Biogas production from palm oil mill effluent and empty fruit bunches by coupled liquid and solid-state anaerobic digestion

Journal Pre-proofs Biogas production from palm oil mill effluent and empty fruit bunches by coupled liquid and solid-state anaerobic digestion Wantanasak Suksong, Wisarut Tukanghan, Kanathip Promnuan, Prawit Kongjan, Alissara Reungsang, Heribert Insam, Sompong O-Thong PII: DOI: Reference:

S0960-8524(19)31534-2 https://doi.org/10.1016/j.biortech.2019.122304 BITE 122304

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Bioresource Technology

Received Date: Revised Date: Accepted Date:

24 September 2019 17 October 2019 18 October 2019

Please cite this article as: Suksong, W., Tukanghan, W., Promnuan, K., Kongjan, P., Reungsang, A., Insam, H., OThong, S., Biogas production from palm oil mill effluent and empty fruit bunches by coupled liquid and solid-state anaerobic digestion, Bioresource Technology (2019), doi: https://doi.org/10.1016/j.biortech.2019.122304

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Biogas production from palm oil mill effluent and empty fruit bunches by coupled liquid and solid-state anaerobic digestion

Wantanasak Suksong a, Wisarut Tukanghan a, Kanathip Promnuan a, Prawit Kongjan b, Alissara Reungsang c, Heribert Insam d and Sompong O-Thong a,e*

a Biotechnology

Program, Faculty of Science, Thaksin University, Phatthalung 93210,

Thailand b Chemistry

Division, Department of Science, Faculty of Science and Technology, Prince of

Songkla University, Pattani, 94000, Thailand c Department

of Biotechnology, Faculty of Technology, Khon Kaen University, Khon Kaen

40002, Thailand d

Institute of Microbiology, University of Innsbruck, Technikerstr. 25, 6020, Innsbruck,

Austria e Research

Center in Energy and Environment, Faculty of Science, Thaksin University,

Phatthalung 93210, Thailand

*Corresponding author e-mail: [email protected]; [email protected]

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Abstract Biogas production of palm oil mill effluent (POME) and empty fruit bunches (EFB) was performed by coupled liquid (L-AD) and solid-state (SS-AD) anaerobic digestion processes. POME was fed to L-AD digester, while mixed of effluent from L-AD and EFB was fed to SS-AD digester. The maximum overall methane production of 60.9 m3-CH4ton1

waste was obtained at an optimal hydraulic retention time of 30 days and an organic

loading rate of 1.66 gVSL-1-reactord-1for L-AD and 6.03 gVSL-1-reactord-1 for SS-AD with L-AD effluent recycling rate of 16.7 mLL-1-reactord-1. The bacterial community in the L-AD reactor was different from the SS-AD reactor, while the archaeal community was similar in both reactors. Synergistaceae, Caldicoprobacteraceae and Lachnospiraceae were increased in the SS-AD reactor. Coupling L-AD and SS-AD is able to increase energy production by 29% and 71% compared to the L-AD and SS-AD alone, respectively, with no outsource SS-AD inoculum required.

Keywords: Solid-state anaerobic digestion, liquid-state anaerobic digestion, biogas production, palm oil mill effluent, empty fruit bunches.

