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“Biocrude” and “Biomethane” from Croton bonplandianum
Resources, Conservation and Recycling, 1 (1988) 111-122
111
Elsevier Science Publishers B.a./Pergamon Press pie - - Printed in The Netherlands
"Biocrude" and "Biomethane" from Croton
bonplandianum D.K. SHARMA* and H.A. MBISE**
Centre of Energy Studies, Indian Institute of Technology, New Delhi 110016 (India) (Received September 4,1987; accepted in revised form April 13, 1988)
ABSTRACT Croton bonplandianum, a latex bearingplant, rich in i~t,ogen,phosphorousand potsuium (NP-K), and whichgrowswild,yieldsgoodquantities (12-30~,) of"biocrude" (definedin the text). This biocrudecan be "hydrocracked"to petroleumoil T! e seedsof this plant yield39% oil which can be a diesel oil substitute. The spent residue after extraction of biocrude can be used for the generation of methane ("biomethane") by anaerobic digestion.The spent residuehas been found to givegoodyields (between4.74-5.66L/ks/day) of biogaswith methane contents from66-72%. The spent residual plant material obtained after digestion, contains enhanced N-P-K contents. This can be utilizedas a fertilizer.
INTXODUCTION Recent declines in oil prices have provided a grace period to search for future sources of oil and natural gas. World reserves of oil and natural gas are limited and these are being depleted fast. No doubt the recent price decline has encouraged their consumption, diminishing energy conservation measures. There is a need to find a nondepletable source of oil and natural gas. Biomass is such a renewable source of ener~. L~.iciferous and resinous plants [ 1-9 ], like heavea and guaule (the rubber tloes), yield "biocrude" by tapping the plant for latex or resin or, alternatively, by solvent extraction. "Biocrude" contains a mixture of hydrocarbons, terpenes, fats, lipi&, steroids, flavonoids, waxes and polyphenols. This mixture (defined here as biocrude) can be "hydrocracked" to get petroleum oil. The main advantage of latex bearing and resinous plants is that they grow wild without much agricultural input or management, they can be grown in arid or semiarid zones, and they do not compete wil.~ food and fibre crops. Rather, in the long run, these would help in the development of these desert areas by generating employment. Laticiferous and resinous plants can be used for the production of both oil *Authorto whomall correspondenceshouldbe addressed. **Presentaddress: Ministry of Energyand Minerals, P.O. Box 2000,Dar-ES-Sa¼m,Tanzania. 0921-3449/88/$03.50
© 1988 Elsevier Science Publishers B.V./Perpmon Press pie
112
and substitute natural gas (methane). After extraction of biocrude from these plants, the spent residue, which is rich in cellulosic material, can be u~ed for the generation of biogas ( ~ t i t u t e natural ~ ) . In the present work, studies have been ~ out on the Croton b o ~ z h k m ~ m (Euphorbiaceae)a ~ d , latex beari~ weed having a fast growth rate. Its growth is an environmental m e ~ and problem in India and these plants have to be removed to clear land. Its utilization for the production of oil and biomethane would help eliminate an environmental hazard. Biomethane can also be converted to methanol throuzh chemical or biochemical routes. The seeds of Croton bonpfand~anum contain about $9% oil It0]. The seeds also contain 3.5% ash which contains K20 (1.4.$%) and PROs (29.8%). This seed oil can be used as a diesel substitute and the spent r e , due obtained after extraction of oil can be compested. EXPBRIMZNTAL
Extraction o( biocr~te Croton b o ~ ~ (C.b.) stems and leaves were crushed and powdered to 40-60 BSS mesh size and dried in a solar drier. Dried leaves or stems (10 g ) were Soxhlet extracted with acetone. The spent residue was Soxhlet extracted with benzene. Similarly, dried C.b. stems or leaves were extracted with methanol followed by hexans, The portion extractable was calculated from the loss in weight of the C,b. items or leaves altar each extraction. At a larpr scale, C,b, leaves or stems (70 g) were taken in a batch extractor (i,e, S L round bottom flask fitted with a reflux condenser) under reflux conditions with methanol C/S0 mL) followed by the extraction of spent residue with hexans under similar conditions. The spent residues were taken as substrates for bioi~ production.
B/oeu product/on Substrates (50 g) were taken each in a one litre digester bottle attached to others (Fig, I), Water (700 mL) was added to each d i p m r , leaving some space for gas formation. Digestsrs were inoculated with cowdun8 inoculum (I00 mL), All the dilpmters were incubated at constant temperature (35-37 °C ). Gas samples were withdrawn daily and analysed for methane and carbon dioxide, usins a 8700 Nucon gas chromatograph. RI~ULTS AND DISCUSSION
Table I shows the chemical composition of the various components, i.e., leaves, stemsend roots of C.b. [ 11]. Roots contained the minimum moisture
113
oAs COLLECTOR I
oossToe
Fig. 1. Experimental set up for one set (out of six) used for the generation of bioges, TABLE 1 Chemical composition of biomems* Co~n~osition of Croton bonplandienum (C.b.) (%)
Moisture Ash Volatil,~ matter (% on dry.basis)
Leaves
Stems
Roots
71 1 76
70 1 79.
43 1.5 65
Cher,fical composition of C.b. (%) Leaves
Cellulose Hemicellulose Lignin Extractive3
Unextracted
Extracted residue
Uneztracted
Extracted residue
49.0 19.8 19.5 19.0
50.0 9.2.0 28.0
46.0 22.5 20.5 12.0
52.0 25.0 23.0
Chemical composition of bagasse (%) Moisture Cellulose HemicelluloN Ligain *Ref.
[11].
