Changes In Acetoclastic Methanogenic Activity Biology Essay


Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.

Microbial Biotechnology and Biosafety Department Agricultural Biotechnology Research Institute of Iran (ABRII) Seed and Plant Improvement Institute's Campus P. O. Box: 31535-1897 Mahdasht Road, Karaj, Iran

Department of Biological Functions and Engineering, Graduate School of Life Science and Systems Engineering. Kyushu Institute of Technology, 2-4 Hibikino, Wakamatsu-ku, Fukuoka, 808-0196, Japan.

A project supported by the Federal Land and Development Authority (FELDA), Malaysia.

*Corresponding author.

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Changes in the acetoclastic methanogenic activity in the 500 m3 closed digester tank (CDT) treating palm oil mill effluent (POME) were studied throughout the 110 days operating period. The acetoclastic methanogenic activity (AMA) test was used to measure the acetoclastic methanogenic activity of the biomass present in relation to the optimal operation of anaerobic digestion along with conventional controlled parameters. Throughout the operating period, the AMA tests were carried out at different organic loading rates (OLR) from 0.5 up to 5.0 kg COD/ m3/ d and hydraulic retention time (HRT) as low as 10 days to observe potential methane production and the performance of the digester. The test was carried out in 100 mL serum vial containing mixture of POME sludge and basal medium ratio 20:80 % (v/v). The AMA values detected in this study were in the reported range of 0.04 to 0.33 g COD/ g VSS/ d which indicates the activity of acetoclastic methanogens in the digester. This test was crucial in determining performance of digester since activity values indicated the active acetoclastic methanogens presents in the CDT and gave a better insight in microbiological view in comparing with conventional parameters measured.

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Keywords: Anaerobic digestion; acetoclastic methanogenic activity; POME

1. Introduction

Anaerobic digestion of palm oil mill effluent (POME) is becoming more attractive recently for the treatment has beneficial products to be utilized. Most of the palm oil mills in Malaysia adopted application of pond/ lagoon system in treating POME since such treatment offers the cheapest technology with minimum maintenance. However, with innovation and advancement of recent technology, the implementations of anaerobic digesters for POME treatment are gaining recognition. A few reports have shown promising applications of bioreactor and process designs in the treatment of POME such as the reversible anaerobic baffled reactor (RABR) (Raof et al., 2005), upflow anaerobic sludge fixed-film bioreactor (UASFF) (Najafpour et al., 2005), modified anaerobic baffled bioreactor (MABR) (Faisal and Unno, 2001), upflow anaerobic sludge blanket reactor (UASB) (Borja and Banks, 1995), thermophilic upflow anaerobic filter (UAF) (Mustapha et al., 2003), anaerobic continuous stirred tank reactor (CSTR) (Tong and Jaafar, 2005) and closed digester tank (CDT) (Yacob et al., 2006). Laboratory and pilot scale studies have been conducted by these researchers intended to evaluate the effectiveness of the technology for POME treatment. In the process of evaluating the performance of any anaerobic bioreactor several parameters were used as indicators among them are volatile fatty acids (VFA) level, organic matter removal, pH, alkalinity, temperature and composition of biogas produced. Another potential test to determine the success of anaerobic digestion is acetoclastic methanogenic activity (AMA). AMA test is a method to determine the activity of acetoclastic methanogenic population in sludge and a bioreactor system (Ince et al., 1997). It is a simple method using substrates such as acetate as carbon and energy sources for the production of methane (Ince et al., 1997: Jawed and Tare,1999) by methanogenic bacteria. This method is important to evaluate the adequate level of activity of active methanogens exists in the anaerobic treatment systems (Ince et al., 1997). As reported by Ince and coworkers (1997), any deterioration of an anaerobic system's performance was due to a change in dominant methanogenic species or a decrease in the quantity of active methanogens or a decrease in their activity. Any imposed stress such as variation in operational and environmental conditions may lead to a change in species types and their relative population levels since each methanogenic group have their own specific activity and growth conditions which directly affected the performance of an anaerobic bioreactor in terms of effluent quality and methane yield (Harper and Pohland, 1986). In this study, the AMA test was used as a tool to establish the relationship between the optimal operation of CDT and acetoclastic methanogenic activity during the POME treatment.

