Magister Research

Study of methane production by methanogenic consortium bacteria with a substrate of Jatropha curcas seed pressed cake

Yati Supriatin, I Nyoman P. Aryantha, Siti Khadijah Chaerun

School of Life Sciences and Technology

Institut Teknologi Bandung

ABSTRACT

The study of biogas production with seed pressed cake of Jatropha curcas as a substrate in a 2.5 L reactor scale aims to know the optimum condition to produce biogas (methane). The research was begun by screening the inoculum source from cow dung, dam sediment, and cow rumen liquid. The potency of single and consortium bacteria to produce biogas with the highest methane concentration was evaluated. After the best microbial agents was determined, the next step was the substrate composition using jatropha seed pressed cake with C/N ratio of 12.6 and jatropha seed pressed cake added with tapioca waste with total C/N ratio of 15. Three variations of inoculums concentrations (5%, 10%, and 20%) with four times repetitions were investigated. The system was inoculated with 30 day old inoculum and observed for 70 days by measuring several physical, chemical, and biological parameters in a batch system reactor. The result shows that natural consortium from Jatiluhur dam sediment produces the highest methane gas concentration. Jatropha seed pressed cake with C/N ratio 12.6 and inoculums concentration of 20% gave the best result. Biogas was produced from 14^th day and then increased until 70^th day with total production of 2.822,25 mL (average 40.31 mL per day). GC analysis showed that methane concentration at day 49^th reached 60.57% and then continued to increase until the 70^th day with a concentration of 74.72%. This value is the highest result compared with other achievements reported in literatures. Aerobic bacteria were isolated in the last process and identified as Bacillus brevis, Bacillus laterosporus, Edwardsiella tarda, Bacillus pasteurii, Bacillus larvae, Bacillus pumilus while, anaerobic bacteria were identified as Desulfotomaculum nigrificans, Alcaligenes faecalis, Desulfotomaculum orientis, Bacillus badius, Bacillus alcalophilus, Bacillus firmus. Biogas was produced the best at a range of pH from 6 - 8. This result can be used as a reference in biogas production with seed pressed cake of J. curcas as a substrate in application scale.

Keywords: biogas, methane, methanogenic bacteria, seed pressed cake, Jatropha curcas.

1. Introduction

The fossil fuel consumption in Indonesia increasingly at several years while production was decreased (Hambali et al, 2006). This problem could be solve with search new alternative energy source. Biogas as gas generated from organic digestion in anaerobic conditions by mixed population of microorganisms is one of alternative energy source, that can be produced from various of wastes. Biogas generally composes of methane (55-65 %), carbondioxide (35-45%), nitrogen (0-3%), hydrogen (0-1%), and hydrogen sulfide (0-1%) (Anunputtikul & Rodtong, 2004). Jatropha seed pressed cake as a bio diesel production waste will be abundant in Indonesia for few years later because developing of bio diesel as one of bio fuel (Hambali et al, 2006). The usage of Jatropha seed pressed cake as substrate to produce biogas had been developed in nicaragua since 1997 (Gubitz, 1997), nevertheless biogas system from Jatropha seed pressed cake have not yet many developed in Indonesia. The aim of this study is to get optimum environment condition in biogas production and the result can be used as a reference in biogas production with Jatropha seed pressed cake as a substrate in application scale.

2. Materials & Method

2.1 Preparation of Substrate

Jatropha seed pressed cake as main substrate in this study were collected from PT Rajawali Nusantara Indonesia Jatitujuh Majalengka. Tapioca waste used as additional substrate was collected from tapioca factory in Tasikmalaya. Total solid (TS), total carbon, total nitrogen, ash, and sulfur content of raw material were determined using standart methods (Oei & Reinhard, 1989).

2.2 Preparation of inoculum source

Inoculum source obtained from cow rumen liquid was collected from animal slaughtering house in Ciroyom, cow dung was collected from dairy cattle ranch in Cimenyan, and Jatiluhur dam sediment deepness 28 metres. Isolation of methanogenic bacteria from 3 inoculum sources have be done to get single methanogenic isolate for compare the biogas production ability with natural consortium bacteria. Isolate screen base on gas production ability (Lay,1994 modified) and than confirm test base on morphology test under fluorescent microscope (Mink & Dugan, 1976). Natural consortium bacteria from three sources kept in a closed container respectively with regular adding a small amount of jatropha seed pressed cake for 14 days and screen base on methane gas production that measured by using gas chromatography at the same time, inoculum source with the higher methane gas production would be kept for 30 days before inoculating the biogas production reactor.

2.3 Biogas production from Jatropha seed pressed cake

The biogas production from Jatropha seed pressed cake was perfomed using the simple-single-state reactor with working volume of 2 liters (L) in batch system. Substrate formulation carried out in two variation that were only Jatropha seed pressed cake with C/N ratio 12.6 and Jatropha seed pressed cake added tapioca waste with mixed C/N ratio 15. Substrate concentration was 10 % (w/v) to water (Hernas, 2007), and inoculum concentration variation was 5%, 10%, 20% (v/v). The biogas fermentation was then operated in four times repetitions at ambient temperature for 70 days by measuring several physical, chemical, and biological parameters.

