750
1768
1 oz _26 __ ^5r^ t __ 31^5
Calculated Pat.a:
Mcthane	 66 .oin Total H l o--gas	 5P .1
n)1 Hio-gas
Per Gram
Wet Weight	 10.9	 12.8
ml ?`ethane
l'cr Gram
WA 1"'eight
i
4,
Table 2: Effect of Swiple Preparation on Rio-gas and Methane Production
From the Anaerobic Decomposition of Alligator Weeds
Chovoed Blended Boi l ed &_ 13lunded
^ _-_--
-- Cum. `Cum.	 _	
---
--	 Cum. -	 _- T_Cum.
No.	 clays Rio-gas	 Methane(rn1.
Rio-Cas Methane Hio-gas Methane
Fl aysed
 -- ^ ) _ _
5 0 0 0 0 0 0
12 45 0 27 0 59
0
20 353 15 307 39
429 '18
14 2 940 503 885 426
1.1.41 478
60 1351 788 1.615 896 1739 750
81 2075 1-363 2015 1196
2344 1064
102
. 226__-___.. 15g..^_._._—_ 26oT	 ^------ -
161 6
--	 —
2( 51
_	
^L,._.
—	
-
_
1
_
2(,6_ 
Cal cul ated, Dai .a :
% Methane
in Total Iii o--gas	 58.1	 62.1	 47' 7
rnl Bio-gas
Per Gram	 10.6
wet weight	 10.9	 1-0.11
mI Methane
Per Grrin	
^^1
I^ et We i ght	
_ __ 
6 
._ . _.__	 --6 -L5 -	 —. 
-7-
Tabl e 3. Rio-gas and Mett.xne Production Form the Anaerobic Decomposition
of Alligator Weeds Under Different Itght Intensitics
iiriKht
	 ht Ordinary T-ab Light Pa r^
- Cum. Cum. --- Ctun.	 - - -- Ciun.	 - _ -- Cun.-
No. rays 1310-Las Methane Rio--gas Mwtliane Rio-gris M.-thane
".1 ap:_ed
_ ^m1Z -_^.__^m^ .—	 l ^-- - -----(rw __^mlj — ---_ - (ml^ ---- —
5 0 0 U U 0 0
12 39 0 145 0 27 0
20 330 17 353 15 357 24
1,2 968 167 9140 503 1067 5 29
60 1-390 789 1351 788 1377 794
81 21.55 1287 2075 1.363 2256 1378
102 2P1, 0 1797 2726 1584 2618 1.784
C-ilc„l at , d
 nat.a:
Methane
^n Total Hio-gas 63.3 SP .l 66.6
1	 Ri o•-gas
r C ram
t.	 '::,-ight 11 ,4 10.9 10.11
thane
Cram
t, ;Ici-ght ` 7.2
 6'3
14: Kffuet of Reducing Agents on Aio-gas and Methane Production
From the Anaerobic Deemposi ► ion of A1]iratur Weeds
Cyst—e
-lne Sulfi de
-
Cum.
No. Pays	 Rio-gas Methane
Cvste.i+e 	 iydr_ochlori_de Cont t.(-,l
Cum.	 Curn.
 Cum.
Rio-gas
	 Mt t 1,:(ne Bi 0-gas MO bane
m1
0	 0 0 0
50	 Y/ 45 0
470	 307 353 15
1145
	 729 9/40 X03
1750	 1124 .1351 188
2420	 .1576 2075 1363
2t3 L0 - -- —
	 r
	- --
	
_
7 2
-
6
- 1 H	 _ 
__. ___
58.1
10.9
.,
M_. (, . 3
i JF' U14
1,13 POOR
PH. -9-
cum. CILM.
Rio-gas Methane
0 0
80 37
634 44'1
121+5 852
1790 1385
21+(?0 1 P22
28.88 1464	 -
I
i
,rah] e 5: Pi o-eas and M(:thane Production Froin the Anaerobic Nvomposi t.i on 	 k
of Allieat.or Weeds at Variou8 pH
(-,,,ntrol(nH 6.6) nH 3 pH 8_
Curti. 
	
Cum. ^Cl,m. um. Cum. Cum.
No . rays Rio-has	 Methane 131 o•-t:aa !- et};ane Ri o•-leas 1`".c thane
M'1 nosed -_
(mt^- 
-^ml^  _ _ no "Al-
5 0	 0 0 0 0 0
12 45	 0 44 0 35 10
20 353
	
1.5 350 25 1+2 5 230
t+ 2 940	 503 972 443 1133 '13^,
60 1351	 788 1300 '115 1242 805
81 2075
	
1363 1800 106, 1660 110'1
_ 102 2726
	 _	 4.— IA75 _ _ '?35 .1022
Calcul at ud T ,, i.a :
% Methane
i n Total
Hi o--gas	 5P.1	 57.8	 64.0	 6P.0
in]. Rio-gas
P(. r Gram
Viet wett;ht	 1.0.9	 10.2	 1.0.1	 11.5
.:,
8 111 Met,},;ine
Per ( ram
Wet we lellt	
_ 6. 3. __  
	
__ 5-19
	
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Bio-gas production from alligator weeds

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