Fractionation of organic substances during anaerobic digestion of farm wastes for biogas generation
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M1RCEN Journal, 1989, 5, 27-42
Fractionation of organic substances during anaerobic digestion of farm wastes for biogas generation
M,M. EI-Shinnawi*, B.S. EI-Tahawy, S.A. El-Shimi & Soheir S. Fahmy Department of Soil Science, Menufiya University, Shibin Elkom and Soils and Water Research Institute, Giza, Egypt
Received as revised 6 July 1988; accepted 28 July 1988
Present day anaerobic digestion processes yielding methane have been established by allowing events which occur in nature, e.g. in sediments, the rumen, and other anaerobic environments, to take place within designed containers. This is to make efficient use of combustible gas evolved as an accessible and cheap source of energy, especially in rural areas. By fermentation of organic matter, the farmer can simultaneously satisfy his needs for fuel and good quality manure from one source, i.e. the farm wastes, besides controlling environmental pollution.
The main digestible components of solid wastes are carbohydrates (cellulose and hemicellulose), proteins and fats. Although these components in themselves are easily digested, they can be present in wastes in such a structural form that makes their availability for biodegradation difficult. This is the case for coagulated and fibrous protein, cellose, and hemicellulose incorporated in lignic complex (Hobson et al. 1974). Rate of methanogenesis depends on the status, type and constituents of the organic matter undergoing anaerobic digestion (Sathianathan 1975; Hobson et al. 1981).
Fermentation of mixed waste materials has proved to be more effective in biogas production than that of a particular material. For instance, Park (1979) showed a synergistic effect on the gas yield when different materials were mixed with each other, e.g. sewage waste on its own gave 0.265 m 3 biogas/kg volatile solids added and
* To whom correspondence should be addressed.
9 Oxford University Press 1989
28 Biogas from farm wastes
weeds alone produced 0.277m3/kg volatile solids fed, but their mixture (50:50) produced 0.387 m3/kg volatile solids added, with an increase of 39%.
Our work here aims to study the biochemical changes of major constituents brought about in mixtures of cow dung with crop residues during the fermentation process for biogas production.
Methods and Materials Materials
Cow dung, rice straw, maize stalks and cotton stalks were employed as substrates. The crop residues were air dried and pulverized, but cow dung was used in its fresh wet status (78% moisture, on the basis of drying at 70~ for 24 h).
Laboratory techniques Treatments including fresh cow dung alone and in combinations with each of the air- dried crop residues were performed. In the animal/plant waste combinations, the ratio 1:1, on the 70~ dry weight basis, was applied. Initial analysis of those mixtures is shown in Table 1. CaCO3 (20 g) was added to each treatment (to serve as a buffer). The ingredients were thoroughly mixed and introduced into 4-1itre laboratory biogas fermenters (Fig. 1). Tap water was added to bring both the total solids concentration to about 8% (Sathianathan 1975) and the working volume to 2 litres. The C/N ratio of the feedstocks was left unmodified. All treatments were run in duplicates; thus 40 fermenters were allocated to satisfy five-interval estimations. The fermenters were then sealed and incubated at 35 ~
For gas sampling---l]-
Polyethylene tube I I
1 2 3 Fermenter Water trap for Receiving excess
gas collection water
Fig. 1 Schematic diagram of the laboratory biogas digestion Unit.
Assays. Measurement of biogas yield and its content of methane was carried out every 2 days as follows:
s of t
30 Biogas from farm wastes
1. biogas, by means of displacement technique, using acidified water (2% H2SO 4) to prevent the solubilization of CO2 (Maramba 1978);
2. methane, by gas-liquid chromatography (Wujick & Jewell 1980), using a flame ionization detector. Gas samples were withdrawn by a 50-ml gas-tight syringe and 0.5 ml injected into a gas chromatograph fitted with stainless-steel column (120 cm 0.2m) packed with 5% (w/w) OV-101 on Chrom-PAW80-100 mesh. Nitrogen, as a carrier gas, was at 28 ml/min. The column oven was at 75~ the injection port at 100~ and the detector at 150~ Results were calculated referring to periodically-made calibration curves of pure methane.