2. Materials and Methods

2.1 Sludges sampling for AMA test

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Test sludge was obtained from the bottom of the CDT treating POME at Serting Hilir mill in the state of Negeri Sembilan, Malaysia. The POME sludge was collected by using silicon tubes and quickly placed into 1 L Schott bottle with modified stainless steel cap. The stopper was modified to maintain anaerobic condition inside the bottles that was flushed with industrial grade 99 % N2 (Malaysian Oxygen Sdn. Bhd.). Microbial activities were kept by incubating the sludge samples at 370C or at room temperature prior use as inoculum or for further analytical purposes.

2.2 Description of Closed Digester Tank

An industrial scale CDT designed by Sumitomo Heavy Industries Ltd., Japan and constructed by FELDA Palm Industries Sdn. Bhd (FPISB), Malaysia was used for this study. The total working volume of the digester is approximately 500 m3. This unit was installed with the resistance temperature detector and pH probe to monitor the temperature and pH of the process respectively. The CDT was operated under mesophilic condition. The pH was controlled between 6.8 and 7.2 which was adjusted automatically using sodium hydroxide (NaOH) concentration 6.25 M. Influent COD was range 30,000 mg/L â€" 90,000 mg/L per day throughout this study. The schematic diagram and start up operation of the CDT was described earlier (Yacob et al., 2006). To begin the operation, the CDT system was fed with POME at an organic loading rate (OLR) of 0.5 kg COD/ m3/d with a HRT of 74.4 days without mixing provided. The VFA concentration in the CDT was determined about 188 mg/L in the CDT, indicating an acceptable concentration of organic levels before commencing the actual operation. These controlled parameters were monitored daily to prevent the souring and deterioration of the CDT.

2.3 Media Preparation

Media solution used for AMA test contains the similar components and the procedures are adopted from Dolfing and Bloemen (1985). The strict anaerobic methods developed by Hungate (1969 were adopted throughout these investigations.

2.4 AMA test unit

The experimental setup is shown in Fig. 1. Media solution containing sodium acetate was dispensed in serum bottles up to 80 mL and purged with N2 gas. Then the serum bottles were capped with butyl rubber stopper and aluminum seal. After autoclaving, the media were cooling down at room temperature prior inoculation with 20 mL of CDT sludge. No vitamin and trace element was added in the test medium since the AMA test was monitored in 24 h reaction and no significant growth of methanogens growth at short period (Dolfing and Bloemen, 1985). The contents of the serum bottle were mixed by swirling manually several times and samples were withdrawn with syringes and needles for zero hour reaction (0 h). Biogas and methane gas production was measured at time intervals (12 h). The entire test was conducted at 37°C and without mixing provided. On completion, the amount of sludge (VSS) remaining in the serum bottle was determined. The remaining VSS and cumulative methane production were used to calculate the methanogenic activity.

2.5 Analytical Methods

Influent and effluent samples were analyzed for chemical oxygen demand (COD) and volatile fatty acids (VFA) once every other day. Suspended solids (SS) and volatile suspended solids (VSS) were carried out at the end of every test. All analyses were carried out according to Standard Methods (APHA, 1992). Methane concentration was determined using a portable methane analyzer (XP-314A, Shin-Cosmos Electrics Co. Ltd, Japan).

3. Results and discussion

AMA test and CDT performance

Table 1 summarized the results on CDT performance in relation to AMA test values. In the first week of operation, the CDT was fed with influent COD of raw POME in the range 27,800 to 69,100 mg/L (OLR= 0.5 kg COD/m3/d). Average VFA and CH4 production determined in the reactor was 182 mg/L and 40 % respectively. The reactor achieved 89 d HRT. The AMA test in the first week was recorded 0.33 g COD / VSS / d. The OLR was then increased to 1.0 kg COD/m3/d on the second week of operation with influent COD ranged from 36,200 to 74,600 mg/L recorded. However, the activity of AMA sharply declined to 0.09 g COD/ g VSS/ d at the second week of bioreactor operation. This indicated that the highest activity value during the first week was attributed to newly transfer fresh POME at the lowest OLR to activate the biomass (Yacob et al., 2006). Then activity sharply decreased probably due to OLR changes that required adaptation period of methanogenic bacteria to suit with a new environment (McCarty, 1964).

