UTILIZATION OF AQUATIC WEED Salvinia molesta AS A RAW MATERIAL FOR BIOGAS PRODUCTION

Salvinia molesta is an aquatic weed having very high growth rate. The high abundance of this species biomass could be utilized as a source of alternative energy. This research was aimed to utilize S. molesta as a substrate for biogas production using batch system in order to reduce euthrofication effect in a lake. Cow manure was used as starter for anaerobic process because containing bacteria. Proximate test was conducted to analyze the chemical characteristics of the aquatic weed. Chemical oxygen demand, total solid, and total volatile suspended solid of weed and manure were analyzed according to the APHA 2012 method. Biogas composition was analyzed with gas chromatography. The results showed that the S. molesta contained high lignin content and low C:N ratio. Hydrolisis and acidification process occured very rapid, resulting in an increase of the weed and manure mixture COD. In the other hand, the decreased of COD removal was caused by the massal death of methanogenesis bacteria. The total solid and total volatile suspended solid content were not significantly decreased. Potential biogas production from S. molesta was 58.16 L.kg-1 with 318.29 mL.gram VS-1day-1 of methane production. This production value was still low or compared to that of cow manure which have been established as biogass materials and need modification to improve the biogas production.


INTRODUCTION
Fossil fuels still dominate the primary energy consumption.Development of renewable energy becomes a priority due to the diminishing of fossil energy sources.One of the new renewable forms of energy that needs to be developed is biogas.Biogas is a product of anaerobic degradation of organic substrates.This process involves microorganism mixture and depends on various factors such as pH, temperature, hydraulic retention time, and the C:N ratio (Yadvika et al. 2003).Indonesia has the potential for bioenergy development by utilizing biomass resources that are very abundant.Biomass used in the biogas production come from various sources, such as aquatic weed.
The use of aquatic plant biomass as an energy source gains a lot of attention (Nipaney and Panholzer 1987;Abbasi et al. 1990;Jain et al. 1992;Wilkie and Evans 2010;Raposo et al. 2011;Koyama et al. 2014).S. molesta is a floating aquatic weed belongs to ferns species.S. molesta is widely found in the slow current rivers, ponds, puddles, marshes and conduit.S. molesta has a rapid growth with a doubling time of 5-41 days and high density (Galam et al. 2015).S. molesta productivity is very high and can reach a dry weight of 57 ton per ha per year (Abbasi et al. 1990).
The use of S. molesta as the raw material of biogas was first investigated by Abbasi and Nipaney (1984), by adding 750 kg of fresh S. molesta to 5250 L of water in a biogas digester.The study showed that after 40 days of the retention time, the average biogas produced was 6.7 L per kilogram of wet weight and the concentration of methane was 40-78%.O'Sullivan et al. (2010) produced biogas using S. molesta substrate in a laboratoryscale digester (batch method) and generated 155 L of biogas per kilogram, with methane concentration as high as 65%.Meanwhile, Mathew (2014) tested biogas formation using S. molesta substrate with batch system and produced 221 L of biogas per kilogram of wet weight.
The above studies suggest that S. molesta has the potential to be developed as a raw material for biogas production.In addition, the utilization of aquatic weed as biogas material can reduce the pollution from eutrophication in lake.This study was aimed to analyze the biodegradability of S. molesta for biogas production through anaerobic process using batch system and to analyze the biogas properties .

Materials
The fresh biomass of S. molesta was collected from the Situ Perikanan, IPB University, and was subsequently cleaned and dried.Proximate analysis based on the SNI method 01-2891-1992 was performed to determine the chemical characteristics of the weed (BSN 1992).S. molesta was used for Chemical Oxygen Demand (COD) analysis and the C:N ratio based on APHA (2012).Inoculum bacteria were obtained from dairy cow manure.Dairy cow manure was selected because it contains complete consortium of methane-producing bacteria (Methanomicrobium, Methanosarcina, Methanococcus, and Methanothrix) and more practical to use than other artificial inoculums (Haryati 2006).Dairy cow manure was obtained from Faculty of Veterinary, Bogor Agricultural University.

Substrate preparation
The biogas substrate was prepared from previously soaked S. molesta weed mixed with water with a ratio of 1:2.The mixture was then shredded in a blender to produce the substrate.

Inoculum preparation
The inoculum was obtained from a mix of dairy cow manure and water (1:1).Twenty four liters of the inoculum were added to the reactor (Figure 1) and were incubated for several days until the pH and the temperature were stable.

Acclimatization
Acclimatization was carried out to familiarize the degrading and methaneproducing bacteria with new environment.Acclimatization was carried out by providing organic materials for 0.5 kg COD.m -3 .day - , or equal to 1.041 L.day -1 (equation 1).Substrate as much as 1.041 L was gradually added every two days to the bioreactor for 28 days.During the acclimatization process, the temperature, pH, and volume of the biogas were measured.

