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World Applied Sciences Journal 35 (3): 344-351, 2017 ISSN 1818-4952 © IDOSI Publications, 2017 DOI: 10.5829/idosi.wasj.2017.344.351 Comparison of Biogas Productionfrom Macroalgae Eucheuma cottonii in Anaerobic Degradation under Different Salinity Conditions Mujizat Kawaroe, 2Salundik, 1Rhojim Wahyudi and 1,4Dea Fauzia Lestari 1,3 Department of Marine Science and Technology, Faculty of Fisheries and Marine Science Bogor Agricultural University, Dramaga, Bogor, Indonesia 2 Department of Animal Productions and Technology, Faculty of Animal Science, Bogor Agricultural University, Dramaga, Bogor, Indonesia 3 Surfactant and Bioenergy Research Center (SBRC), Bogor Agricultural University, Baranangsiang, Bogor, Indonesia 4 Center for Environmental Research, Bogor Agricultural University, Dramaga, Bogor, Indonesia 1 Abstract: Eucheuma cottonii (E.cottonii) is one of the most widely cultivated macroalgae species in Indonesian marine culture. Some of the harvested biomass has been rejected for utilization by industry, however its utilization as a feedstock for biogas production is deemed as a feasible alternative use. In the present study, the chemical characteristics of the substrates, acclimatization process of the anaerobic digestion inoculum and biogas production from E. cottonii at different salinity levels (0 and 29 ppt) were analyzed using the batch method. Biogas production was carried out in a 30L digester made of fiber with an additional modification gauge. The lignin content, carbohydrate content and C/N ratio were 2.77±0.083%, 63.89±1.162% and 23.13, respectively. Generally, the pH ranges during acclimatization were lower than batch process. The range for 0 ppt digester were 5.9-7.2 in acclimatization and 7-7.8 in batch process, in other hand the range for 29 ppt digester were 6.1-7.3 in acclimatization and 7.1-8 in batch process. The highest daily biogas production on 0 ppt digester was 2.54 mLg 1VS and in 29 ppt digester was 2.05 mLg 1VS. These productions were not significantly different between two treatments. Cumulative biogas and methane volume of 0 ppt treatment were higher than 29 ppt. Key words: Keywords Eucheuma cottonii Salinity INTRODUCTION Batch Biogas Methane Anaerobic South of Sulawesi Province. This area has the same characteristics as coastal areas in Indonesia, but has less electricity and freshwater availability. Therefore, the challenge is how to convert the rejected macroalgae into biogas without a fresh water mixture and washing before anaerobic degradation process. Previous research was conducted by Kawaroe et al. [1] in the laboratory using fresh water to wash and mix with the substrate to reduce the sand and mud contents of the substrate.Due to fresh water limitations in the field, the washing and mixing process used local seawater in that area. Biogas is produced from anaerobic degradation by methane-producing bacteria [2,3,4], Gram-negative basil bacteria [5], or Gram-positive bacteria [6]. Eucheuma cottonii (E. cottonii) is a cultivated macroalgae species that grows in Indonesia and it is used as an export commodity. Sometimes the harvest product does not qualify as food grade export criteria because of its low quality, then will be rejected. The rejected product opens an opportunity to develop alternative biogas energy from biomass. This product can be collected and stored for biogas source especially in coastal areas where biogas can be utilized by fishermen for electricity and household necessities. One of the most famous cultivation areas of E. cottonii is PuntondoVillage in the Takalar Region, Corresponding Author: Mujizat Kawaroe, Department of Marine Science and Technology, Faculty of Fisheriesand Marine Science Bogor Agricultural University, Dramaga, Bogor, Indonesia. 344 World Appl. Sci. J., 35 (3): 344-351, 2017 The biodegradation process is divided into four stages: hydrolysis, acidogenesis, acetogenesis and methanogenesis [3]. The lignin content is one of the most influential factors in the biodegradation process. Lignin is composed of heterogeneous and complex carbohydrates and is, like cellulose, a component of the cell wall in plants; lignin is difficult for bacteria to decompose [7]. The lignin content in macroalgae is relatively lower (15% w) than that in terrestrial plants (15-20%) [8, 9, 10] and according to Kawaroe et al. [13] lignin content in E. cottonii is 0.85%. In addition, E. cottonii biomass contains 35.6-78.3% carbohydrate [11,12]. Therefore, this biomass has good potential as a substrate for anaerobic biodegradation and biogas production [13]. This research was focused on different salinity of mixture water for anaerobic degradation. There were two salinity compared in the process, 0 ppt (fresh water) and 29 ppt (seawater). The observations concerned on chemical characteristic of E. cottonii as a substrate, acclimatization process of the inoculum and substrate and biogas