Stems
8,0 49.5 28.5 15.0 6.0
114
(43%) and leaves and stems contained about 70%. The volatile matter in C.b. were 65-76% and about 1% ash. The plant components were subjected to acetone extraction, followed by benzene extraction of the spent residue, Table 2 shows the results. Acetone is expected to extract the polar components such as polyphenols, chlorophyll, etc., whereas, benzene extracts mostly the nonpolar and less polar chemical components, such as, terpenoids, steroids and hydrocarbons, There were more acetone extractives (AE) than the benzene extractivee (BE). The total AE + BE represented the biocrude yields. The yield of biocrude from the leaves was more than that from the stems. Leaves yielded 20% bioerude, whereas stems yielded only 8.1%. In fact, leaves contain more latex than the stems and that could be the reason for higher biocrude yields. The solvent for extraction ofbiocrude from C.b. was changed tp methanol followedby the hexane for the residue. Methanol extracts the pokr components whereas, non-polar components are extracted in hexane. This solvent system provides a better fractionation between polar and nonpolar extractivee, as the difference between the polarities of methanol and hexane is more than that between acetone and benzene. In fact, the methanol extractivee (ME) were found to be more than the AE. Overall, biocrude yield through the methanol-hexane solvent system was more than that through acetone-benzene system (Table 2). Leaves yielded about 30% biocrude through methanol-hexane extraction, which was more than that (I4%) obtained through acetone-benzene extraction, Stems yielded 9% biocrude through acetone-benzene solvent system and 12% through methanol-hexane solvent system. Table 3 shows the biocrude yield from some of the promising latex bearing plants. It can be seen that C.b. (latex bearing plant) yields 18,2% biocrude which is the maximum amongst the potential petrocrops taken for comparison. Only Pergularia e~cnsa yields biocrude equivalent to this, The use of solar concentrators can be made for heating the extractors. The use of solar energy for heating these reactors has been demonstrated in our laboratory [ 11 ] and can reduce the processing cost by 20%. Table I shows that C,b, leaves and stems contain 42% and 46% cellulose and TABLE 2
Sompk
Moisture contents (%)
Extraction (% on dry basis ) !
Acetone
Benzene
Total
Methanol
Hexane
Total
14,0 9.1 i0.0
20,0 8.1 12.2
10.2 3.5 6.0
30.2 11.6 18.2
qs
Leav~ Ste~ W~de plant
71 70 66
1~,0 8,0 9.0
2,0 1,1 1.0
115 TABLE 3 Comparison of biocrude yields of C.b. and of some potential petro-crops Species
Croton bonplandianum Colotropis gigantea Calotropis procera Hemidesmas indicus Pergularia eztensa Euphorbia neriifolia Pedilanthus tithymaloides
Moisture
70.0 78.0 53.0 45.0 89.0 87.0 83.0
Extraction (%) on dry basis Acetone
Benzene Total
Methanol
Hexane
Total
9.0 7.8 7.5 5.3 8.0 10.6 7.5
1.0 0.7 0.6 0.6 0.7 0.6 0.6
12.2 5.1 5.2 9.0 14.1 7.0 7.0
6.0 2.8 1.9 3.0 4.0 6.0 3.0
18.2 7.9 7.1 12,0 18.1 13.0 10.0
10.0 8.5 8.1 5.9 8.7 11.2 8.1
19.5% and 22.5% hemicellulose, respectively. The percentage of these cellulosic contents is obviously more in the spent residue obtained after biocrude extraction than these in the original C.b. Thus, these could be good substrates for biogas generation, where cellulose, hemiceUulose, fats and proteins can be converted biochemically, through methanogenic bacteria, to methane and carbon dioxide. Common materials used for biogas generation are: animal manure, agrowastes, industrial wastes and night soil. Weeds have a comparatively favorable C/N ratio for biogasification. Thus, these are quite suitable for methane generation [12 ]. In the present work, the use of various components of Croton bonplandianum (weed) before and after extraction of biocrudehas been made. The following substrates were taken: extracted leaves (EL), dried unextracted leaves (DUL), dried extracted stems (DES), unextracted stem (UES), a mixture of extracted stem and bagasse (1:1) (SB) and unextracted wet leaves (UWL). Cowdung inoculum (100 mL or 5% W/V in an IL digester) was used for the anaerobic digestion of these substrates. A group of microorganisms act on the organic substrates to convert the biopolymeric materials (cellulose, hemicellulose, fat, etc.) into soluble monomers, which become substrates for the microorganisms in the second stage, in which the soluble organic compounds are converted to organic acids. These organic acids are then converted to methane and carbon dioxide by the methanogenic bacteria. Table 4 shows the cumulative volume of biogas generation per day, from 3 to 42 days of digestion (of 50 g biomass substrate). There was no significant biog~s productiozl during the first two days. Figure 2 shows the total gas production (mL) at different digestion times (days). Minimum biogas generation was from the unextracted wet leaves (UWL) (2725) mL) as these contained minimum hemicellulosic mid cellulosic contents because of high moisture contents (Table 1). Unextrncted leaves produced more biogas than the extracted leaves upto 40 days. But the biogas production after 42 days was more in the
116 TABLE 4
Cumubtivevolumeofbio~s~
(mL) ( f m m 3 / 1 2 t 8 6 t o 13/1/67)
Dilpmer containing ( m L )
2
1
3
C.bJ~ves U,ntmmd C . ~ C.b.mm U n n t m m d C.b.mm UnHm~M'C~b,mm+ba~Ne
------
------
Unntmct~
--
--
b~h C.b,bam
1~
14
7
5
70
250
100
300 400
800 700 MO
850 '725 650
180 300 100
87~
650
9~
400 250
525 88O
600 480
15
16
17
19
19
--
--
200
.6
9
10
1175 1200 1600 2350 1600 1150
1275 1475 1725 1400 1800 2150 2200 3050 3400 285033603800 2060 2700 33OO 1250 1400 1450
20
21
22
4175 5480 5775 6075 848O 1975
48?6 4678 6100 6125 6475 68?8 7278 6150 6450
--' ---
2125
--
32
33
8
4
950 IlOO 900 IlOO 800 IlOO 1400
1850
850 IlOO 780 1500
U
23
12
24
~sns.
