Enhanced volatile fatty acid production and microbial population analysis in anaerobic treatment of high strength wastewater

Journal of Water Process Engineering 33 (2020) 101058

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Enhanced volatile fatty acid production and microbial population analysis in anaerobic treatment of high strength wastewater

T

J.X. Lima, Y. Zhoub, V.M. Vadivelua,* a b

School of Chemical Engineering, Universiti Sains Malaysia, Engineering Campus, 14300 Nibong Tebal, Penang, Malaysia School of Civil and Environmental Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798, Singapore

A R T I C LE I N FO

A B S T R A C T

Keywords: Volatile fatty acid Acidogenic Anaerobic digestion Microbial analysis

Volatile fatty acid (VFA) is the intermediate product of the anaerobic digestion. Generally, the amount of acidogenic and methanogenic microorganisms will affect the output VFA concentration in a wastewater treatment process. In this study, sequencing batch reactor (SBR) was used to enhance VFA production during the anaerobic treatment of palm oil mill effluent. The system managed to suppress the growth of methanogens and increased VFA output by elevating the growth of acidogens. The SBR managed to achieve stable accumulation of VFA throughout the operation. It was found that the VFA produced increased from 7300 mg/L to 10,500 mg/L; however, the VFA composition remains unchanged. The balanced composition of short-chain VFA could be used as the feed for the production of biodegradable polymer. Microbial analysis revealed that methanogens (Methanosaeta, Pseudomonas, and Bacteroides) were eliminated from the system. In addition, VFA-producing bacteria (Lactobacillus, Olsenella, Aeriscardovia, Pseudoramibacter, and Atopobium) were found to be the dominant bacteria in the reactor.

1. Introduction Traditionally, wastewater treatment has been focused on the removal of nutrients and organic content in wastewater. In recent years, however, the focus has shifted toward the recovery of nutrients and valuable by-products during wastewater treatment. For example, many studies managed to utilize the organic content of wastewater for the production of biogas and polyhydroxyalkanoate (PHA), a biodegradable polymer, through the wastewater treatment process [1–3]. Wastewater of high chemical oxygen demand (COD), especially agricultural wastewater, has high potential for the mass production of PHA and biogas due to the abundance of carbon content [4,5]. The conventional biological treatment of high organic content wastewater includes anaerobic and aerobic processes. In anaerobic treatment, a sequence of processes occurs, namely, hydrolysis, acidogenesis, and methanogenesis. Wastewater will first undergo hydrolysis to breakdown the complex organic content (e.g., carbohydrates, proteins, lipids) to simpler organic compounds such as simple sugars, amino acids, and long-chain fatty acids. In the second phase (acidification phase), the simpler organic compounds undergo acidogenesis to produce organic VFAs, alcohol, and carbon dioxide. In the third phase (methanogenic phase), the products of acidogenesis are used by methanogenic microorganisms to produce methane, carbon

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dioxide, and hydrogen [6]. In the subsequent aerobic treatment, the aerobic microorganisms utilize oxygen to oxidize the organic content of the wastewater carried over from the anaerobic treatment to meet discharge requirements. Although the conventional treatment system successfully minimizes the organic content, as well as nutrient content, it misses the opportunity to harness useful nutrients and by-products during the treatment. Therefore, it is beneficial to revise the treatment system to maximize the potential recovery of resources. One of such highly valuable intermediate products during the wastewater treatment is VFA. During the acidogenesis process, VFA will be produced. However, these VFA will be converted into methane gas in the following methanogenic phase. Methane gas is proven as an energy source, which can be converted into electricity, but not all treatment plants have the facility to convert the energy source into electricity. Alternatively, VFA can be used as the substrate to produce PHA. PHA is a biodegradable plastic monomer accumulated naturally inside certain microorganisms involved in wastewater treatment. To utilize VFA as a substrate to accumulate PHA inside microorganisms, the production of VFA has to be optimized. The operating conditions play an important role in optimizing the production of VFA. Among the parameters proven to have an effect on promoting VFA are hydraulic retention time (HRT), solid retention time (SRT),

Corresponding author. E-mail address: [email protected] (V.M. Vadivelu).

https://doi.org/10.1016/j.jwpe.2019.101058 Received 19 August 2019; Received in revised form 4 November 2019; Accepted 6 November 2019 2214-7144/ © 2019 Elsevier Ltd. All rights reserved.