Biogas production using batch method
The biogas was produced using batch method.The production was done by removing the cow manure from the digester as much as half of the work volume.S. molesta substrate was then in equal volume as the slury.S. molesta substrate introduced in this stages was 7 L.The production was carried out for 53 days, started on day 47 of 99 total research days.During the biogas production stage, the pH, temperature, and volume were recorded every day.The Chemical Oxygen Demand (COD), Total Solid (TS), Total Volatile Suspended Solid (TVSS) were analyzed every seven days using APHA method (2012).The biogas composition was analyzed using gas chromatography.The COD data of the substrate and the slurry were used to calculate COD removal (CODr) using equation 2 and equation 3.
The gas composition was expressed as percentage.These were are used to calculate the volume of methane produced.= volumetric COD removal (g/day); = COD concentration in the substrate (g.L -1 ); = COD concentration of outflow/ slurry (g.L -1 ); = the flow rate of substrate (L per day).
The processing of S. molesta biomass into biogas was performed through anaerobic digestion.The anaerobic digestion is affected by environmental conditions such as pH and temperature, as well as chemical characteristics of the organic material.The analysis showed that S. molesta had a water content of 95.66% (Table 1).The result obtained was comparable with that of Mani (1998).Sufficient water content will help the biodegradation process.Lipid, carbohydrate, and protein are organic compounds that hydrolyzed by microorganisms (Chang et al. 2010).Those three organic compounds have a positive correlation with the amount of methane producted.The content of lipid, protein, and carbohydrate in S. molesta were relatively low (Table 1).Moozhiyil and Parauf (1986) found that the more mature the growth phase of S. molesta, the less protein content.Meanwhile carbohydrate content in S. molesta was 1.62%.Generally, aquatic plants contain low carbohydrate content.
The lignin content of S. molesta in this study was 17.11% (Table 1).This value was not much different from the result obtained by Mani (1998), which was 15.94%.Lignin is a complex organic compound and is resistant to degradation (Speece 1996).This property due to the basic structure of lignin, which forms a phenyl propane based complex threedimensional polymer compound (Kirk and Farrell 1987).The average of lignin content in S. molesta was 10.8 to 17.5% (Moozhiyil and Parauf 1986).Fifteen percents of lignin content is already enough to inhibit biodegradation (Pfeffer and Khan 1976).
The C:N ratio in S. molesta was 6.87 (Table 1).This value was below the minimum value of C:N ratio for optimal biogas production.Optimum C:N ratio for anaerobic digestion ranged from 20 to 30 (Mathew et al. 2014).A very high C:N ratio indicates the excessive nitrogen consumption by methanogenic bacteria thus a little nitrogen can react with carbon.This will impact on the low production of the gas.On the other hand, if C:N ratio is too low, it can lead to the accumulation of ammonia thus the pH will exceed 8.5 (Abbasi and Abbasi 2012).

pH dan Temperature
The performance of biogas installations can be controlled by studying variations in the parameters such as pH and temperature.pH is an important parameter that affects microbial growth during the process of anaerobic degradation.The pH value during the acclimatization process fluctuated but showed improvement, while the temperature condition tend to be stable in the range of 5.8 to 6.5 (Figure 2).The pH decreased at the early production, then increased and stable in the range of 6 to 7.7 (Figure 2).The stable pH value during the process of organic loading showed that the equilibrium was already achieved.The pH condition in the digester should be kept within the optimum range to produce biogas (6.8 to 7.2) (Yadvika et al. 2003).Temperature has a great influence in the production of biogas.The temperature during the acclimatization process ranged fluctuated between 25°C and 30°C.The temperature during on biogas production process using batch method ranged from 27°C to 32°C (Figure 3).The temperature was in accordance with the results obtained by Mital (1996) where anaerobic bacteria activities are most active in the mesophilic temperature range (20-45°C).

Chemical Oxygen Demand, Total Solid, and Total Volatile Suspended Solid
Anaerobic biodegradation conditions can be deserved from the change of COD, TS and TVSS.The COD value increased from 3248.42 mg/L to 10335.45 mg/L in the last observation day.Chemical Oxygen Demand (COD) represents the total amount of oxygen required to oxidize organic materials chemically, into CO 2 and H 2 O.The COD continued to increase during the process of anaerobic degradation due to rapid hydrolysis and acidogenesis (Li et al. 2011).CODr percentage indicates the amount of organic material degraded during anaerobic degradation process.High CODr, indicates high biogas production (Kawaroe et al. 2015).A decrease in CODr every week indicates less-than optimal degradation of organic material in the digester.There was rapid decreasing of COD from day 50 to 57, because of weed and cow manure degradation by anaerobic bacteria.
The Total Solid (TS) and Total Volatile Suspended Solid (TVSS) for 53 days of biogas production process was shown in Table 2. Overall, the value of TS and TVSS decreased, but increased on day 78 and 92.The CODr percentage for 8 weeks was shown in Table 2.The highest CODr was obtained in the first week (71.80%).The decreasing of CODr value was in accordance with the length of production time, with the lowest value was observed in the last week (10.27%).LCODr or volumetric removal of COD removal shows the removal COD value in the unit of volume.LCODr value was directly proportional to the COD removal.
The TS value decreased from the first day until the last day of observation.An increase occurred in the value of TS on day 78.This was presumably due to the addition of suspended matter from bacterial biomass (USGS 2003).
A decrease in TVSS during the process of anaerobic degradation was not significant.