Ext~cted C,b,l~v~ Ext~
1925
C.~mm
8800
8880
Unexmcted C,b,m
4050
44?5
Umxtm~tedC.b.mm+b~lm~ Unex~h~hC.b.lum
3650
4000
1500
1525
25 ~xtm~tedC.b~m U n n t m c t ~ l C,b, b a v n ~ t r a c t e d C,b,stem Unextmcted C,b,#em
UnexLn~c~l C , b , ~ m + ~ Unox~ ~ C.hJ~vm
-"
-"
---
----
--
34 marinated C.~iRvm UnexU~ctMIC.b.luvH ~ t m ~ t K I C.~ttem UnRtqwt~l C.b.mm UnR~t~/C.b.~tm+b~me U~xtm~ted bmh C,I~IRm
35
-------
8178 8600 4278 5175 4680 1628
4100 4400
o
i
5575
--
--
1725
--
--
~7
28
29
6175 7480 63~ 8800 ?800
6475 7600 86~ 8600 7780
6675 6975 71 ?800 8000 ~OO 8878 9 1 ~ 9225 8 8 0 0 9 0 0 0 9200
--*
--*
--
--
?900 8050
8250
--
--
2400
~
36
~i
38
40
§175 83?8 8 / 0 0 8800 100115 10175 10575 10578 9950 10150 10184) 10300 8600 8750 8900 9080
30
39
31
41
3335
5175 6300 69?5 7680 7000 2725
42
???S
79?5
--
--
--
9125
9475
8600
8684)
--
--
--
9150
9150
'
'
*
--
--
--
--
--
--
11075 11325 10850 11150 9 8 8 0 9880
' R R d l n p not takem
case of extracted loaves (9475 mL) than that from unoxtracted leaves (9150 mL). This showed that the biops generation from leaves is reduced after extraction of biocrude. Fatty components are removed from the leaves during biocrude extraction, which result in the initial dropping of biogas yields up to 40 days, but after 40 days, the biogas generation was more in the extracted leaves. This is thought due to the digestion of cellulose becoming predominant through solubiUsation and microbial degradation in the prolonged process of biogas generation. Therefore, after 40 days the gas yield from extracted leaves containing comparatively more cellulosic contents (Table I) is enhanced. Stems showed more b i o ~ production tha~i leaves, which could be due to the fact that stems contain more cellulosic and hemicellulosic contents than
117 x 103 14
. . . . ............ ......... ..I
O 1: Extracted CB leaves O 2: Unextrocted CB leaves D 3: Extracted CO stem D ¢ : Unextracted CB stem O S: Unextracted CB stem • bagasse D 6: Unextracted fresh CB leaves
~w
w
iO
Average fermentation temp: 35-37 'C Total solids: 7 % Cow clung inoculum: S %
5
~
a
W ij
6
Q, J 0
~--~ 0
I 6
i
I 16
i
I 2~
i
I
,
)2
I &0
REI'ENTIOH Tilde (days)
Fig. 2. Cumulative volume of biogas production at different retention times.
leaves (Table 1). Biogas generation from extracted stems (11335 mL) was more than that from unextracted stems (11150 mL). Statistical analyses of the data obtained on the biogas generation from different substrates was done and the conclusions drawn from the data were confirmed [11]. This showed that biocrude extraction was a beneficial pretreatment for biogas generation. There could be two reasons for this. One could be that extraction breaks the plant cells by extracting the cytoplasmic fluid, i.e. latex extractives. Action of solvents (hexane and methanol) under reflux conditions breaks the cells to extract the biocrude from cytoplasm. In fact, latex is accumulated mostly in specialized cells or vessels known as lactifere [18]. In stems, latex is stored in the rings of lactifers in the bark. Extraction of latex (biocrude) from the stem (bark) exposes the hemicellulose, cellulose and lignin. As cytoplasmic fluid (latex) is removed by solvents from plant tissues, cells collapse, and cell wall components, such as, cellulose, lignin, hemicelhlose and proteins, are disrupted and their association is disintegrated. Moreover, extraction increases the surface area and porosity of the biomass, alJowing the better accessibility for bacteria and methanogenic bacterial enzymes. Another reason could be that biocrude extraction increases the percentage of cellulosic contents (Table 1 ) in the spent residue. Further work on finding these reasons is currently underway. Table 5 shows the overall average of biogas production rate, expressed as
118 TABLE 5 Ovendl averqe of biops production rate in a one litre disester Disesterc~with:
DI: F,xtracted C.b. leavn D~: Unextracted C.b. l~veJ D~: ~tmcted C.b. item D4: Unextlm~C.b,e~m D~: Unez~ct, dC.b. item+~ D6: Unextt~cted fresh C.t~ IMvu
Dif~tion Totaivolume mL/h m L / d ~ L/ks/d~v (Bmedon time (days) (mL) (Based onS0g lkS sub#rate subltrate) 40 40 40 40 40
9475 91S0 11825 11150 10880
35
2725
9.87 9.53 I!.79 11.61 11.33
236.90 228.70 282,96 278.64 271.96
4.74 4.57 5.66 5.57 5.44
3,55 85.20 1.70
mLlh, mL/day (based on 50 g of biomass) and L/kg/day. Average hourly, daily or by weight daily rates were maximum in the extra©ted stems of C.b.: 11.79 mL/h, 282.96 mL/day, 5.66 L/kg/day, respectively. This was followed by the rate for unextra©ted stem of C.b. The biogas production rate for the mixture of bagasse and extracted C.b. was less than that for the unextra©ted stem,
Rutes of biogas generation from leaves were less than those from stems. Minimum rates could be seen from wet leaves (3.55 mL/h, 85.20 mL/day and 1.70 L/kg/day) and the biosas generation rate for extracted leaves was more than that for the unextracted leaves. This follows from the same reasoning as given earlier for blobs generation. These studies showed that extraction of biocruds renders the spent residue more susceptible to anaerobic digestion. The mixture of bagasse and extracted C.b. stems showed less biogas yields (10880 mL) than the extracted (11325 mL) or unextracted C.b. (11150 mL). Table 1 shows the composition of bagasse. Bagasse was added on the premise that this would be digested smoothly and thus, would help in the digestion of Croton bonp/endianum, but this did not happen. It seems C.b. is better substrate for biops generation than bagasse. In fact, extracted C.b. stem contains more holocellulosethan bagasse, though bagasse contained more of these than the unextracted C.b. stem. Moreover, C/N ratio in weed is 26:1, which is optimum ratio for biopsification by anaerobic digestion (using methanogenic bacteria) [ 12 ]. This could be the reason for the better oiu~tLicatlon results from C.b. stems. A mixture of latex bearing plant, such as Euphorbia tiruealli Linn. with cowdung (1/1 W/W), has been reported [ 14 ] to give higher output of total gas by anaerobic digestion than that from cowdung alone. Even more methanogenic activity was observed in the digestion where a mixture of Eu-
119
phorbia tirucaUi and cowdung was used as compared to that from cowdung alone. The yields of biogas from extracted and unextracted C.b. stem was found to be more than that from agrowastes and was comparable to that from cowdung [ 11]. Further work on the studies of biogas generation using mixtures of Croton bonplandianum, water hyacinth and bagasse is being extended. Figure 3 shows the methane contents in the biogas per week. Methane contents from all the substrates studied were found to increase with increase in the digestion time. Maximum methane contents (72%) were found in the extracted stem of C.b., followed by 70% from extracted leaves of C,b. Unextracted stem of C.b. showed the minimum methane contents of 62%, which were less than those from the mixture of extracted stem of C.b. and bagasse (66%). Wet leaves showed a greater methane content (68%) than those in the weekly biogas from unextracted leaves. Statistical analyses of the data obtained were done [ 11 ] and supported the above results. The mixture of extracted C.b. stem
. . . . . . . . .