Chemical analysis. 1. Total solids (TS) in the slurry samples dried for 24h at 70~ according to the
recommendations of the American Public Health Association (APHA 1976). 2. Volatile solids (VS) by burning the 70~ samples at 650~ to constant weight
(APHA 1976). 3. Total volatile fatty acids (VFAs), by steam-distillation of the slurry effluent
acidified with HzSO 4 to pH 1, then back-titration with NaOH (Neish 1952). 4. Individual VFAs by gas-liquid chromatography (Wujick & Jewell 1980). Effluent
of liquid slurry was acidified with H3PO 4 and left to stand for 60 min at 4~ then centrifuged at 1000 rev/min for 10 min. The supernatant was acidified to pH 1 and centrifuged at the same speed for 5 min. A sample (5 ~xl) was injected into the gas chromatograph (specifications are described above) running at 160~ for column oven and 210~ for each of injection port and detector. Results were calculated referring to periodically-made calibration curves of pure major fatty acids.
5. Analyses carried out according to the methods given by Chapman & Pratt (1961): In the liquid slurry: NH4~-N, by micro-steam distillation in alkaline medium.
In the slurry dried at 70~ for 24h: organic carbon by the method of Walkely & Black using H2SO 4 + K2Cr207 and total N by the Kjeldahl method.
6. Analyses for the major organic constituents, performed following the methods recommended by Kononova (1966): Water-soluble substances by loss of weight of the 70~ samples subjected to addition of distilled water and filtration three times.
Crude protein by multiplying the difference between the total N and NH~--N contents by 6.25. Fats, by extracting the 70~ samples using ethanol-benzene mixture (1:1 v/v) for 12-20 h in a Soxhlet apparatus. Carbohydrate fractions: Hemicelluloses, by hydrolyzing the residue remaining after removal of water-soluble substances using 0.3M HC1 for 3h. Reducing sugars were then determined in the hydrolysate by phenol-sulphuric acid technique. Data resulting were multiplied by 0.9 to give the hemicellulose content. Cellulose, by hydrolyzing the reesidue as before using 14 ra HzSO 4 for 2.5 h. The mixture was then diluted with distilled water and left to stand for 5 h. Reducing sugars were measured in the hydrolysate and their data were multiplied by 0.9 to give the cellulose content. Lignin, by drying the residue, as for cellulose to a constant weight at 105~ then ashed in a muffle furnace at 550~ Loss of weight represented the lignin content.
M. M. El-Shinnawi, B. S. El-Tahawy, S. A. El-Shimi & Soheir S. Fahmy 31
Results and discussion
Biogas and its methane component Production of biogas and its methane component measured every 2 days during the biomethanation of cow dung and its mixtures with crop residues is illustrated in Fig. 2. Evolution of gas showed fluctuating levels during the experimental duration. Appreciable amounts of gas were produced from days 7 to 35 of incubation. The highest biogas peak was detected for cow dung + maize stalks (CD+M) (1.61itres/ litre over 2 days) and followed in descending order by cow dung + rice straw (CD+R), cow dung + cotton stalks (CD+C) and cow dung (CD) alone. However, peaks of methane content mostly did not follow those of biogas. Flammable gas started after 6-9 days of incubation, depending on the type of biomass.
Cumulative production of each of biogas and methane through the fermentation course of the various feedstocks is shown in Fig. 3. Top total volumes of both gases were gained by CD+M which produced 171itres biogas and 81itres methane/litre fermented matter, whereas CD+R, CD+C, and CD alone followed, respectively.
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1.0 J 0.8
(a) I- (b)
i I ."
~ii\.%.; 9 "...
.! I I I
I I [ " I [ 15 30 45 60 75 0 15 30 45 60 75
Fermentation time (days)
Fig. 2 Production of biogas and its methane component during fermentation of cow dung and its mixtures with crop residues. (a) cow dung, (b), cow dung + rice straw, (c) cow dung + maize stalks, (d) cow dung + cotton stalks. - - Biogas, -... methane.