Change of physical parameters involved odor and texture slurry, flame test. Change of chemical parameters involved COD used bi chromate method (Oei & Reinhard,1989), Measurement of pH used pH meter (Uchida KT-1A), total gas volume used water displacement method (Anunputtikul & Rodtong,2004), and biogas composition used gas chromatography (Shimatdzu, GC8A, Japan) equipped with column Porapak- Q and Mulsife 5A, injector temperature and detector temperature 70⁰C, column temperature 40⁰C, detector type Thermal Conductivity Detector, gas Argon as the carrier gas with speed 30 cc/minute. Change of C/N ratio during fermentation measured by using Khjedahl method for nitrogen content analysis whereas carbon total content analysis by counted difference of dry weight & ash weight (Oei & Reinhard, 1989).

Biological parameters involved change of aerobic and anaerobic bacteria population by using FDA method (Green et al., 2005 modified), bacteria cell enumeration used plate count method (Cappucino & Sherman, 2005), Isolation and identification aerobic and anaerobic dominant culture able bacteria that cultured at seventh day and 70th day used pour plate method and used nutrient agar media (Cappucino & Sherman, 2005). Quantitative Analysis of methanogenic bacteria had be done at seventh day, 30th day and 70th day used viable count method (Lay, 1994 modified).Reactor design showed at figure 1

3. Result & Discussion

3.1 Raw material for biogas production

Jatropha seed pressed cake was bio diesel production waste that produced from Jatropha curcas seed. Some physical and chemical compositions of Jatropha seed pressed cake were analyzed (Table 1).

Table 1 physical and chemical compositions of substrate

No	Composition (% dry weight)	Jatropha seed pressed cake	Tapioca waste
1.	moisture	10.94	12.67
2.	carbon	84.20	77.393
3.	Nitrogen	6.68	0.393
4.	S	4.53	0.0047
5.	TS	92	80.09
6.	Ash	6.7	2.7

3.2 Biogas production from Jatropha seed pressed cake

Result of methanogenic bacteria isolation & screening state was obtained only one isolate bacterium, because of only one isolate that produced, so this research not enables use isolation result as inoculum. Then the research was more developed at usage natural consortium as inoculum. The biggest methane gas production in natural consortium bacteria screening process was achieved by natural consortium bacteria that indigenous in Jatiluhur dam sediment. It happened because Microorganism variety was very height in sediment and it be caused of sediment supports aerobic micro environment forming anaerobic condition (Maier et al., 2003). Methane gas that produced from sediment could be more than cow rumen liquid. According to Madigan et al. (2003) cow rumen liquid had more CO₂ composition that was 65% than CH₄ that only reach 35%, this condition happen because acetotroph methanogenic bacteria in rumen slow growth whereas retention time for the happening of acetate conversion become CH₄ is short time & sintrophyc bacteria fatty acid degrader not play main role in rumen because fatty acid will be adsorb by intestinal wall and enter to circulation system (Madigan et al., 2003).The result of treatment state was shown in figure 2 &3.

the treatment which higher CH₄ gas production was achieved by C/N ratio 12.6 and inoculum concentration 20 %, composition of methane gas more stabilizes from 49th day till 70th day that is the average of 60.57 % ± 0.205 s/d 74.72% ± 0.256 with total gas production 2.822,25 mL and the average of production biogas per day 40.31 mL.

In C/N ratio substrate 12.6 the system more balance between non methanogenic bacteria & methanogenic bacteria activity compare in C/N ratio substrate 15. Change of pH during fermentation process was decreased at initial pH 8 became average 4-5, moreover in C/N ratio 15 pH decreased drastically became 2-3, it cause methane formation was obstructed.

When a biogas plant is newly started, the acid formation become active first, reducing the pH, then methanogenic bacteria start using these acid, increasing the pH back to neutral (Fulford, 1988) , but it not happen in C/N ratio 15 because of acid forming to much for methanogenic bacteria could consumption (Burke,2001), finally organic matter in C/N ratio substrate 15 was not convert in CH₄ gas. In this study result that in C/N ratio 12.6 CH₄ gas production at 42^nd day decreased drastically, it caused by accumulating CO₂ gas as result of aerobic bacteria metabolism that achieved 2,4 x 10⁷ CFU/mL sample , but after that CH₄ gas production increased until 70^th day. This result appropriate with result of methanogenic bacteria quantitative analysis that the amount of methanogenic bacteria achieve 5.1 x 10³ cell/mL then continued to increased until 16 x 10³ cell/mL at 70^th day (figure 4).

The result of aerobic & anaerobic bacteria population show that aerobic⁷ CFU/mL sampel while bacteria population achieved 10 anaerobic bacteria population reaches 10⁴ CFU/mL sample. Aerobic bacteria were isolated in the last process and identified as Bacillus brevis, Bacillus laterosporus, Edwardsiella tarda, Bacillus pasteurii, Bacillus larvae, Bacillus pumilus while, anaerobic bacteria were identified as Desulfotomaculum nigrificans, Alcaligenes faecalis, Desulfotomaculum orientis, Bacillus badius, Bacillus alcalophilus, Bacillus firmus.

Conclusion

1. Inoculum that produce highest methane gas production is natural consortium bacteria from Jatiluhur dam sediment with the average of methane gas production that produced is 7.163 % ± 0.689.

2. Substrate that produce highest methane gas is Jatropha seed pressed cake with C/N ratio 12.6 with result 74.72% ± 0.256.

3. Inoculum concentration that produce highest methane gas is inoculum concentration 20%, composition of methane gas more stabilizes from 49th day till 70th day that is the average of 60.57 % ± 0.205 s/d 74.72% ± 0.256 with total gas production 2,822,25 mLs and the average of production biogas per day 40.31 mL

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