OI : Extracted CB kczves 0 2 : Unextracted C8 leaves O3 : Extracted CB stem ----..---Or, • Unextracted CB stem . . . . . . . . . . . . . OS : Unextractecl CB stem. bagasse - - - - - - - - - - O6 : Unextracted fresh CB leaves
20r
\
1
i/ /
i
\ I
,\
\
I
#
g~
!
I
I
I
I
2
3
4
S
WEEK NUMBER
6
120 70
60
SO
~0 .|
f }0
I
x O! Etl~Jcted CO leaves
o O~ Ue,ettmc~ted CBINveJ 0 0]1 Eelmcted CO s l ~ I O~ tNxlm¢led CB Item • OS U~tncl~l CB shm.l~gaue • D6 Ue~tmct~ fre~ CB leaves
|0
I
I
I
I
I
I
DAYS
Fig,3, (a), Weeklybiogasproduction:(b), Methanecontentsin blobs. and bas~se showed hiilher methane contents in the biogas than those in the biolla8 from extracted or unextracted C.b. stem in the first 4 weeks. This showed some advantap of usin8 basasse along with C.b. stem, i.e. the use of bagasse enhances methane yields in the first 4 weeks. These studies are being extended further. Althoush the b i o ~ yield from leaves was less than those from the stem of C.b., still the methane contents in biops were more from the leaves, especially in the first four weeks. This could be due to the fact that the cellulose in the lignocellulosicbiomu8 of leaves has loose lignin-~ellulose chemical bonds and is easily solub'dized and the action of methanopnic bacteria is faster in leaves which are softer than stems. Above studies had shown that extracted C.b. stem 8ive8 the maximum biosas yields. Table 6 shows the methane contents in biops pnerated every week from the extracted C.b. stem. The methane contents increased from 2'7% in the first week to 72% in the sixth week. The methane percentap did not show maxima or minima. Rather it showed all increase with an increase in dipstion time. This is due to the fact that as the dipmtion time incumses, the reaction of methanogenic bacteria also increases, to convert more and more of the acids to methane. Also, more COs is
121 TABLE 6 Relationship between wee,kilt c u ~ extracted C.b. stem
~, ,~ ;ota! volume of biogas and methane content (%) from
Week no.
Total volume (Litres)
Methane (%)
1 2 3 4 5 6
0.800 3.050 2.625 2.400 11.275 0.225
27 46 54.8 58.4 69.3 72.0
converted to CH, over an extended incubation period, thus reducing the CO~ concentration and increasing the CH4. The spent residue of C.b. obtained after the biogesification was found to be enriched in potash, nitrogen and phosphorus, because of three reasons: (1) extraction of biocrude raises the N-P-K contents in the spent residue; (2) conversion of cellulose through biogasification also raises the N-P-K contents in the spent residue; and (3) the original C.b. is rich in N-P-K which makes it suitable for composting. Thus, the spent residue of C.b. obtained after biogasification can be utilized as fertiliser/soil conditioner, with or without chemical fertilizers. CONCLUSION
The studies reported here show that about 12-30% yields of biocruds can be obtained from the leaves and stems of Croton bonp~m~mnum, along with the extraction of 39% of diesel fuel substitute from its seeds. Therefore, Croton bonp~m~i~num appear~ to be a potential candidate for "petrofarming". The biocrude obtained can be hydrocracked to get oil. The spent residue obtained from the extraction of biocrude from C.b. can be utilized for the generation of biogas. The methane content in the biogas is between 66-72% and can be used as a substitute natural gas. Methane obtained can be converted to methanol under high temperature ( 400-600 ° C ) and high pressure (5-10 MPa ) with copper or nickel catalysts [ 15 ]. Methane can also be converted to methanol through biochemical routes under milder conditions by inhibiting the methanol dehydrogenase enzyme by using iodoacetic acid in the anaerobic digestion process [ 16,17 ]. Therefore, petrocrops could form an alternate and renewable source of oil, natural gas and methanol. Since the extracted stems produced more bioges than the unextracted stems, this indicates that extracted ~tems are better substrates for biogas generation. Further work is being undertaken to study anaerobic digestion of latex alone.
122 In some countries latex bearing plants are being commercially used for biogas generation [ 11 ]. It would be worthwhile to recover biocrude before charging these to digesters for biogas generation. ACKNOWLEDGEMENTS This work was carried out as project work of Mr. H.A. Mbise, under the United Nations University Programme in Renewable Energy Systems at the Centre of Energy Studies, Indian Institute of Technology, New Delhi, 110 016, India.
REFERENCES I 2 3 4 5 6 7 8 9 10 !! 12 13 14 15 16 17
Calvin,M., 1977. Energy Res., 1: 290. Hillen,L.W., Wake, L.V. and Warren, D.R., 1980.Fuel, 59: 446. Hall,D.O., 1982. Experientia, 38: 3. Bhatia, V.K., Srivastava, G.S., Garg, V.K., Gupta, V.K., Rawat, S.S. and Singh, J., 1983. Fuel, 62: 953. Erdman,M.D. and Erdman, B.A., 1981. Economic Botany, 35: 467. Sharma,D.K. and Babu, C.R., 1984. Fuel Sci. & Technol., 3: 49. Sharma,D.K. and Prasad, R., 1986. BiomMs, 11: 75. Srivastava,G.S., 1986. Proc. Nat. Workshop on Petrocrops, New Delhi. Bhatia,V.K., 1986. Proc. Nat. Workshopon Petrocrops, New Delhi. The Wealth of India, 1950. Paw Materials, Vol. 11, Published by C.S.I.R., New Delhi. Mbise,H.A., 1987. Petroleum Extraction U~ing Solar Energy and Production of Biogas or Alcohol t~m Petrocrops. Project Report of United Nations University Programme on Renewable Sources of Energy, Submitted in liT, Delhi, February. Guide Book on Btogas Development, 1980. Energy Resources Development Series No. 21 United Nations, New York. Introduction to Plant Biochemistry, 1983. T.W. Goodwin and E.I. Mercer (Eds.), Second end,, Pergmon Preu, UK, P~Mekaran,P., Swaminathan, K.R. and Krishanavani, S., 1986. Pro¢. Nat. Solar Energy Convention, Madurai, India, Sept. 12-15. Hunter,N.R., Geuer, H.D., Morton, L.A. and Prakaah, C.B., 1984. Pro¢. 4th int. Syrup. on A|cohoi Fuels, Ottawa. Sharma,D.K., Personal Communication. Sharma,D,K,, Unpublished Results.