production of rejected E. cottonii under different salinity conditions (0 ppt and 29 ppt). material reduced the inhibition factor in the anaerobic degradation process [14,15], while the starter inoculum was derived from a manure and water mixture (1:2) with different salinity levels (0 ppt and 29 ppt).Water is an important element for biological process on biogas production. The composition should be sufficient to support degradation activity. In this research the composition of water was more than manure itself. The foods of cow were dry weeds, these food condition determine the type of the manure (texture and solidity). The manure was compact and needed much water to make perfect dissolved substrate. The substrate and starter were acclimatized as an inoculum in as much as 24 L of a working volume of the digester. The purpose of acclimatization was to adapt the bacteria to the macroalgae substrate to produce highquality biogas. The inoculum was observed until it reached a neutral pH and produced biogas. Next, 1.2 L of the substrate per day was loaded in each digester under different salinity conditions, followed by releasing the same amount of slurry. The loading volume was determined by multiplying the loading rate of 0.5 kg CODL 1day 1 by the working volume of the digester and then dividing by the chemical oxygen demand (COD) value of the macroalgae substrate used. During acclimatization, the inoculum was stirred and analyzed by measuring the pH, temperature, salinity and biogas production every day. The biogas composition was analyzed once per week. MATERIALS AND METHODS Research Location: The research was conducted from September 2014 to April 2015 at Puntondo Village, Takalar Region, South of Sulawesi Province. Macroalgae samples were taken from the rejected product of the harvest. The samples were then analyzed at the Chemical Oceanography and Animal Science Laboratory of Hasanudin University and Police Forensic Laboratory of Makassar. Fresh manure source was taken from cattle resident farm around research site in Puntondo.During the anaerobic process, two digesters with a 30 L capacity made from fiber that were equipped with two tanks as the substrate input (inlet) and sludge output (outlet) were used. The digesters could be easily moved and had a modified gauge for biogas production sampling. The gas volume was determined by water displacement from a onegallon water tank connected to the experimental digester using the batch method. Anaerobic Biodegradation Using Batch Method: The process of anaerobic biodegradation was started by removing half of the slurry volume on digesters (12 L) and then by adding the same amount of substrate under different salinity conditions to each digester. The substrate contained 4 kg of E. cottonii and 8 L of water (0 ppt and 29 ppt). The substrate input was loaded once and stirred every day gently. The mixing helped to homogenize substrate and we should keep anaerobic condition inside digester. The design of digester is also important to make anaerobic condition during biogas production process. The position of inlet and outlet should be equal with upper past of main digester. The substrate in inlet and slurry in outlet will cover the holes of main digester to make sure there is no air comes in and out of digester.The pH, temperature, salinity, biogas and methane production volume were measured every day, while the biogas composition and COD were measured once per week. Substrate and Inoculum Preparation: The substrate was prepared by soaking macroalgae under water with different salinity conditions. In the first reactor, macroalga was mix with fresh water (0 ppt). While in the second reactor, macroalga was mix with saline water (29ppt). Both are in the ratio 1:2. The leaching and dilution of raw 345 World Appl. Sci. J., 35 (3): 344-351, 2017 Data Analysis: The rejected substrate of E. cottonii was cleaned from mud and sand and then, the blend was used in the juice form. The water, ash, carbohydrates, lipid, protein and nitrogen contents were analyzed [16]; the lignin content was measured according to Van Soest [17] and the total organic carbon content was measured according toWalkey and Black [18]. A refractometer, digital pH meter and thermometer measured the salinity, pH and temperature, respectively. The biogas volume was calculated by measuring the water volume removed from the gallon tank. The biogas sample was accumulated in a plastic sample bag and was then analyzed in a laboratory by gas chromatography. The substrate sample was also collected to measure the COD and then, it was analyzed [19]. ANOVA General Linear Model analyzed data statistically using Minitab version 14 to compare 0 ppt and 29 ppt treatment. Table 1: Proximate analysis of Eucheuma cottonii. Composition Euchema cottonii Water content (%) 16.39±0.23 Ash content (%) 14.39±0.035 Lipid (%) 1.03±0.007 Carbohydrate (%)* 63.17±1.605 Protein (%) 5.33±0.011 Lignin (%) 