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1. Introduction The worldwide demands for palm oil are continuously increasing (Silalertruksa et al., 2017). In the palm oil mill extraction process, palm oil accounts for only 20% of the total biomass. The remaining 80% of biomass is often discarded as waste. It is common that one tonne of fresh fruit bunch releases almost 0.5–0.75 tons of palm oil effluent (POME) and 0.2-0.3 tons of empty fruit bunches (EFB). The crude palm oil (CPO) extraction process generates a large amount of palm oil mill effluent (POME) with 0.5-0.75 m3tonne-1 oil palm fresh fruit bunches. POME is a combination of wastes, which are discharged from three sources of extraction process such as clarification wastewater (60%), sterilizer condensate (36%), and hydro-cyclone wastewater (4%) (Ahmed et al., 2015). In recent years, the further utilization of palm oil mill residues for biogas production has been focused only on POME. It is calculated that around 28 m3 of biogas may be generated from one m3 of POME in the treatment plant under mesophilic (30-40 ˚C) conditions. Liquid anaerobic digestion process (L-AD) is the most suitable approach for its treatment. However, a large amount of methanogenic effluent is generated from the L-AD process, which is commonly treated in an oxidation pond (Awalludin et al., 2015). Besides the disadvantage of the large area required, the escaping greenhouse gases (GHGs) pose a serious environmental problem of this treatment system (Choong et al., 2018). Suksong et al. (2019) found that the liquid effluent from anaerobic digestion of POME may serve as a suitable inoculum for the solid-state anaerobic digestion (SS-AD) of EFB in terms of cellulose degradation efficiency, biogas production, and microbial activity.

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EFB has high moisture content and is not suitable for use as fuel in biomass power plants due to its high K content (20.5 gkg-1 EFB) resulting in slag on the surface of the water tubes and grate floor in the combustion chamber. Fast accumulation of K crust on tube surface hinders heat transfer worsens fuel economy (Tan et al. 2018). Therefore, solidstate anaerobic digestion of EFB in a single step treatment would simplify the technical and economic requirements for the transformation of solid wastes into biogas and compost. Solid-state anaerobic digestion may produce 70 m3 CH4tonne-1 EFB with using liquid effluent sludge as inoculum under batch condition. The methane production of the SS-AD process was 2-3 times higher per unit fresh mass than the L-AD process (Suksong et al. 2016). However, the SS-AD process required a large amount of inoculum for start-up and operation. The solid digestate from SS-AD is inefficient as inoculum due to its high content of undigested organic materials such as lignin with low microbial activity (Meng et al. 2019). Inoculating L-AD digested sludge to a semi-continuous SS-AD reactor fed with EFB doubled methane production compared to recycled SS-AD digested sludge (Suksong et al. 2019). Therefore, the SS-AD process of EFB with recycling effluent of POME-biogas reactor as inoculum would simplify the technical and economic requirements for the transformation of POME and EFB into biogas and reduce the amount of effluent from a POME-biogas reactor. Estevez et al. (2014) found that anaerobic digestion of Salix viminalis with recycling effluent from a liquid biogas reactor at a ratio of 1:1 (w/v) increased methane yield up to 21%. The liquid AD effluent could improve the preservation of microbes, nutrients, and buffer capacity. Some studies showed that SS-AD might suffer from nitrogen deficiencies when using lignocellulose biomass as substrates, which liquid

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effluent recycling could offset by supplementing nitrogen to the SS-AD reactor (André et al. 2018; Xu et al. 2018). Therefore, this study investigated the coupling of an L-AD and an SS-AD reactor for biogas production from POME and EFB. We hypothesize that (i) the stability of an SSAD reactor increases upon recycling of methanogenic effluent from an L-AD reactor; (ii) the overall output increases by coupling L-AD and SS-AD; (iii) the coupling may be set in operation by making use of existing digesters. The stability of coupling L-AD and SS-AD was evaluated by methane production, biodegradation testing and microbial community analysis to understand the coupling L-AD and SS-AD process.

2. Materials and methods 2.1 Feedstocks and inoculums The basic characteristics of the palm oil mill effluent (POME) and empty fruit bunches (EFB) are shown in Table 1. POME, EFB, and anaerobically digested sludge were collected from the crude palm oil extraction factory at the Pitak palm oil company, Trang province, Thailand. EFB was screw pressed to remove oil and moisture. EFB were cut to size < 0.5 cm. POME and EFB were stored at 4°C before use. For concentrating, the anaerobically digested sludge inocula were sedimented by gravitation and the supernatant was discarded to concentrated the sludge. The sludge with a volatile solids (VS) content of 75-80 gL-1 was used as an inoculum in both the AD process (Suksong et al. 2016). 2.2 Experimental design for coupled L-AD and SS-AD process