111
Elsevier Science Publishers B.a./Pergamon Press pie - - Printed in The Netherlands
"Biocrude" and "Biomethane" from Croton
bonplandianum D.K. SHARMA* and H.A. MBISE**
Centre of Energy Studies, Indian Institute of Technology, New Delhi 110016 (India) (Received September 4,1987; accepted in revised form April 13, 1988)
ABSTRACT Croton bonplandianum, a latex bearingplant, rich in i~t,ogen,phosphorousand potsuium (NP-K), and whichgrowswild,yieldsgoodquantities (12-30~,) of"biocrude" (definedin the text). This biocrudecan be "hydrocracked"to petroleumoil T! e seedsof this plant yield39% oil which can be a diesel oil substitute. The spent residue after extraction of biocrude can be used for the generation of methane ("biomethane") by anaerobic digestion.The spent residuehas been found to givegoodyields (between4.74-5.66L/ks/day) of biogaswith methane contents from66-72%. The spent residual plant material obtained after digestion, contains enhanced N-P-K contents. This can be utilizedas a fertilizer.
INTXODUCTION Recent declines in oil prices have provided a grace period to search for future sources of oil and natural gas. World reserves of oil and natural gas are limited and these are being depleted fast. No doubt the recent price decline has encouraged their consumption, diminishing energy conservation measures. There is a need to find a nondepletable source of oil and natural gas. Biomass is such a renewable source of ener~. L~.iciferous and resinous plants [ 1-9 ], like heavea and guaule (the rubber tloes), yield "biocrude" by tapping the plant for latex or resin or, alternatively, by solvent extraction. "Biocrude" contains a mixture of hydrocarbons, terpenes, fats, lipi&, steroids, flavonoids, waxes and polyphenols. This mixture (defined here as biocrude) can be "hydrocracked" to get petroleum oil. The main advantage of latex bearing and resinous plants is that they grow wild without much agricultural input or management, they can be grown in arid or semiarid zones, and they do not compete wil.~ food and fibre crops. Rather, in the long run, these would help in the development of these desert areas by generating employment. Laticiferous and resinous plants can be used for the production of both oil *Authorto whomall correspondenceshouldbe addressed. **Presentaddress: Ministry of Energyand Minerals, P.O. Box 2000,Dar-ES-Sa¼m,Tanzania. 0921-3449/88/$03.50
© 1988 Elsevier Science Publishers B.V./Perpmon Press pie
112
and substitute natural gas (methane). After extraction of biocrude from these plants, the spent residue, which is rich in cellulosic material, can be u~ed for the generation of biogas ( ~ t i t u t e natural ~ ) . In the present work, studies have been ~ out on the Croton b o ~ z h k m ~ m (Euphorbiaceae)a ~ d , latex beari~ weed having a fast growth rate. Its growth is an environmental m e ~ and problem in India and these plants have to be removed to clear land. Its utilization for the production of oil and biomethane would help eliminate an environmental hazard. Biomethane can also be converted to methanol throuzh chemical or biochemical routes. The seeds of Croton bonpfand~anum contain about $9% oil It0]. The seeds also contain 3.5% ash which contains K20 (1.4.$%) and PROs (29.8%). This seed oil can be used as a diesel substitute and the spent r e , due obtained after extraction of oil can be compested. EXPBRIMZNTAL
Extraction o( biocr~te Croton b o ~ ~ (C.b.) stems and leaves were crushed and powdered to 40-60 BSS mesh size and dried in a solar drier. Dried leaves or stems (10 g ) were Soxhlet extracted with acetone. The spent residue was Soxhlet extracted with benzene. Similarly, dried C.b. stems or leaves were extracted with methanol followed by hexans, The portion extractable was calculated from the loss in weight of the C,b. items or leaves altar each extraction. At a larpr scale, C,b, leaves or stems (70 g) were taken in a batch extractor (i,e, S L round bottom flask fitted with a reflux condenser) under reflux conditions with methanol C/S0 mL) followed by the extraction of spent residue with hexans under similar conditions. The spent residues were taken as substrates for bioi~ production.
B/oeu product/on Substrates (50 g) were taken each in a one litre digester bottle attached to others (Fig, I), Water (700 mL) was added to each d i p m r , leaving some space for gas formation. Digestsrs were inoculated with cowdun8 inoculum (I00 mL), All the dilpmters were incubated at constant temperature (35-37 °C ). Gas samples were withdrawn daily and analysed for methane and carbon dioxide, usins a 8700 Nucon gas chromatograph. RI~ULTS AND DISCUSSION
Table I shows the chemical composition of the various components, i.e., leaves, stemsend roots of C.b. [ 11]. Roots contained the minimum moisture
113
oAs COLLECTOR I
oossToe
Fig. 1. Experimental set up for one set (out of six) used for the generation of bioges, TABLE 1 Chemical composition of biomems* Co~n~osition of Croton bonplandienum (C.b.) (%)
Moisture Ash Volatil,~ matter (% on dry.basis)
Leaves
Stems
Roots
71 1 76
70 1 79.
43 1.5 65
Cher,fical composition of C.b. (%) Leaves
Cellulose Hemicellulose Lignin Extractive3
Unextracted
Extracted residue
Uneztracted
Extracted residue
49.0 19.8 19.5 19.0
50.0 9.2.0 28.0
46.0 22.5 20.5 12.0
52.0 25.0 23.0
Chemical composition of bagasse (%) Moisture Cellulose HemicelluloN Ligain *Ref.
[11].