2.77±0.083 Total Organic Carbon (%) 26.14±0.097 Nitrogen (%) 1.13±0.014 C/N rasio 23.13 * by difference inhibition occurred when the lipid content was greater than 30% under anaerobic degradation. On the other hand, high protein content in the substrate is not recommended because of the high risk of inhibition by ammonia [26, 27, 28]. The carbohydrate content of E. cottonii in Indonesia commonly ranges between 35.6-78.3% [11]. Its polysaccharide content are composed to carbohydrates, such as floridian starch and xylenes, which are easily decomposed by bacteria [29] and they potentially produce biogas by anaerobic degradation [30]. The lignin content in E. cottonii was reported to be 2.77±0.083%, which is categorized as a low content (<7%), simplifying the degradation of raw materials [13,15,31,32] such as polysaccharides, cellulose and hemicelluloses by bacteria[2,24,33] to produce high methane. A high content of lignin (>15%) can inhibit biodegradation [10,34]. The C/N ratio value was 23.13, which is categorized as a normal and balanced range for anaerobic biodegradation, whose optimum C/N ratio is 20-30 [35, 36, 37]. According to Wu et al. [38] and Wang et al .[39], the balanced condition will increase methane production 11-16 times and produce low concentrations of total ammonium nitrogen (TAN) and free ammonia.Low C/N ratios can promote the increasing of ammonia concentrations because nitrogen is released and accumulated in the form of ammonia, exceeding the concentration required to inhibit microbial growth and anaerobic degradation. In the same way, high C/N ratios can increase the growth of the methanogen population because its protein requirements are met; thus, there is no reaction with the residual carbon content of the substrate and less biogas is produced[1,37]. RESULTS AND DISCUSSIONS Chemical Characteristics of E. cottonii: The water content of E. cottonii was 16.3±12:23%. A high water content of the substrate enhances the growth of microorganisms under degradation [20]. The ash content was 14.39±0.035% (Table 1). According to Matanjun et al. [21] this species has a fairly high ash content.The ash consisting of several major minerals, such as sodium, potassium, calcium and magnesium. Minerals such as calcium and magnesium are needed to improve the growth of microbes and have a positive impact on the granulation and sodium role in the formation of ATP and NADH [22]. Chen et al. [14] and Soto et al. [23] reported that the medium concentration encourages microbial growth in anaerobic digesters, but excessive concentrations can diminish the growth, causing inhibition or toxicity. According to Chen et al. [14], the optimum concentration of Ca2+ and Na+ ions for methane synthesis from acetate was found to be 200 mgL 1 and 230 mgL 1respectively, whereas a concentration of 8,000 mgL 1of either ion inhibited the process. The organic content consists of lipids, carbohydrates and proteins of macroalgae, whichare hydrolyzed by microorganisms in order to produce methane [24]. This species has the highest levels of carbohydrates (63.89%± 1,162), with 0.01±1.30% of lipids and 5.33%±0.01 of proteins. The lipid content is within the range of most macroalgae (1-3%) [21]. Cirne et al. [25] reported that no inhibition of methane production occurs if the lipid concentration is greater than 18%; however, Inoculum and Substrate Acclimatization Process: The acclimatization process for the inoculum was indicated by a stable or neutral pH value in the digester. Both treatments had a different day to reach stable pH. 346 World Appl. Sci. J., 35 (3): 344-351, 2017 The increases in the pH value in both the digesters showed effective anaerobic degradation and that the bacteria adapted to the substrate. According to Igoni et al. [42], the anaerobic process works well when the pH levels are between 6 and 8. Substrate should be continually loaded to acclimate and adapt manure bacteria to the macroalgae substrate under different salinity conditions. The pH was non- significantly different between 0 ppt and 29 ppt digester on acclimatization process. The pH range in 0 ppt digester was 5.9-7.2, while in 29 ppt digester was 6.1-7.3. On the other hand there was a significant difference on pH fluctuation in time between starting and ending time of acclimatization process. During acclimatization process, cumulative production of biogas was 13.75mLg 1VS in 0 ppt digester and 4.72mLg 1 VS in 29 ppt digester (Fig. 2). While the cumulative methane volume were 7.94 mLg 1 VS in 0 ppt digester and 2.38 mLg 1VS in 29 ppt digester. There were significantly different of cumulative biogas and methane production between 0 ppt and 29 ppt digester. Both cumulative volume in the 29 ppt digester was found to be less than that that of the 0 ppt digester due to the adaptation process of the microorganisms to the salinity levels. High salinity can inhibit anaerobic activity and produce a low methane content [43]. According to Chen