Journal of Water Process Engineering 33 (2020) 101058

J.X. Lim, et al.

return sludge, the SRT of the reactor was equal to the HRT of the biomass in the reactor. The HRT was kept constant throughout the study. The reactor was operated at room temperature (29 ± 1 °C). The pH of the medium was monitored throughout the process (varying from 4.8 to 5.5). The POME was the only feed to the reactor.

temperature, pH, and the availability of micronutrient in the treatment system [7–9]. In general, methanogens have longer generation time than facultative anaerobic and aerobic microorganisms. An anaerobic digestion unit that includes methane gas generation usually has SRT more than 12 days, to allow methanogen to grow [10,11]. Earlier studies suggested that an anaerobic reactor operated at shorter SRT (lower than 10 days) could achieve an VFA increase of about 10–25 % by eliminating the methanogen [12]. However, conventional anaerobic digester favours high SRT value to achieve higher COD removal efficiency, provides buffer capacity for resistance against shock loadings, and maintain population of mixed culture. However, studies on enhanced VFA production and microorganisms responsible for the VFA enhancement are still limited. It was previously found that bacteria of phylum Firmicutes is dominant in anaerobic reactor [13,14]. Nevertheless, information on the relationship between the microbial community and the output VFA composition is still scarce. The complete reaction in anaerobic digestion will yield methane, carbon dioxide, and hydrogen. To avoid the complete reaction, the methanogenesis process need to be ceased. The elimination of methanogenesis can be carried out by limiting the growth of methanogens through the reduction of retention time of biomass in the reactor. As such, the slower growing methanogen will be washed out from the system. Furthermore, the characterization of the microorganisms in enhanced VFA production has not been fully investigated. It is generally accepted that the abundance of both acidogenic and methanogenic microorganisms are predominant factors in the output VFA concentration. Thus, in this study, the shift of the microbial community, associated with the enhancement of VFA production, will be investigated.

2.3. Monitoring the acidogenic treatment of POME in SBR The operation of the SBR-treating POME was monitored through the reactor performance and VFA accumulation for a period of 30 days. 2.3.1. Analytical method The SBR was operated with constant reaction time, influent feed volume, and influent feed COD. Liquid-phase mixed liquor samples were withdrawn from the SBR to evaluate the performance of the reactor. The samples were used to measure mixed-liquor suspended solids (MLSS), mixed-liquor volatile suspended solids (MLVSS), VFA, and COD according to the American Public Health Association (APHA) standard method [15]. The performance of the SBR was determined on the basis of COD removal as well as biomass concentration (MLVSS). 2.3.2. VFA extraction and quantification VFA was analyzed using gas chromatography (GC). Mixed-liquor samples were collected from the SBR. The mixed-liquor samples were centrifuged at 4500 rpm for 15 min to separate the liquid phase and solid phase content. The supernatant was filtered using a syringe filter (ø 22 mm and mesh size of 0.45 μm) and then injected into the GC instrument. The inlet flame ionizer was set to 200 °C while the detector temperature was set to 220 °C. The GC column was able to measure a range of volatile fatty acid content, including acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, and heptanoic acid.

2. Material and methods

2.3.3. Gas collection and analysis Gas samples were collected from the SBR by using 1 L Supelco’s Gas Tedlar sampling bag. The collected samples were tested in a Gas Chromatography equipment (Agilent 7820A). Porapak N column was used to separate carbon dioxide, carbon monoxide, hydrogen, methane, oxygen, and nitrogen. A flow of argon as carrier fixed at 40 mL/min was used for sample elution in Porapak N. The gas chromatograph injector temperature was set at 140 °C. The initial and final temperatures of the oven were set at 140 °C and 200 °C, respectively. The total analysis time was 25 min for each complete injected sample analysis.

2.1. Palm oil mill effluent (POME) and seed sludge The POME used in this study was collected from the effluent of Hilltop Palm Oil Mill, Bagan Serai, Perak and stored in a cold room at a temperature of 4 ± 1 °C. The required volume of POME was acclimatized to room temperature (28 ± 2 °C), filtered to remove debris, before it was fed into the sequencing batch reactor (SBR). The seed sludge was obtained from the anaerobic pond of the same palm oil mill. Table 1 shows the characteristics of the POME used in this study. 2.2. Operation of reactor