Stems
8,0 49.5 28.5 15.0 6.0
114
(43%) and leaves and stems contained about 70%. The volatile matter in C.b. were 65-76% and about 1% ash. The plant components were subjected to acetone extraction, followed by benzene extraction of the spent residue, Table 2 shows the results. Acetone is expected to extract the polar components such as polyphenols, chlorophyll, etc., whereas, benzene extracts mostly the nonpolar and less polar chemical components, such as, terpenoids, steroids and hydrocarbons, There were more acetone extractives (AE) than the benzene extractivee (BE). The total AE + BE represented the biocrude yields. The yield of biocrude from the leaves was more than that from the stems. Leaves yielded 20% bioerude, whereas stems yielded only 8.1%. In fact, leaves contain more latex than the stems and that could be the reason for higher biocrude yields. The solvent for extraction ofbiocrude from C.b. was changed tp methanol followedby the hexane for the residue. Methanol extracts the pokr components whereas, non-polar components are extracted in hexane. This solvent system provides a better fractionation between polar and nonpolar extractivee, as the difference between the polarities of methanol and hexane is more than that between acetone and benzene. In fact, the methanol extractivee (ME) were found to be more than the AE. Overall, biocrude yield through the methanol-hexane solvent system was more than that through acetone-benzene system (Table 2). Leaves yielded about 30% biocrude through methanol-hexane extraction, which was more than that (I4%) obtained through acetone-benzene extraction, Stems yielded 9% biocrude through acetone-benzene solvent system and 12% through methanol-hexane solvent system. Table 3 shows the biocrude yield from some of the promising latex bearing plants. It can be seen that C.b. (latex bearing plant) yields 18,2% biocrude which is the maximum amongst the potential petrocrops taken for comparison. Only Pergularia e~cnsa yields biocrude equivalent to this, The use of solar concentrators can be made for heating the extractors. The use of solar energy for heating these reactors has been demonstrated in our laboratory [ 11 ] and can reduce the processing cost by 20%. Table I shows that C,b, leaves and stems contain 42% and 46% cellulose and TABLE 2
Sompk
Moisture contents (%)
Extraction (% on dry basis ) !
Acetone
Benzene
Total
Methanol
Hexane
Total
14,0 9.1 i0.0
20,0 8.1 12.2
10.2 3.5 6.0
30.2 11.6 18.2
qs
Leav~ Ste~ W~de plant
71 70 66
1~,0 8,0 9.0
2,0 1,1 1.0
115 TABLE 3 Comparison of biocrude yields of C.b. and of some potential petro-crops Species
Croton bonplandianum Colotropis gigantea Calotropis procera Hemidesmas indicus Pergularia eztensa Euphorbia neriifolia Pedilanthus tithymaloides
Moisture
70.0 78.0 53.0 45.0 89.0 87.0 83.0
Extraction (%) on dry basis Acetone
Benzene Total
Methanol
Hexane
Total
9.0 7.8 7.5 5.3 8.0 10.6 7.5
1.0 0.7 0.6 0.6 0.7 0.6 0.6
12.2 5.1 5.2 9.0 14.1 7.0 7.0
6.0 2.8 1.9 3.0 4.0 6.0 3.0
18.2 7.9 7.1 12,0 18.1 13.0 10.0
10.0 8.5 8.1 5.9 8.7 11.2 8.1
19.5% and 22.5% hemicellulose, respectively. The percentage of these cellulosic contents is obviously more in the spent residue obtained after biocrude extraction than these in the original C.b. Thus, these could be good substrates for biogas generation, where cellulose, hemiceUulose, fats and proteins can be converted biochemically, through methanogenic bacteria, to methane and carbon dioxide. Common materials used for biogas generation are: animal manure, agrowastes, industrial wastes and night soil. Weeds have a comparatively favorable C/N ratio for biogasification. Thus, these are quite suitable for methane generation [12 ]. In the present work, the use of various components of Croton bonplandianum (weed) before and after extraction of biocrudehas been made. The following substrates were taken: extracted leaves (EL), dried unextracted leaves (DUL), dried extracted stems (DES), unextracted stem (UES), a mixture of extracted stem and bagasse (1:1) (SB) and unextracted wet leaves (UWL). Cowdung inoculum (100 mL or 5% W/V in an IL digester) was used for the anaerobic digestion of these substrates. A group of microorganisms act on the organic substrates to convert the biopolymeric materials (cellulose, hemicellulose, fat, etc.) into soluble monomers, which become substrates for the microorganisms in the second stage, in which the soluble organic compounds are converted to organic acids. These organic acids are then converted to methane and carbon dioxide by the methanogenic bacteria. Table 4 shows the cumulative volume of biogas generation per day, from 3 to 42 days of digestion (of 50 g biomass substrate). There was no significant biog~s productiozl during the first two days. Figure 2 shows the total gas production (mL) at different digestion times (days). Minimum biogas generation was from the unextracted wet leaves (UWL) (2725) mL) as these contained minimum hemicellulosic mid cellulosic contents because of high moisture contents (Table 1). Unextrncted leaves produced more biogas than the extracted leaves upto 40 days. But the biogas production after 42 days was more in the
116 TABLE 4
Cumubtivevolumeofbio~s~
(mL) ( f m m 3 / 1 2 t 8 6 t o 13/1/67)
Dilpmer containing ( m L )
2
1
3
C.bJ~ves U,ntmmd C . ~ C.b.mm U n n t m m d C.b.mm UnHm~M'C~b,mm+ba~Ne
------
------
Unntmct~
--
--
b~h C.b,bam
1~
14
7
5
70
250
100
300 400
800 700 MO
850 '725 650
180 300 100
87~
650
9~
400 250
525 88O
600 480
15
16
17
19
19
--
--
200
.6
9
10
1175 1200 1600 2350 1600 1150
1275 1475 1725 1400 1800 2150 2200 3050 3400 285033603800 2060 2700 33OO 1250 1400 1450
20
21
22
4175 5480 5775 6075 848O 1975
48?6 4678 6100 6125 6475 68?8 7278 6150 6450
--' ---
2125
--
32
33
8
4
950 IlOO 900 IlOO 800 IlOO 1400
1850
850 IlOO 780 1500
U
23
12
24
~sns.