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The reactor was further supplied with higher OLR to monitor the reactor behavior. There were fluctuations in AMA values observed when the system introduced to higher OLR, (OLR 1- 3.0 kg COD/ m3/d). However, VFA levels recorded for this period was in the acceptable range for methanogens growth (182- 208 mg/L). The AMA values were recorded low at this OLR may be due to less substrates introduced into the CDT resulted minimal amount of intermediates produced and short chain fatty acids like propionic, formic and butyric acids were readily being consumed by hydrogen oxidizing methanogens before being converted into acetate. Despite the significant reduction in activities of acetoclastic methanogens to as low as 0.04 g COD/ g VSS/ d at 2.5 kg COD/m3/d methane concentration was still recording high ≥ 50% emitted from CDT. This might have been due to the production of methane from hydrogen-utilizing methanogens (Jawed and Tare, 1999).

The reactor was supplied with OLR 3.5 kg /m3/d for 10 days. The influent COD concentration determined was in the range from 45,700 to 62,300 mg/L. It is clear from Figure 2 showed that increases the OLR from 3.0 to 3.5 kg COD/ m3/ d caused the accumulation of VFA (318 mg/L). These observations are attributed to the increase of OLR and concentration of COD loads into the CDT which resulted in VFA accumulated (Najafpour et al., 2005). At this stage, the CDT has achieved average 16 d HRT with 52 % methane produced and reduction in VSS content. Reduction in VSS content was believed the active biomass had been washout during feeding of influent since increasing of OLR has resulted in the higher amount of POME being introduced. AMA value determined at this stage was 0.13 g COD/ g VSS/ d. It was reported that, acetoclastic methanogens especially Methanosaeta sp. was known as obligate acetate consuming methanogens, because of the low threshold of substrate concentration and longer period of doubling time as long as 12 days (Taconi, 2004). Then it shows beyond sign of recovery with increased in activity (0.13 g COD / g VSS/ d). This may be probably due to stable microbial equivalent achieved by systems after CDT was supplied with POME at OLR 3.5 kg COD /m3/d for 10 days.

The VFA value was increasing when supplied with higher OLR and methane production was recorded 55%. At the end of CDT operation, VFA concentration detected high with 977 mg/L and COD removal efficiency was reduced to 92% (Figure 3). These signs has showed the bioreactor has reached a critical level in which VFA concentration reached near to 1000 mg/L and reduction of COD removal efficiency (Yacob et al., 2006). Despite recording a high of VFA concentration, average methane production was 55% from this stage. The activity values were kept remained at 0.1 g COD/ g VSS/d until the end of operation in which CDT was supplemented with POME at OLR 5.0 kg COD/ m3/d.

The fixed amount of sludge / microorganisms to be tested in the experiments is the important factor. In this test, 20 mL of sludge is supplied in 80 mL synthetic medium with final volume 100 mL and approximately content 0.1 -0.2 g of VSS. This test was used in order to determine potential methane production capacity of the CDT system throughout the operation thus allowing suitable OLR to be applied. Acetoclastic methanogens was the importance microbes that converted acetate to methane in the most anaerobic digesters and about 70% of the methane is formed from acetate and the remaining 30% was from CO2 and hydrogen (McCarty, 1964: Lay et al., 1998). It should be noted that AMA tests only measure the CH4 production from acetate, generally referred to as the acetoclastic methanogenic activity and does not include methane produced by hydrogen-utilizing methanogenic bacteria. However, the sludge obtained from this study not only consists of acetate consuming bacteria, but it also has hydrogen-utilizing methanogens that converting hydrogen and other methylated compounds to methane (Jawed and Tare, 1999).

4. Conclusion

Biogas pilot plant treating POME was successfully performed. From our observation, the AMA test results have shown a better insight into microbiological behaviour and could be used as a tool to determine optimum or maximum organic loading of anaerobic digestion systems. The activity values obtained from this study are within those generally reported, ranging from 0.1-1.0 g COD/g VSS/d. The highest OLR achieved during the study period was 5.0 kg COD /m3 /d with 55%methane concentration at less than 10 days HRT. Our future study of POME treatment will focus on microbial composition, activity and their distribution compared to conventional parameters.


The project was sponsored by FELDA Palm Industries Sdn. Bhd. (FPISB), Malaysia and Japan Society for Promotion of Science (JSPS). The authors would like to thank the management of Serting Hilir Palm Oil Mill for their co-operation and kindness throughout the study.