2.4. DNA extraction and sequencing data analysis

2.2.1. SBR start-up and operation A 12-L SBR with an effective working volume of 8 L was used. The height over diameter ratio of the SBR was 6.67. The feed was pumped into the SBR via a peristaltic pump (Cole-Parmer Masterflex 7523-20), and the effluent was discharged using a magnetic valve. The SBR was operated in a 24-hr cycle with three phases, which included a filling, anaerobic reaction, and decanting phase. The times allocated for each phase were 15 min, 23 h 30 min, and 15 min, respectively. In each cycle, 1.6 L of POME was fed into the SBR and 1.6 L of the treated effluent was taken out during the decanting phase, leading to an HRT of 5 days. Since there was no settling phase in this SBR and no

Biomass samples were obtained from the seed sludge and enrichment SBR. DNA was extracted from the collected biomass using an automated DNA extraction kit (MP Biomedicals, Singapore). Primer 515 F (50-GTGCCAGCMGCCGCGGTAA-30) and 806R (50-GGACTACN NGGGTATCTAAT-30) were used for high-throughput sequencing, targeting the V4 region of 16S rRNA genes. Bacterial and archaeal 16S rRNA genes were amplified by polymerase chain reaction (PCR). All PCR reactions were carried out with Phusion® High-Fidelity PCR Master Mix (New England Biolabs). The amplification of 16S rRNA/18SrRNA/ ITS genes of distinct regions (16SV416SV3/16SV3/-V4/16SV4-V5, 18S V4/18S V9, ITS1/ITS2, Arc V4) took place using a specific primer (e.g. 16S V4: 515 F-806R, 18S V4: 528F-706R, 18S V9: 1380F-1510R, et. al) with a barcode. The same volume of 1× loading buffer (contained SYB green) was mixed with PCR products, and electrophoresis was conducted on 2 % agarose gel for detection. Samples with a bright main strip between 400–450 bp were chosen for further experiments. PCR products was mixed in equidensity ratios. Then, the mixed PCR products were purified using the Qiagen Gel Extraction Kit (Qiagen, Germany). The libraries were generated with the NEBNext® Ultra™ DNA Library Prep Kit for Illumina and quantified via Qubit and Q-PCR. After removing the adapter primer, barcodes, and low-quality sequences, the

Table 1 Characteristics of Palm Oil Mill Effluent (POME). Parameter

Unit

Range

Average Value

COD NH3-N COD:N ratio MLVSS VFA pH

mgCOD/L mg/L – mgVSS/L mg/L

30800–35970 262–297 – Negligible 6978–7621 4.5–4.7

33400 280 595:5 Negligible 7300 4.6

2

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removed from the system in terms of methane gas. The lack of the methanogenesis stage in the SBR was closely related to the microbial population in the mixed culture. Various group of bacteria are needed to enhance the VFA concentration in the SBR for PHA production. There were two major groups of microorganisms present in the anaerobic digestion reactor, namely acidogens and methanogens. The acidogens utilized the organic content (carbohydrates, proteins, and lipids) and produced short-chain fatty acids (SCFA), hydrogen, and carbon dioxide. In contrast, the methanogens utilized the SCFA produced by the acidogens to produce methane and carbon dioxide. A biological wastewater treatment unit needs both acidogens and methanogens to perform complete anaerobic treatment. The growth of both acidogens and methanogens is affected by the operational parameters of the anaerobic reactor such as SRT, HRT, pH, and temperature. In SBR, the growth of acidogens was faster than the growth of methanogens. This could be observed in Fig. 1, where the VFA concentration increased rapidly and reached the plateau stage. The average VFA production was 43.8 % of the original VFA content in raw POME. Wong et al. (2014) [16] used anaerobic reactor to produce methane gas. Their study showed that the POME of 12,170 mgCOD/L·day-1 organic loading rate was able to accumulate about 2584 mg/L (as acetic acid) VFA, which was lower than the findings of this study in terms of percentage VFA produced per influent COD. Increasing the HRT of reactor might be good for VFA accumulation since the contact time of microorganisms and feed increases [17]. However, it was also suggested that the reactor operating at prolonged HRT could lead to stagnant accumulation of VFA [18]. Lim et al. [18] had observed that the yield of VFA at HRT of 4, 8, and 12 days were in the range of 0.26–0.32, 0.34–0.37, and 0.36–0.39 mgVFA/mgCOD, respectively, showing that the prolonged HRT did not affect VFA accumulation significantly. Some of the VFA was converted to methane gas and H2 through methanogenesis. In this study, the VFA yield in the SBR was about 0.41 ± 0.05 mgVFA/mgCOD at an HRT of 5 days, which was higher than the reported value by Lim et al. [18]. Table 4 shows the concentration and composition of VFA in the feed and in the SBR on Day 1 and 30. On the first day of the operation of the SBR, the concentration of acetic acid and propionic acid were reduced slightly. Meanwhile, the concentration of butyric acid, valeric acid, caproic acid, and heptanoic acid increased. Once the SBR acclimatized and reached steady-state (Day 30), the concentration of all the detected VFA species had increased. Yet, the composition of the VFA species present in the SBR was balanced and did not change significantly compared to the initial composition in the feed. However, the VFA accumulated through complete anaerobic digestion generally had a higher acetic acid composition than the other VFA (propionic, butyric, valeric, caproic, and heptanoic acid). Previously, it was reported that microorganisms could accumulate PHA at around 80 wt% (in the form of polyhydroxybutyrate (PHB)) when the VFA, consisting of high acetic acid composition, was used as the feed [19]. However, it is well known that a biopolymer with only one type of PHA (scl-homopolymer) in its composition, such as PHB, has limited usage as it is brittle and stiff [20]. In comparison, PHA of variable short-chain-length monomers (sclcopolymer), such as polyhydroxybutyrate-co-valerate (PHB-co-HV), is more desirable because it is less crystalline, has a lower melting point, is easier to mold, and is tougher than PHB [21]. VFA, which contains both even- (acetic and butyric acids) and odd-numbered (propionic and valeric acids) carbon sources, is essential for the production of such biopolymers [22]. Thus, the balanced composition of VFA in this study could be the better option for PHA production because the balance oddand even-numbered VFA can produce PHB-co-HV polymer.