Ext~cted C,b,l~v~ Ext~
1925
C.~mm
8800
8880
Unexmcted C,b,m
4050
44?5
Umxtm~tedC.b.mm+b~lm~ Unex~h~hC.b.lum
3650
4000
1500
1525
25 ~xtm~tedC.b~m U n n t m c t ~ l C,b, b a v n ~ t r a c t e d C,b,stem Unextmcted C,b,#em
UnexLn~c~l C , b , ~ m + ~ Unox~ ~ C.hJ~vm
-"
-"
---
----
--
34 marinated C.~iRvm UnexU~ctMIC.b.luvH ~ t m ~ t K I C.~ttem UnRtqwt~l C.b.mm UnR~t~/C.b.~tm+b~me U~xtm~ted bmh C,I~IRm
35
-------
8178 8600 4278 5175 4680 1628
4100 4400
o
i
5575
--
--
1725
--
--
~7
28
29
6175 7480 63~ 8800 ?800
6475 7600 86~ 8600 7780
6675 6975 71 ?800 8000 ~OO 8878 9 1 ~ 9225 8 8 0 0 9 0 0 0 9200
--*
--*
--
--
?900 8050
8250
--
--
2400
~
36
~i
38
40
§175 83?8 8 / 0 0 8800 100115 10175 10575 10578 9950 10150 10184) 10300 8600 8750 8900 9080
30
39
31
41
3335
5175 6300 69?5 7680 7000 2725
42
???S
79?5
--
--
--
9125
9475
8600
8684)
--
--
--
9150
9150
'
'
*
--
--
--
--
--
--
11075 11325 10850 11150 9 8 8 0 9880
' R R d l n p not takem
case of extracted loaves (9475 mL) than that from unoxtracted leaves (9150 mL). This showed that the biops generation from leaves is reduced after extraction of biocrude. Fatty components are removed from the leaves during biocrude extraction, which result in the initial dropping of biogas yields up to 40 days, but after 40 days, the biogas generation was more in the extracted leaves. This is thought due to the digestion of cellulose becoming predominant through solubiUsation and microbial degradation in the prolonged process of biogas generation. Therefore, after 40 days the gas yield from extracted leaves containing comparatively more cellulosic contents (Table I) is enhanced. Stems showed more b i o ~ production tha~i leaves, which could be due to the fact that stems contain more cellulosic and hemicellulosic contents than
117 x 103 14
. . . . ............ ......... ..I
O 1: Extracted CB leaves O 2: Unextrocted CB leaves D 3: Extracted CO stem D ¢ : Unextracted CB stem O S: Unextracted CB stem • bagasse D 6: Unextracted fresh CB leaves
~w
w
iO
Average fermentation temp: 35-37 'C Total solids: 7 % Cow clung inoculum: S %
5
~
a
W ij
6
Q, J 0
~--~ 0
I 6
i
I 16
i
I 2~
i
I
,
)2
I &0
REI'ENTIOH Tilde (days)
Fig. 2. Cumulative volume of biogas production at different retention times.
leaves (Table 1). Biogas generation from extracted stems (11335 mL) was more than that from unextracted stems (11150 mL). Statistical analyses of the data obtained on the biogas generation from different substrates was done and the conclusions drawn from the data were confirmed [11]. This showed that biocrude extraction was a beneficial pretreatment for biogas generation. There could be two reasons for this. One could be that extraction breaks the plant cells by extracting the cytoplasmic fluid, i.e. latex extractives. Action of solvents (hexane and methanol) under reflux conditions breaks the cells to extract the biocrude from cytoplasm. In fact, latex is accumulated mostly in specialized cells or vessels known as lactifere [18]. In stems, latex is stored in the rings of lactifers in the bark. Extraction of latex (biocrude) from the stem (bark) exposes the hemicellulose, cellulose and lignin. As cytoplasmic fluid (latex) is removed by solvents from plant tissues, cells collapse, and cell wall components, such as, cellulose, lignin, hemicelhlose and proteins, are disrupted and their association is disintegrated. Moreover, extraction increases the surface area and porosity of the biomass, alJowing the better accessibility for bacteria and methanogenic bacterial enzymes. Another reason could be that biocrude extraction increases the percentage of cellulosic contents (Table 1 ) in the spent residue. Further work on finding these reasons is currently underway. Table 5 shows the overall average of biogas production rate, expressed as
118 TABLE 5 Ovendl averqe of biops production rate in a one litre disester Disesterc~with:
DI: F,xtracted C.b. leavn D~: Unextracted C.b. l~veJ D~: ~tmcted C.b. item D4: Unextlm~C.b,e~m D~: Unez~ct, dC.b. item+~ D6: Unextt~cted fresh C.t~ IMvu
Dif~tion Totaivolume mL/h m L / d ~ L/ks/d~v (Bmedon time (days) (mL) (Based onS0g lkS sub#rate subltrate) 40 40 40 40 40
9475 91S0 11825 11150 10880
35
2725
9.87 9.53 I!.79 11.61 11.33
236.90 228.70 282,96 278.64 271.96
4.74 4.57 5.66 5.57 5.44
3,55 85.20 1.70
mLlh, mL/day (based on 50 g of biomass) and L/kg/day. Average hourly, daily or by weight daily rates were maximum in the extra©ted stems of C.b.: 11.79 mL/h, 282.96 mL/day, 5.66 L/kg/day, respectively. This was followed by the rate for unextra©ted stem of C.b. The biogas production rate for the mixture of bagasse and extracted C.b. was less than that for the unextra©ted stem,
Rutes of biogas generation from leaves were less than those from stems. Minimum rates could be seen from wet leaves (3.55 mL/h, 85.20 mL/day and 1.70 L/kg/day) and the biosas generation rate for extracted leaves was more than that for the unextracted leaves. This follows from the same reasoning as given earlier for blobs generation. These studies showed that extraction of biocruds renders the spent residue more susceptible to anaerobic digestion. The mixture of bagasse and extracted C.b. stems showed less biogas yields (10880 mL) than the extracted (11325 mL) or unextracted C.b. (11150 mL). Table 1 shows the composition of bagasse. Bagasse was added on the premise that this would be digested smoothly and thus, would help in the digestion of Croton bonp/endianum, but this did not happen. It seems C.b. is better substrate for biops generation than bagasse. In fact, extracted C.b. stem contains more holocellulosethan bagasse, though bagasse contained more of these than the unextracted C.b. stem. Moreover, C/N ratio in weed is 26:1, which is optimum ratio for biopsification by anaerobic digestion (using methanogenic bacteria) [ 12 ]. This could be the reason for the better oiu~tLicatlon results from C.b. stems. A mixture of latex bearing plant, such as Euphorbia tiruealli Linn. with cowdung (1/1 W/W), has been reported [ 14 ] to give higher output of total gas by anaerobic digestion than that from cowdung alone. Even more methanogenic activity was observed in the digestion where a mixture of Eu-
119
phorbia tirucaUi and cowdung was used as compared to that from cowdung alone. The yields of biogas from extracted and unextracted C.b. stem was found to be more than that from agrowastes and was comparable to that from cowdung [ 11]. Further work on the studies of biogas generation using mixtures of Croton bonplandianum, water hyacinth and bagasse is being extended. Figure 3 shows the methane contents in the biogas per week. Methane contents from all the substrates studied were found to increase with increase in the digestion time. Maximum methane contents (72%) were found in the extracted stem of C.b., followed by 70% from extracted leaves of C,b. Unextracted stem of C.b. showed the minimum methane contents of 62%, which were less than those from the mixture of extracted stem of C.b. and bagasse (66%). Wet leaves showed a greater methane content (68%) than those in the weekly biogas from unextracted leaves. Statistical analyses of the data obtained were done [ 11 ] and supported the above results. The mixture of extracted C.b. stem
. . . . . . . . .