32 Biogas from farm wastes
== t -
"O o o. 6
0 ~..-~... v - 15
9 / . . j , . . .~ . . . .A . - . .~- . .A . . .~- .~ i
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.,,~" .~::" +0" / '
.~ . . "
I I I I 30 45 60 75
Fermentation time (days)
Fig. 3 Cumulative biogas and methane production during fermentation of cow dung and its mixtures with crop residues. Biogas, .-.. methane. O, cow dung; x, cow dung + rice straw; A, cow dung + maize stalks, 9 cow dung + cotton stalks.
The above results are attributed to the type of raw biomass. For instance, molecular composition of the constituents of the plant materials undergoing digestion is of significance, e.g. the high lignin content of cotton stalks (Table 1) retards its biodegradation. The higher content of water-soluble substances and the lower C/N ratio of maize stalks makes them superior to rice straw. Moreover, the presence of growth-promoting substances, most probably in plant materials, enhances the microbial population - - thus mixing the cow dung with crop residues is beneficial for methanogenesis (Chengdu 1979; Hobson et al. 1981; Han 1982).
Total and volatile solids Contents of both TS and VS of the digesting materials decreased with time (Fig. 4). Such decreases are due to the bioconversion of the organic substances into gases (CH4 and CO2 ) and water. The extent of disappearance for both solids followed the order: CD+M>CD+R>CD+C>CD. Sharp depressions occurred within the first month
M. M. El-Shinnawi, B. S. EI-Tahawy, S. A. El-Shimi & Soheir S. Fahmy 33
~ 65 g g ~ 6O
~ 55 P, o
- \ \
\ "~ .~..o-. . . . . .
~k,~ '~'x~ - ~ ~' ,o -x
I I I I I 15 30 45 60 75
Fermentation time (days)
Fig. 4 Changes of total and volatile solids during fermentation of cow dung and its mixtures with crop residues. Total solids, ---- volatile solids. e, cow dung; x, cow dung + rice straw; A, cow dung + maize stalks, 9 cow dung + cotton stalks.
and thereafter the rate of solids diminution slowed down. This correlated with the changes in the bacterial growth dynamics, which bloomed at the early stages but then steadily tended to decline by advancing the process duration as a result of nutritional exhaustion and/or accumulation of by-products.
Production rates of biogas and its methane component, calculated at the end of the digestion course of the various feedstock materials, are listed in Table 2. Figures of biogas indicated that CD+R gave the highest rate for VS added (2891itres/kg) and followed by CD+M, CD+C, and CD, respectively, whereas CD+C attained the maximum level for VS consumed (l1791itres/kg); CD+R, CD+M and CD were successively lower. CD+R was the highest producer of methane for VS added (1371itres/kg); CD+M, CD and CD+C followed descendingly, whilst CD surpassed all of the tested biomass in relation to the VS consumed and gave 636 litres/kg and
s of fe
t 75 d
te of p
M. M. El-Shinnawi, B. S. El-Tahawy, S. A. El-Shimi & Soheir S. Fahmy 35
followed by CD+M, CD+R and CD+C, respectively. Consequently, mixing the cow dung with the crop residues increased the amounts of biogas produced but decreased the methane content on the basis of VS consumed. This is attributed to the high cellulose content of the plant materials (National Academy of Sciences, 1981). The overall loss of VS ranged between 18 and 25%.
Again, the nature of feedstocks governed the rate of disappearance of VS in the digesters. The C/N ratio, contents of both water-soluble substances and lignin, and initial VS content appeared to determine the extent of breakdown of the organic matter, as mentioned above. The lower C/N ratio of cow dung might contribute to the high rate of methane generated (for VS consumed), according to Hill (1979) who noted that the percentage of methane gas increased with decreasing C/N ratio of the feedstocks.
Volatile fatty acids Total VFAs. The contents of total VFAs was increased within the first 15 days of incubation, and thereafter was severely diminished (Fig. 5). CD exhibited the greatest VFA formation throughout, while CD + R, CD + C and CD +M followed, respectively. Amounts of VFAs formed by CD during the first 2 weeks, reached 93 mEq/litre).
t~ E 7O tD3 C
~ 50 W
E ~ 40 i f ) < l.J_
- - ao
I I I I I o 15 30 45 60 75
Fermentation time, days
F ig . 5 Changes of total volatile fatty acids during fermentation of cow dung and its mixtures with crop residues, e, cow dung; x, cow dung + rice straw; A, cow dung + maize stalks, 9 cow dung + cotton stalks.