Gas Production
Anaerobic degradation of organic material will produce gases, particularly CH 4 and CO 2 .The production rate of biogas was observed and measured daily.Biogas production for 53 days was shown in Figure 4.The biogas production showed a fluctuative result with the highest production was observed at day 47 to day 55 and began to decline on day 57.
The degradation process began on day 47 to day 99.The highest biogas production was achieved at day 53 (5.05 L) and the lowest production was on the day 90 (1.59 L).Biogas production during the process tend to decrease.Total production of biogas from anaerobic activity for 53 days of observation was 174.48 L. Based on the cumulative volume, the rate of gas production in S. molesta was 3.29 L.day -1 .If it is seen from the amount of biomass used (3 kg), then the potential for the gas production that can be produced from 1 kg S. molesta is 58.16L.kg -1 .
Table 3 shows biogas production time percentage.Biogas contained several gases.The gas concentrations measured in this study was methane (CH 4 ) and carbon dioxide (CO 2 ), because both of the gases are the largest constituent of biogas.Besides these two gases, there are also byproduct gases such as hydrogen, nitrogen, oxygen, and ammonia with a small amount.
The process of anaerobic degradation produced biogas up to 174.48 L. The value was higher than the result obtained by O' Sullivan et al. (2010), which was 155 L and Abbasi et al. (1992) which 137 L. Based on the cumulative biogas production, the potential for biomass production per kilogram of S. molesta was 58.16 L. Abbasi and Nipaney (1991) examined the potential of biogas from Salvinia sp. and obtained biogas with a concentration range from 48 to 410 L.kg -1 .The  volume of biogas generated in this study was also within that range.
The main component of biogas was methane (CH 4 ) and carbon dioxide (CO 2 ).The highest percentage of methane in this study was 54%.Methane composition in biogas is 50-70% (Soerawidjaja 2009), and carbon dioxide was 30-40% (Rasi 2009).Methane concentration tend to decrease and biogas composition was dominated by carbon dioxide (Table 3).It can be related to the inhibition of methanogenesis by CO 2 accumulation.Methane concentration increased again on day 99.Due to adaptation of degrading bacteria the structure and chemical compounds in S. molesta substrate.The percentage of methane produced is enough for combustible gas.The decreasing in viscosity of the slurry indicates anaerobic digestion is occuring The acidogenesis and hydrolysis process run very rapid, it was suspected that there was formation of total ammonia nitrogen (TAN) and accumulation of volatile fatty acid (VFA).TAN and VFA are intermediate products that could inhibit anaerobic digestion process (Yen and Brune 2007).Formation of acid reduced the pH during acidogenesis stage in the beginning of biogas production process (Figure 4).Low pH condition was not conducive for the growth of methanogenic bacteria, resulting in the death of microorganisms.Undigasted microbial biomass as well as their particles and organic materials affected the TS and TVSS values (Table 2).
Total volatile suspended solid is an organic matter that can evaporate or can be converted into biogas.Production of CH 4 and CO 2 can be determined based on the content of TVSS (Table 4).Weekly volume of CH 4 produced by TVSS value.CH 4 production continued to decline, while CO 2 tend to be stable.The amount of substrate that could potentially be converted into methane can be estimated through the level of volatile solid.S. molesta is  The results obtained in this study was comparable to that of Hansen et al. (2004).

CONCLUSION
The biomass of S. molesta could be used for biogas production.The total production for 53 days was 174.48 L, with daily biogas production was 3.29 L. Potential production per kilogram of S. molesta was 58.16 L with methane concentration of 54% indicating that S. molesta potentially can be used as a substrate for biogas raw material.Based on the TS, TVSS, and COD conditions during the production process, batch system was not effective as a method to degrade S. molesta.
convertion into biogas was determined by calculating the amount of biogas produced (L) or cumulative volume divided by the number of observation day COD

Figure 3
Figure 3 Temperature during the process of (a) acclimatization, (b) biogas production.

Table 1
Characteristics of S. molesta chemical constituents

Table 2
The value of TS, TVSS, COD removal and CODr volumetric

Table 4
Hansen et al. (2004)an CO 2 based on TVSS -based waste because it has a high lignin content.Hansen et al. (2004)used cellulose-based waste as a substrate for paper bags production.Average production of methane from cellulose-based waste was 379 mL CH4.gram.VS -1 during 50 days the production process.Average production of methane from S. molesta was 318.29 mL CH 4 .gram.VS -1 for 53 days of the production process.