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Continuous L-AD and SS-AD reactors were carried out in horizontal CSTR reactors with a working volume of 5 L (Fig 1). In both reactors were operated at a temperature of 40 ºC by circulating hot water inside a coil surrounding each reactor. The L-AD reactor was started with 30% (v/v) of methane-producing sludge. The 33.3 mL L-1d-1 of POME was fed to the reactor for 21 days until a working volume of 5 L was reached. After that, the LAD reactor was operated at hydraulic retention times (HRT) of 30 days and an organic loading rate (OLR) of 2.14 gCODL-1d-1 with a POME at a feeding rate of 33.3 mLL-1d-1. The SS-AD reactor was started with 30% (v/v) of methane-producing sludge. The 11.1 gVSL-1d-1 of EFB and 22.2 mLL-1d-1 of L-AD effluent were mixed and added to the reactor for 21 days until the working volume of 5 L was reached. After that, the SS-AD reactor was operated for 30 days of hydraulic retention time (HRT) with an organic loading rate (OLR) of 6.03 gVSL-1d-1. At different stages of the SS-AD reactor operation, the LAD effluent recycling rate was 22.2(R1), 16.7(R2), 11.1(R3), 16.7(R4), and 0.0(R5) mLL1d-1,

respectively. As a control, R5 was run without L-AD effluent recycling. A gas counter

was installed in each reactor to record the gas amount automatically. The coupled L-AD and SS-AD reactors were routinely monitored for pH, gas production and gas composition. 2.3 Analytical methods The compositions of POME and EFB were analyzed for moisture content, total solids, volatile solids, total Kjeldahl nitrogen, pH, and lipid contents according to APHA (2012). Elemental compositions of the POME and EFB were performed in a CHNS/O Analyzer, (Flash EA 1112 Series, Thermo Scientific, Netherlands) with dynamic flash combustion at 900 ºC for C, H, N and S, and at 1060 ºC for O (Lesteur et al., 2010).

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Chemical oxygen demand and ammonium nitrogen contents were determined by the closed reflux colorimetric test (Merck test kit, 500-10,000 mg∙L-1) and spectroquant ammonium cell test (Merck test kit, 0.013-3.86 mg∙L-1), respectively. The lignin, cellulose, and hemicellulose contents were determined according to the procedures proposed by Van Seot et al. (1991). The total alkalinity of digested sludge was measured according to APHA (2012). The liquid digested sludge was collected by a syringe (1 ml) and filtered through a nylon membrane (0.2 µm) (Raposo et al., 2015). Filtrated samples were acidified to pH 3– 3.2 with 30% (v/v) phosphoric acid (Raposo et al., 2015) for volatile fatty acids (VFAs) analysis by a gas chromatograph GC-17A (Shimadzu, Kyoto, Japan) with a stabilwax®-DA fused silica column connected to a flame ionization detector (FID) (Suksong et al., 2019). The biogas composition (H2, N2, CH4, and CO2,) was measured with a gas chromatograph GC-8A (Shimadzu, Kyoto, Japan) equipped with thermal conductivity detector (TCD) connected to a 1-meter stainless steel column packed with Shin Carbon (60/80 mesh) (Suksong et al. ,2016). 2.4 Microbial community analysis Digested sludge samples from L-AD and SS-AD reactors were analyzed by 16S rRNA sequencing using MiSeq technology. The genomes of digested sludge from L-AD and SS-AD reactors were extracted for DNA using a PowerSoil DNA kit (MO BIO, Carlsbad, CA, USA) according to the manufacturer’s instructions. The V3–V4 regions of the bacterial and archaeal 16S rRNA gene fragments were amplified by polymerase chain reaction (PCR) with the forward primers:5’-TCGTCGGCAGCGTCAGATGTGTATA AGAGACAGCCTACGGGNGGCWGCAG-3’ and reverse primers :5’GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAAT

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CC-3. The qPCR Quantification Protocol Guide of Illumina Sequencing platforms (KAPA Library Quantification kits) was used for the quantification of the purification product. The paired-end (2×300 bp) sequencing was performed by Macrogen Inc, using the Illumina Miseq platform (San Diego, CA, USA). The phylogenetic affiliation of each sequence was analyzed with the ribosomal database project (RDP) and NCBI nucleotide database search against the non-metagenomes and non-environmental sequences in the BLAST homology (Tatusova et al., 2016). The complete linkage clustering method using CLC Genomics Workbench with an error probability <0.05 was used for clustered the sequences each sample. The 16S rRNA gene sequence was grouped into operational taxonomic units (OTUs) by using 97% identity of the sequence as a cutoff, and the OTUs table was generated for each sample and used for statistical analysis (Suksong et al., 2019). 2.5 Calculations and statistical analysis The modified Buswell equation (Eq. 1) was used for estimating the theoretical methane yield of POME (Eq. 2) and EFB (Eq. 3) (Buswell and Mueller, 1952). Biodegradation efficiency was calculated by the theoretical methane yield of substrate and methane yield of each experiment (Eq. 4) (Raposo et al., 2011). The energy yield from biogas of the coupled L-AD and SS-AD processes was calculated by using an energy value of 36 kJ∙L-1 CH4 described by O-Thong et al. (2012). The SPSS software (version 17.0; Spss Inc., Chicago, IL, USA) was used for statistical analysis of the results by analysis of variance (ANOVA) with a P value at 0.05. 4a ― b ― 2c + 3d )H2O 4

Ca HbOcNd +(

4a + b ― 2c ― 3d )CH4 8

→(

C27 H42O13N + 10H2O →15CH4 + 12CO2 + NH3

4a + b ― 2c ― 3d )CO2 8

+(a ―(

+ d NH3 Eq.1

Eq.2

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Eq.3

C64 H48O38N + 34H2O →28CH4 + 36CO2 + NH3 Biodegradation efficiency (%) =

Theoretical methane yield (ml g ―1VS) Methane yield of experiment (ml g ―1VS)

× 100

Eq.4

3. Results and Discussion 3.1 Compositions of palm oil mill effluent and empty fruit bunches The POME consisted of 95.1% water and 4.98% total solids with a volatile solids content of 4.26% (Table 1). The chemical oxygen demand of POME was the main organic source for anaerobic microorganisms with accounted for 63.9 gL-1, indicating that POME had a high potential for biogas production (Choong et al., 2018). The theoretical methane production of POME was 576  m3CH4tonne-1VS. POME was acidic with a pH as low as 4.42 and showed high total volatile fatty acid contents (1,274 mgL-1). Empty fruit bunches (EFB) are a solid waste residue after fruit removal from fresh fruit bunches (FFB). EFB accounted for 23% of the total FFB (Derman et al., 2018). EFB had a high moisture content of 61.6% (Table 1). EFB was composed of cellulose (44.1% of TS), hemicellulose (26.1% of TS), and lignin (29.8% of TS). EFB had a low nitrogen content (0.13%), resulting in a C:N ratio of 64:1. EFB accounted as low C:N ratio with respect to an optimum of 40:1-25:1 for the anaerobic digestion (Li et al., 2018; Suksong et al., 2016). Adjusting the C:N ratio of EFB to 30:1 with urea increased methane production from EFB by 25% (Suksong et al., 2016). EFB had a high potential for methane production with a theoretical methane yield of 438 m3 CH4 tonne-1VS, corresponding to 158 m3 CH4∙tonne-1 fresh EFB.