36 Biogas from farm wastes
This value is above the limits reported by McCarty and McInerney (1961) (35 mEq/ litre), Hobson et al. (1975), (10 mEq/litre), Kroeker et al. (1979) (30-60 mEq/litre), and even Velsen & Lettinge (1979)(85 mEq/litre). This may explain the least gas amounts produced by CD (Fig. 3), as a result probably of lowered activity of methanogens.
Since CD is a rich source of active bacterial agents with their energy and nutritional requirements, as well as possessing high contents of protein and fats (Table 1), its digestion results in the generation of large amounts of VFAs by means of the acid- forming bacteria. Decline in VFAs occurring by increasing the incubation time is a result of their consumption by methane-producing bacteria.
Individual VFAs. Changes in the individual fatty acids liberated during the incubation period of the different fermenting feedstocks are illustrated in Fig. 6. It is clear that acetic acid was the prime short-chain fatty acid produced in all cases. Its maximum appeared on day 15 for all biomass tested. This is probably due to intensive bacterial action on long-chain fatty acids. CD gave the highest value for acetic acid production, followed by CD+M, CD+R, and CD+C, respectively. The highest amount of such acid detected was 4070mg/litre, with propionic and butyric acids occupying the
N 2100 E
~ 0 Cr~
E ~ 4200
g 3500 0
15 30 45 60 75
- (b )
0 15 30 45 60 75
Fermentation time, days
Fig. 6 Changes in individual fatty acids during fermentation of cow dung and its mixtures with crop residues. (a) cow dung, (b) cow dung + rice straw, (c) cow dung + maize stalks, (d) cow dung + cotton stalks.e, Acetic acid; x, propionic acid; A, butyric acid; 9 valeric acid; I , caproic acid.
M. M. El-Shinnawi, B. S. El-Tahawy, S. A. EI-Shimi & Soheir S. Fahmy 37
second-and third-most abundant VFAs. Valeric and caproic acids appeared in very low amounts in the plant/animal waste combinations only, which means that their formation in the digests is confined to the plant origin. After 45 days, butyric, valeric and caproic acids had mostly disappeared, but acetic and propionic remained detectable until the end of the experiment. Probably the initial amounts of acetic, propionic and butyric acids which were present in the plant/animal waste combinations were derived from the CD portion and consequently, the plant portion showed lower efficiency than the animal waste in the formation of the three major fatty acids. This accords with the earlier reports of Ghosh & Klass (1978) and Mclnerney & Bryant (1981).
Nitrogen forms Figure 7 shows the changes that took place for both ammoniacal nitrogen and total nitrogen during the fermentation course of the various feedstocks. Peaks of NH] -N content appeared at 15 days; thereafter the values declined. This trend is due to liberation of the mineral N form via putrefaction and deamination of the nitrogenous organic compounds during the first days of incubation, then consumption of such forms by the methane-forming bacteria in the latter period. In this regard, Bryant (1974) and Mclnerney & Bryant (1981) reported that ammonium serves as the main N source for methanogens. Quantities of NH] -N detected exhibited the order: CD>CD+C>CD+M>CD+R. Such a pattern mostly followed the initial C/N ratio of the biomass fed (Table 1). The complex composition of cotton stalks made them inferior to the maize stalks, despite the lower C/N ratio of the formers (Table 1). The greatest NH] -N level gained in the present study did not exceed 300mg/litre. This level is still safe. In this connection, McCarty & Mclnerney (1961) reported that concentrations of NH~--N between 50 and 200 mg/litre have a beneficial action on the anaerobic process, and that levels up to 1500mg/litre have no adverse effect.
The content of total nitrogen showed a slight diminution over time for CD, whereas the other treatments revealed inconsiderable decreases. Amounts of total N observed in Fig. 7 reflected the initial values of the different feed materials (Table 1), where: CD>CD+C>CD+M>CD+R. Decreases occurred are through N loss via volatiliz- ation of NH3, as well as to evolution of nitrogenous gases through denitrification of any nitrates probably present at feeding time. However, the overall N loss was about 8% for CD, whereas it ranged between 1 and 3% for the other treatments.