Table 2 The operating conditions and characteristics of effluent of SBR after steadystate. Parameter

Unit

Average Value at Steady-State

Temperature Qin VR OLR COD in Reactor NH3-N COD:N ratio MLVSS F/M ratio VFA pH

°C L/day L mgCOD/L·day mgCOD/L mg/L – mgVSS/L mgCOD/mgVSS mg/L

29 1.6 8.0 6680 25700 264 473:5 1220 5.48 10,500 5.13

resulting sequences were performed on an Illumina MiSeq platform by Novogene Inc. (China). Operational taxonomic units (OTUs) were categorized at a 97 % similarity threshold. The taxonomic assignment of OTUs was performed by using the SILVA Database. 3. Results and discussion 3.1. The performance of SBR 3.1.1. Start-up of SBR Raw POME without dilution was used as feed for the SBR. In general, a wastewater treatment unit needs about 3–4 SRT to achieve steady-state condition [11]. Steady-state condition implies that the generation of biomass (represented by MLVSS) and the biomass wasted in the reactor are the same. In this study, it took about 12 days for the reactor to achieve steady-state with an average MLVSS value of 1220 mg/L (stable MLVSS in the reactor). Once the reactor achieved steady-state, solid, liquid, and gas samples were taken from the reactor for analysis, and the average values were presented in Table 2 and Table 3. In this study, the SBR achieved a COD removal of 23 % because the organic content in the feed did not undergo the complete anaerobic treatment process. Fig. 1 shows the profile of MLVSS and VFA in the SBR throughout the operation. In this study, the input concentration of the VFA in the SBR was 7300 mg/L. The total VFA concentration in the SBR increased to about 8530 mg/L in the first 5 days. Meanwhile, the total VFA concentration after 15 days (3 HRT) of operation managed to reach a concentration of about 10,500 mg/L. Beyond this point, the total VFA concentration level in the SBR was stable, around 10,500 ± 500 mg/L throughout the operation. Although the biomass concentration in the reactor experienced a reduction, it did not reduce the performance to accumulate VFA; instead, the total VFA concentration remained at about 10,500 mg/L. On the other hand, methane was not detected in the gas collected from the SBR (Table 3). The absence of methane in the output gas has proven that the SBR carried out a partial anaerobic wastewater treatment where it did not undergo the methanogenesis stage. Thus, the low COD removal (23 % COD removal) in this reactor suggested that the COD (organic component) in the reactor was transformed from a complex organic substance into simpler molecules (VFA) but not Table 3 The gas composition in SBR after steady-state. Gas Component

Average volume percentage, (%)

Standard deviation, (%)