et al. [14], inoculum and substrate adaptation are required to maintain the microorganism viability from inhibitors, such as salinity in the digester. Increasing the pH and biogas production showed that there was a successful acclimation process. The optimum pH level for acidogenic bacteria is 5-6.5, while the optimum pH level for methanogen bacteria is 6.5-8.0 [44]. Fig. 1: Daily biogas production in different salinity (0 ppt and 29 ppt) during acclimatization process. Anaerobic Degradation Using the Batch Method: Biogas production during the acclimatization process showed that the macroalgae substrate could be degraded and adapted to different salinity conditions (0 ppt and 29 ppt). The biogas volume increased after substrate loading until the 57th day and then decreased until the 71st day, similar to that in methane production. The production of biogas was fluctuated during anaerobic process, but there was non-significantly difference between the treatments. The highest production on 0 ppt digester was 2.54 mL g 1VS and in 29 ppt digester was 2.05 mL g 1VS. Constant conditions until the 71st day were caused by decreasing the amount of substrate degraded by bacteria and by reducing the amount of biogas production[27,45]. Fig. 2: Cumulative biogas volume, methane volume and pH during acclimatization process.(A) Salinity at 0 ppt. (B) Salinity at 29 ppt. The 0 ppt digester was stabilized on the day 20th in the pH range of 5.9 to 7.2. Biogas production at 0 ppt digester started on the day 7th, with as much as 0.004mLg 1VS, while the 29 ppt digester was established on the day 27th in the pH range of 6.1 to 7.3 and started producing biogas on day 8th with as much as 0.02mLg 1VS (Fig. 1). According to Bruhn et al.[15] and Demirbas [40], high salinity levels can retard the anaerobic process. The depletion of the pH value indicates acidification or organic acid accumulation in the digester [41]. 347 World Appl. Sci. J., 35 (3): 344-351, 2017 Fig. 3: Daily biogas volume in different salinity digester during batch system. Fig. 5: Chemical oxygen demand and cumulative methane volume from E. cottoniidegradation.(A) Salinity at 0 ppt. (B) Salinity at 29 ppt. process. According to Chen et al. [14], microorganisms or degrading bacteria can maintain viability at certain concentrations, exceeding the inhibition concentration of factors after they have adapted and produced a higher volume of methane. Additionally, polymerase chain reaction-denaturing gradient gel electrophoresis (PCRDGGE) analysis conducted by Zhang et al. [46] and Patil et al. [47] showed a change in the bacterial community interaction and that of archaea under high salinity concentrations and that their interaction can intensify anaerobic degradation. The increases in biogas production were influenced by the low lignin content and high carbohydrate content.The low lignin content assists bacteria in the degradation to organic material and then in the production of high biogas and methane contents [2,13,15,31]. The high carbohydrate content in macroalgae species also affects biogas production. MacroalgaeE. cottonii contains polysaccharides and cellulose [21], which can be easily decomposed by bacteria [29,48]. Fig. 4: Cumulative biogas volume, methane volume and pH during batch method.(A) Salinity at 0 ppt. (B) Salinity at 29 ppt. The cumulative biogas and methane volumes in the 0 ppt digester were 91.94mL g 1VS and 81.62mLg 1VS, respectively, higher than those with a 29 ppt salinity, whose volumes were 91.12mLg 1VS and 73.40mLg 1VS, respectively (Fig. 4). Both cumulative biogas and methane volume were significantly different between 0 ppt and 29 ppt. This difference was caused by the salinity concentration level, which inhibits bacterial activity[43]. Otherwise, the difference in this range would be not significant because bacteria would not have adapted to the salinity level (29 ppt) during the acclimatization 348 World Appl. Sci. J., 35 (3): 344-351, 2017 REFERENCES The pH value tends to be variable from the 1st until the day 50th and then, it remains constant until the day 71st (Fig. 4). This fluctuation indicates the degradation process of organic material, while the constant value is caused by the substrate reduction inside the digester. The pH range in both digesters was 6.9-7.7 and 7.0-8.0. These values are categorized as conditions for anaerobic degradation. According to Igoni et al. [42], the optimum pH range for anaerobic biodegradation is 6-8. Chemical oxygen demand (COD) is an indicator for oxygen requirement in organic material oxidation contained in the substrate. This value can also determine the methane production volume from anaerobic degradation. A COD reduction occurred from the initial day until the last day of the batch method (Fig. 5). The range value of COD removal in the 0 ppt digester was 3.45-6.17 gL 1, while in the other digester was 3.055.17gL 1. 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