Organic fractions Water-soluble substances: Changes in the content of water-soluble substances of the various substrates exhibited fluctuating values with incubation time increasing and decreasing but with no consistent trend being noted. CD+M and CD+R attained the highest figures, followed by CD+C and CD, respectively (Fig. 8). It is recognized that the water-soluble organic substances comprise mono- and di-saccharides, simple fatty and amino acids, and some tannins. These compounds are subject to contradictory processes, namely, the production by the depolymerization of macromolecules on the one hand, and the consumption by the fermentative bacteria for energy and carbon demand, on the other. The balance between the two processes is controlled by the composition of biomass. For instance, the lower C/N ratio of CD (Table 1) resulted in
38 Biogas from farm wastes
- ! E 0
~ 1250 0 0
.1 .~ I I _ _~ L . __
750 ~, - ~ . ,
- - - - - - X
5oo ~ d'~--. L ~ ~ .} .~ L I
15 30 45 60 75 Fermantat ion t ime, days
~'ig. 7 Changes of ammoniacal nitrogen and total nitrogen eontertts during the fermentation course of cow dung and its mixtures with crop residues, o, cow dung; x, cow dung + rice straw; A, cow dung + maize stalks, 9 cow dung + cotton stalks.
a higher exhaustion rate of the water-soluble Substances, comparing to the other treatments. Likewise, the higher lignin content of CD +C resulted in lower production rate of such substances. Content of the water-soluble substances ranged between 11.5 and 15.2%
Protein: Protein content of the different biomass tested revealed no notable alterations throughout the experimental duration, except for CD treatment which resulted in a decrease of about 8% (Fig. 8). In regard to protein content the sequence
M. M. El-Shinnawi, B. S. El-Tahawy, S. A. EI-Shimi & Soheir S. Fahmy 39
l Water-sol. subs.
16~ . . ~
14 ~ ~
12 g ,
9 - Protein
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2 9 l 1
I I I I _ _L . . 15 30 45 60 75
27"51 Hemicellulose 22.5:1; ~ -
,"~ \ \ i12.5 t
Lignin 18 E - -~-o_~_o o o
16 A 9 ~
0 15 30 45 60 75 Fermentation time, days
Fig. 8 Changes of organic fractions during fermentation of cow dung and its mixtures with crop residues. t , cow dung; x, cow dung + rice straw; A, cow dung + maize stalks, 9 cow dung + cotton stalks.
of the fermenting materials reflected their original values (Table 1) where the order was: CD>CD+C>CD+M>CD+R.
The decrease recorded for CD protein is correlated with the N loss (Fig. 7). Otherwise, the stability of protein content of most treatments is due to the equilibrium between putrefaction of the original protein and the construction of bacterial protein. Moreover, incorporation of protein into complexes with plant substances, especially lignin, reduces its biodegradability (Waksman 1952).
Fats: The fat content of the fermenting materials showed slight decreases with the incubation time. CD produced the greatest decrease whereas the other combinations were close to each other (Fig. 2). The overall loss of fats in the digesters was 19% for CD and 45-48% for the other combinations.
Hemicelluloses: Contents of these carbohydrates showed sharp depression within the first month of the fermentation course of the various feedstuffs; thereafter the rate of
40 Biogas from farm wastes
diminution slowed down (Fig. 8). CD surpassed the other treatments in hemicellulose content throughout the experimental duration. Among the other biomass, CD+M had least value, whilst CD+R and CD+C were similar. The overall loss of hemicellulose was 32% for CD and 41-51% for the plant/animal waste combinations.
Hobson et al. (1981) noted that hemiceUulose-degrading bacteria isolated from piggery-waste digesters were similar in numbers to those of cellulolytic bacteria but they appeared to be predominantly of one species, which could be a reflection of the fact that hemicellulose does not require the complex enzyme system of the celluloses for its hydrolysis.
Cellulose: Cellulose contents of the fermenting materials dropped severely during the first month, then steadily to the end of the experiment (Fig. 8). Within the first month, the order of cellulose content continued following the initial sequence (Table 1), i.e. CD+M>CD+R>CD+C>CD; thereafter no definite trend could be stated except for CD+C which surpassed the others in keeping the highest content of cellulose at those terminal intervals. The overall losses of cellulose were 23, 28, 35, and 44% for CD, CD+C, CD+R, and CD+M, respectively.