Carbon dioxide Hydrogen Oxygen Nitrogen Methane

60.9 3.3 7.3 28.5 Not detected

± 6.6 ± 0.7 ± 1.9 ± 5.1 –

3.2. Microbial population shift in SBR In this study, samples were collected for microbial analysis at different stages. The analyzed microbial population were listed in Table 5 3

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Fig. 1. The MLVSS concentration and VFA concentration profile in anaerobic reactor (SBR) operating at SRT = HRT = 5 days; mesophilic temperature, 29 ± 1 °C; and no pH control.

to the growth of bacteria in the phyla group of Actinobacteria and Firmicutes but unsupportive to the growth of the phyla group of Bacteroidetes and Proteobacteria. This might be due to the acidic environment in the SBR. Most of the bacteria associated with the phyla Actinobacteria and Firmicutes in this study are gram-positive bacteria, so they are more tolerable of acidic conditions than gram-negative bacteria. In that sense, the gram-positive bacteria could imply several countermeasures toward acidic conditions, such as alteration of cellmembrane composition, changing metabolic pathways, extrusion of protons, protection of macro-molecules, and generation of alkali [26]. The information about the phyla group is valuable to understanding the activity of microorganisms. This study showed that some of the anaerobic bacteria in the phylum Firmicutes and Actinobacteria, such as Lactobacillus and Olsenella, were able to adapt to the operating condition and, thus, became the dominant group in the reactor. The mentioned groups were able to grow and produce VFA as a product of their biochemical activity in the SBR, similar group of microorganism was found to produce VFA through fermentation of acidic wastewater [27,28]. The production of organic acids reduces the pH in the SBR and leads to acidification in the reactor. The acidic condition was hostile to some dominant species in the seed such as Methanosaeta (phylum Methanosarcinales), Pseudomonas (phylum Proteobacteria), and Bacteroides (phylum Bacteroidetes), causing their population in the SBR to decline. As the population of acid-utilizing methanogens declined, less VFA was converted to methane, instigating the accumulation of VFA in the reactor. The cycle of acid generation and methanogen inhibition continued and, eventually, the genus Methanosaeta was eliminated from the SBR. The dominance of Actinobacteria and Firmicutes suggested that they are the major bacteria that contributes to the accumulation of VFA in the SBR. Bacteria such as Bacteroidetes and Clostridium secretes the hydrolytic enzymes needed for the degradation of carbohydrates, proteins, and lipids [29]. Corresponding to the first step in anaerobic digestion, the availability of Bacteroidetes in this study suggests that the hydrolysis stage was not the limiting process in the SBR. In the second step of anaerobic digestion, long-chain fatty acids are broken down to shorterchain fatty acids. The bacteria in the phyla group of Firmicutes are related to the conversion of simple sugars into organic acids. Bacteria of classes Clostridia (phylum Firmicutes) were found to degrade organic matter to produce various organic acids. Meanwhile, some Actinobacteria were found to be the bacteria that contribute to propionic acid accumulation [29]. In the SBR, the bacteria of phyla Firmicutes and Actinobacteria were abundant. Firmicutes such as Lactobacillus were able to produce lactic acid through the Embden-Meyerhof pathway while Actinobacteria such as the Bifidobacteriaceae family and Coriobacteriaceae family were able to convert the lactic acid into acetic acid and propionic acid. Furthermore, the presence of Pseudoramibacter (phylum