OI : Extracted CB kczves 0 2 : Unextracted C8 leaves O3 : Extracted CB stem ----..---Or, • Unextracted CB stem . . . . . . . . . . . . . OS : Unextractecl CB stem. bagasse - - - - - - - - - - O6 : Unextracted fresh CB leaves
20r
\
1
i/ /
i
\ I
,\
\
I
#
g~
!
I
I
I
I
2
3
4
S
WEEK NUMBER
6
120 70
60
SO
~0 .|
f }0
I
x O! Etl~Jcted CO leaves
o O~ Ue,ettmc~ted CBINveJ 0 0]1 Eelmcted CO s l ~ I O~ tNxlm¢led CB Item • OS U~tncl~l CB shm.l~gaue • D6 Ue~tmct~ fre~ CB leaves
|0
I
I
I
I
I
I
DAYS
Fig,3, (a), Weeklybiogasproduction:(b), Methanecontentsin blobs. and bas~se showed hiilher methane contents in the biogas than those in the biolla8 from extracted or unextracted C.b. stem in the first 4 weeks. This showed some advantap of usin8 basasse along with C.b. stem, i.e. the use of bagasse enhances methane yields in the first 4 weeks. These studies are being extended further. Althoush the b i o ~ yield from leaves was less than those from the stem of C.b., still the methane contents in biops were more from the leaves, especially in the first four weeks. This could be due to the fact that the cellulose in the lignocellulosicbiomu8 of leaves has loose lignin-~ellulose chemical bonds and is easily solub'dized and the action of methanopnic bacteria is faster in leaves which are softer than stems. Above studies had shown that extracted C.b. stem 8ive8 the maximum biosas yields. Table 6 shows the methane contents in biops pnerated every week from the extracted C.b. stem. The methane contents increased from 2'7% in the first week to 72% in the sixth week. The methane percentap did not show maxima or minima. Rather it showed all increase with an increase in dipstion time. This is due to the fact that as the dipmtion time incumses, the reaction of methanogenic bacteria also increases, to convert more and more of the acids to methane. Also, more COs is
121 TABLE 6 Relationship between wee,kilt c u ~ extracted C.b. stem
~, ,~ ;ota! volume of biogas and methane content (%) from
Week no.
Total volume (Litres)
Methane (%)
1 2 3 4 5 6
0.800 3.050 2.625 2.400 11.275 0.225
27 46 54.8 58.4 69.3 72.0
converted to CH, over an extended incubation period, thus reducing the CO~ concentration and increasing the CH4. The spent residue of C.b. obtained after the biogesification was found to be enriched in potash, nitrogen and phosphorus, because of three reasons: (1) extraction of biocrude raises the N-P-K contents in the spent residue; (2) conversion of cellulose through biogasification also raises the N-P-K contents in the spent residue; and (3) the original C.b. is rich in N-P-K which makes it suitable for composting. Thus, the spent residue of C.b. obtained after biogasification can be utilized as fertiliser/soil conditioner, with or without chemical fertilizers. CONCLUSION
The studies reported here show that about 12-30% yields of biocruds can be obtained from the leaves and stems of Croton bonp~m~mnum, along with the extraction of 39% of diesel fuel substitute from its seeds. Therefore, Croton bonp~m~i~num appear~ to be a potential candidate for "petrofarming". The biocrude obtained can be hydrocracked to get oil. The spent residue obtained from the extraction of biocrude from C.b. can be utilized for the generation of biogas. The methane content in the biogas is between 66-72% and can be used as a substitute natural gas. Methane obtained can be converted to methanol under high temperature ( 400-600 ° C ) and high pressure (5-10 MPa ) with copper or nickel catalysts [ 15 ]. Methane can also be converted to methanol through biochemical routes under milder conditions by inhibiting the methanol dehydrogenase enzyme by using iodoacetic acid in the anaerobic digestion process [ 16,17 ]. Therefore, petrocrops could form an alternate and renewable source of oil, natural gas and methanol. Since the extracted stems produced more bioges than the unextracted stems, this indicates that extracted ~tems are better substrates for biogas generation. Further work is being undertaken to study anaerobic digestion of latex alone.
122 In some countries latex bearing plants are being commercially used for biogas generation [ 11 ]. It would be worthwhile to recover biocrude before charging these to digesters for biogas generation. ACKNOWLEDGEMENTS This work was carried out as project work of Mr. H.A. Mbise, under the United Nations University Programme in Renewable Energy Systems at the Centre of Energy Studies, Indian Institute of Technology, New Delhi, 110 016, India.
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Calvin,M., 1977. Energy Res., 1: 290. Hillen,L.W., Wake, L.V. and Warren, D.R., 1980.Fuel, 59: 446. Hall,D.O., 1982. Experientia, 38: 3. Bhatia, V.K., Srivastava, G.S., Garg, V.K., Gupta, V.K., Rawat, S.S. and Singh, J., 1983. Fuel, 62: 953. Erdman,M.D. and Erdman, B.A., 1981. Economic Botany, 35: 467. Sharma,D.K. and Babu, C.R., 1984. Fuel Sci. & Technol., 3: 49. Sharma,D.K. and Prasad, R., 1986. BiomMs, 11: 75. Srivastava,G.S., 1986. Proc. Nat. Workshop on Petrocrops, New Delhi. Bhatia,V.K., 1986. Proc. Nat. Workshopon Petrocrops, New Delhi. The Wealth of India, 1950. Paw Materials, Vol. 11, Published by C.S.I.R., New Delhi. Mbise,H.A., 1987. Petroleum Extraction U~ing Solar Energy and Production of Biogas or Alcohol t~m Petrocrops. Project Report of United Nations University Programme on Renewable Sources of Energy, Submitted in liT, Delhi, February. Guide Book on Btogas Development, 1980. Energy Resources Development Series No. 21 United Nations, New York. Introduction to Plant Biochemistry, 1983. T.W. Goodwin and E.I. Mercer (Eds.), Second end,, Pergmon Preu, UK, P~Mekaran,P., Swaminathan, K.R. and Krishanavani, S., 1986. Pro¢. Nat. Solar Energy Convention, Madurai, India, Sept. 12-15. Hunter,N.R., Geuer, H.D., Morton, L.A. and Prakaah, C.B., 1984. Pro¢. 4th int. Syrup. on A|cohoi Fuels, Ottawa. Sharma,D.K., Personal Communication. Sharma,D,K,, Unpublished Results.