Bacteria which hydrolyse cellulose vary in the amount of cellulose-hydrolysing enzyme (cellulase) which they secrete and thus in their rate of attack on any particular cellulosic material (Hobson et al. 1981). The lower degradability of some cellulosic materials is not only conferred by the orderly and close arrangement of the cellulose molecular structures, but by the presence of substances inherently resistant to microbial enzymes: waxes, lignin and even inorganic materials such as silica. Han and Anderson (1975) reported that the lignious structure within an organic complex tends to shield cellulose. Accordingly, the cellulose/lignin ratio (Table 1) might determine, to some extent, the rate of loss of the cellulose fraction from the examined feedstocks.
Lignin: No significant changes were detected for lignin contents throughout digestion of the various feedstocks (Fig. 8). The sequence of the tested materials continued the initial order (Table 1), i.e. CD+C>CD+M>CD+R>CD. The overall loss of lignin ranged from 0.03 to 0.6%. Lignin is almost entirely undegraded in anaerobic digesters by virtue of its complex structure (Hobson et al. 1981).
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Generation rate of biogas and its methane component, as well as changes of major organic fractions during anaerobic digestion of fresh cow dung alone and in combination with each of air-dry rice straw, maize stalks, and cotton stalks at a ratio of 1:1 (on the basis of 70~ dry weight of either source) have been monitored in laboratory fermenters for 75 days at 35~ Mixtures of cow dung + maize stalks produced the highest cumulative volumes of both biogas and its methane component; i.e. 17.9 and 8.31/1 fermented material respectively, cow dung alone surpassed all of the tested biomass regarding the yield of methane production in relation to the volatile solids consumed which gave 6361/kg; the other materials came in the succession: cow dung + maize stalks, cow dung + rice straw and cow dung + cotton stalks. Acetic, propionic, and butyric were the major detectable fatty acids formed during the digestion course. Cow dung excelled the other treatments in amounts of such acids produced. Combination between cow dung and crop residues resulted in reducing the formation of fatty acids and NH~- and loss of nitrogen, but enhanced the disappearance of volatile solids, fats, hemicellulose and cellulose. The lignin content remained unchanged.
Fractionnement de substances organiques pendant la digestion anaerobie de d~chets de ferme pour la gendse de biogaz La vitesse de gen6se du biogaz et son contenu en m6thane, ainsi que les modifications des principales fractions organiques pendant la digestion ana6robie a 6t6 examin6e en fermenteurs
42 Biogas from farm wastes
de laboratoire pendant 75 jours h 35~ pour les bouses bovines fraiches, seules et en m61ange 1:1 (sur la base du poids sec ~ 70~ soit avec la paille de riz s6ch6e au soleil, les fhnes de mais ou les tiges de cotonniers. Le m61ange de bouses de vache et de fftnes de ma'is a produit le volume cumulatif le plus 61ev6 tant de biogaz que de son constituant, le m6thane, notamment respectivement 17.9 et 8.31/1 de mat6riel ferment6. Les bouses de vache ont surpass6 toutes les biomasses test6es quant au rendement de la production de m6thane par rapport aux solides volatils consomm6s qui donna 6361/kg; les autres mati~res viennent dans l'ordre d6croissant: bouses de vache plus f~nes de mais, bouses de vache plus paille de riz et bouses de vaches plus tiges de cotonniers. Les acides gras principaux d6tect6s et form6s pendant le cours de la digestion 6taient l'acide ac6tique, l'acide propionique et l'acide butyrique. La biom6thanisation de bouses de vache a exceU6 par rapport ~ celle d'autres substrats quant aux quantit6s produites de ces acides. Le m61ange de bouses de vache et de r6sidus agricoles a r6sult6 dans la diminution de la formation d'acides gras et d'ammonium et de la perte en azote mais dans l'augmentation de la disparition des solides volatils, des graisses, de l'h6micellulose et de la cellulose. Le contenu en lignine est rest6 inchang6.