as Day 0 (seed sludge + POME) and Day 30. At Day 0, the microbial population consisted of about 48.0 % archaea and 52.0 % bacteria. About 99.98 % of the identified archaea was assigned to the order Methanosarcinales, which belongs to the phyla Euryarchaeota. On the other hand, the identified bacteria were affiliated with the phyla mentioned: Proteobacteria (31.0 %), Bacteroidetes (12.0 %), Firmicutes (6.0 %), Actinobacteria (2.0 %), and Unclassified phyla (13.5 %). On Day 30, it was observed that the total archaea had been reduced from 48.0 % to 0.005 %, while the total bacteria increased from 52.0%–99.99 %. At this stage, the bacteria in the reactor was assigned to Actinobacteria (49.0 %), Firmicutes (41.0 %), Bacteroidetes (6.0 %), and Proteobacteria (3.0 %). In general, the relative abundance of methanogenic archaea in the SBR had been reduced to a very small amount. The observation of the microbial shift suggested that the microorganism communities in the SBR were highly affected by the characteristics of the operating conditions. Previous studies have suggested that acidogens are more dominant under low SRT conditions (SRT < 8 days), while methanogens are more dominant under conditions of more than 10 days [23]. Therefore, an SRT lower than 8 days does not favor the growth of methanogens. In biological wastewater treatment units, microorganisms of phyla Methanosarcinales are able to produce methane gas as the end product of their biochemical activity [24,25]. In this study, after the acidogenic reactor reached steady-state condition (30 days), the amount of methanogen in the reactor was reduced to a negligible amount. The low population of methanogenic archaea agrees with earlier observations that suggested that there was no methane gas detected in the SBR. This shows that the applied operating condition in the SBR successfully suppressed the growth of methanogens, perhaps due to the fact that the methanogens has a slower growth rate than acidogens, and methanogens are more dominant at SRT > 10 days [23]. Since the methanogens are the major converter of organic acids into methane gas, the incomplete anaerobic treatment of wastewater often leads to the acidification of the treatment unit, causing process failure in the system. However, this study took advantage of the acidification in the reactor to enhance the VFA production through eliminating the consumption of VFA by methanogens. Therefore, manipulating the SRT of the treatment unit is the key factor to influencing the VFA accumulation in the reactor. This prevents the generated VFA from being used by methanogens as a carbon source. The relative abundance of Actinobacteria and Firmicutes increased significantly during the acclimatization stage. The relative abundance of bacteria of phyla Actinobacteria increased from 2.0%–49.0 % while the bacteria of phyla of Firmicutes increased from 6.0%–41.0 %. On the other hand, the relative abundance of Proteobacteria decreased from 31.0%–3.0 %, while that of Bacteroidetes decreased from 12.0%–6.0 %. The results show that the operational condition applied was favorable 4

Journal of Water Process Engineering 33 (2020) 101058

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Fig. 2. Comparison of the bacteria and archaea communities in a) seed sludge and b) SBR based on relative abundance. The affiliation of the bacterial communities was also assessed at the class level. Fig. 2 shows the comparison of the bacteria and archaea communities in seed sludge and SBR based on the percentage population. In the seed sludge, almost all of the identified archaea is of the class Methanomicrobia. Meanwhile, the major classes of identified bacteria in the feed are Gammaproteobacteria, Bacteroidia, Clostridia, and Bacilli. Once the SBR had adapted to the operating condition, the major classes of the bacteria detected in the reactor were Coriobacteriaceae, Unidentified actinobacteria, Bacilli, Clostridia, Bacteroidia, and Alphaproteobacteria.

information about the function of the communities in the reactor. In the seed sludge, the dominant genera were Methanosaeta (48 %), Pseudomonas (28 %), and Bacteroides (8 %). The population of genus Methanosaeta was high, which is common due to the seed sludge that

Firmicutes) in the SBR also explained the accumulation of VFA since the genus was able to convert fermentable carbohydrates into various organic acid. At the genus level, the bacteria detected can provide valuable 5

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Table 4 The composition of VFA in feed and SBR. Feed

Acetic acid Propionic acid Butyric acid Valeric acid Caproic acid Heptanoic acid Total VFA

Day 1

Day 30

Concentration*

Composition**

Concentration*

Composition**

Concentration*

Composition**

9.90 9.90 15.60 17.50 16.70 8.10 77.70

12.74 12.74 20.08 22.52 21.49 10.42

9.20 9.49 19.19 19.24 18.39 9.12 84.59

10.83 11.22 22.68 22.75 21.74 10.78

14.36 14.49 23.55 23.88 23.18 11.44 110.90

12.95 13.07 21.24 21.53 20.90 10.32

* values stated are in mmol/L. ** values stated are in percentage.

and acetic acid [32]. Due to the increase in carbon dioxide gas concentrate in the earlier observation, it was believed that the genus Lactobacillus in this study underwent the heterofermentative process as it grew in the SBR. Several other genera such as Olsenella, Aeriscardovia, Atopobium, and Unidentified Coriobacteriaceae are the major bacteria population in the SBR. The mentioned genera were classified in the family Coriobacteriaceae [33,35]. Similar to genus Lactobacillus, the members of the Coriobacteriaceae family are also able to convert carbohydrates to acetic acid and lactic acid as the end-product of metabolic activity [32]. In addition, genus Pseudoramibacter was also present in the SBR. The genus can produce a variety of organic acids such as formate, acetate, butyrate, and caproate as the end-product of fermentation in the presence of glucose [36].

Table 5 The number of 16S rRNA gene copies of total archaea and bacteria in SBR. Target Group

Total Archaea Methanobacteriales Methanomicrobiales Methanosarcinales Methanococcales Total Bacteria Proteobacteria Bacteroidetes Firmicutes Actinobacteria Unclassified phyla

16S rRNA gene Relative Abundance (%) Day 0

Day 30

26814 – – 26727 (48.0) – 28364 17142 (31.0) 6435 (12.0) 3351 (6.0) 1118 (2.0) 4

3 – – 3 (0.005) – 55164 1680 (3.0) 3492 (6.0) 22,586 (41.0) 27,236 (49.0) –

4. Conclusions was originated from the anaerobic reactor in actual palm oil mill. Under anoxic conditions, where the light source and alternate electron acceptors other than carbon dioxide are lacking, the members of the genus Methanosaeta were capable to catabolize acetate into methane and carbon dioxide [29]. They could be the major cause for the decrease in the acetate concentration in anaerobic reactors. The absence of the genus Methanosaeta in the SBR explained the build-up of acetate without being utilized. The genus with the second highest population in the seed sludge was Pseudomonas. Most species in this genus are aerobic bacteria, which utilized oxygen as the electron acceptor. Some of the species in the genus Pseudomonas were able to convert carbohydrate monomers of more than four carbon chains into polyhydroxyalkanoates during growth [30]. None of the members in genus Pseudomonas can tolerate acidic conditions, with total inhibition occurring at pH 4.5 [30]. The third largest genus in the seed sludge was Bacteroides. This genus is anaerobic and saccharolytic [31]. The major end-product of their growth through fermentation are succinate and acetate [31]. The population of Bacteroides was low compared to that of Methanosaeta and Pseudomonas, perhaps due to sources that do not suit the optimum growth condition (37 °C, pH 7.0) [31]. Meanwhile, after achieving steady-state in the SBR, the population percentage of the bacteria of genera Lactobacillus, Olsenella, Aeriscardovia, Pseudoramibacter, Atopobium, Unidentified Coriobacteriaceae, and Prevotella increased significantly. These genera share some common characteristics as most of them are obligate anaerobic or facultative anaerobic bacteria, gram-positive, tolerant to mildly acidic environments, mesophilic bacteria, and able to produce organic acids from fermentable carbohydrates [32–34]. The genus Lactobacillus was the largest genus in the SBR. The group is strictly fermentative and able to utilize carbon sources fermentatively in two ways. During the homofermentative process, a carbon source was utilized to produce lactic acid. Meanwhile in the heterofermentative process, glucose was converted to lactic acid, carbon dioxide, ethanol,

This study shows that the microbial population is crucial to the accumulation of VFA. The microbial population shift analysis indicates that the operating condition of the SBR favors the growth of acidogens and limits the growth of methanogens. The anaerobic bacteria of genera Lactobacillus, Olsenella, Aeriscardovia, Pseudoramibacter, and Atopobium managed to prevail and generate VFA in the SBR. Meanwhile, the operating condition did not favor the growth of Methanosaeta, Pseudomonas, and Bacteroides. Additionally, the SBR managed to increase the VFA accumulation to about 10,500 mg/L total VFA, which is an accumulation of about 43.8 % of VFA in the feed. The output of the SBR had a balanced composition of VFA, which is suitable for production of scl-copolymer. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements The Universiti Sains Malaysia is thankfully acknowledged for funding this research through Bridging Grant Scheme (Grant No. 304.PJKIMIA.6316120) and Research University Grant Scheme Grant No. 1001.PJKIMIA.8014066). References [1] E.C. Koutrouli, H. Kalfas, H.N. Gavala, I.V. Skiadas, K. Stamatelatou, G. Lyberatos, Hydrogen and methane production through two-stage mesophilic anaerobic digestion of olive pulp, Bioresour. Technol. 100 (2009) 3718–3723. [2] J. Nikodinovic-Runic, M. Guzik, S.T. Kenny, R. Babu, A. Werker, K.E.O. Connor, Chapter Four - carbon-rich wastes as feedstocks for biodegradable polymer (polyhydroxyalkanoate) production using bacteria, in: S. Sima, M.G. Geoffrey (Eds.), Advances in Applied Microbiology, Academic Press, 2013, pp. 139–200.

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