3.2 Biogas production performance of coupled L-AD and SS-AD processes

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3.2.1 Performance of L-AD for biogas production from POME The POME was investigated for biogas production under the L-AD process. The LAD reactor was operated at an OLR of 1.66 g VSL-1d-1 and an HRT of 30 days under mesophilic condition (40˚C). An average methane production rate from the start-up stage was 0.42 L CH4L-1reactor d-1, corresponding to a methane production of 12.7 m3-CH4m-3POME. The methane production rate is shown in Fig. 2a. In steady-state conditions, the maximum methane production rate was 0.63 L-CH4∙L-1-reactor∙d-1, which was observed at day 78. The maximum methane yield and methane production were 293 mL CH4g-1-COD and 20.9 m3-CH4m-3-POME. The methane yield of POME was inlined with 271 mL CH4g1

COD reported by O-Thong et al. (2016) and 310 mL CH4g-1-COD reported by Najafpour

et al. (2006). The methane and carbon dioxide concentration in the biogas was 60.7-65.9% (v/v) and 33.2-38.7% (v/v), respectively. During steady-state, biogas production was stable and COD removal exceeded 80%. The characteristics of liquid effluent from the L-AD reactor are shown in Table 2. The final pH was stable at 7.5-7.9 throughout either experiment without pH regulation in the L-AD reactor. The final alkalinity, VFAs, and ammonia nitrogen were 8,166 mg-CaCO3L-1, 109.3 mgL-1, and 800 mg-NH4+L-1, respectively which could be suitable for maintaining the methanogen activity in the AD reactor (Lui et al. 2013).

3.2.2 Effect of liquid-AD effluent recycling rate on biogas production from EFB in SSAD reactor

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Liquid effluent of the L-AD reactor was recycling to SS-AD reactor as inoculum for biogas production from EFB, which the L-AD effluent recycling rates of 22.2, 16.7, and 11.1 mLL-1 reactord-1. Liquid effluent recycling is a common method used to enhance inoculation efficiency in SS-AD by improving the mass transfer of inoculum to feedstock (Yang et al. 2015). The optimum L-AD effluent recycling rate was 16.7 mL L-1 reactord-1 (Fig. 3a) with a maximum methane production rate of 0.81 L-CH4L-1-reactord-1in stage R2. The methane production rates of stage R-1 with L-AD effluent recycling rate of 22.2 mLL-1 reactord-1, stage R-3 with L-AD effluent recycling rate of 11.1 mLL-1 reactord-1 and stage R5 without L-AD effluent recycling were 0.50, 0.48 and 0.61 L-CH4L-1reactord-1, respectively. The optimum L-AD effluent recycling rate was confirmed by stage R4 (16.7 mLL-1 reactord-1) with a maximum methane production rate of 0.73 mLL-1 reactord-1. These results show that liquid effluent recycling significantly affects the methane production rate. The effect may be attributed to a priming effect (Insam and Markt, 2016) induced by a more balanced nutrient supply. Similar trends were observed by Lu et al. (2013), who found that a 1:1 leachate recirculation rate significantly increased VS degradation and methane production from fresh soybean meal. In contrast, low L-AD effluent recycling rate of 11.1 mLL-1-reactord-1 resulted in high solids (190-200 g-TSL-1), high ammonia (5,746 mg NH4+L-1), and high volatile fatty acids (7,576 mgL-1) accumulation in SS-AD reactor with low biodegradation efficiency of 14-23% (Table 3). Xu et al. (2013) also observed high VFAs accumulation and low methane production in SSAD reactors at low volumes of liquid effluent were recirculated (3:1-5:1; substrates:inoculum ratio). The present study shows that low (R3) and without (R5) L-AD

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effluent recycling resulted in high VFAs concentrations of 7,576 and 2,200 mgL-1, respectively. Xu et al. (2013) found that larger amounts of liquid effluent as inoculum (more than 33%) could provide buffering capacity to the SS-AD reactor, helpful to stabilize the AD process. Corresponding, the VFAs concentrations of stage R1, R2, and R4 were low, with a concentration of 178, 370, and 337 mgL-1, respectively. Moreover, elevated ammonia concentrations were observed during stage R3 and stage R5 with a concentration of 5,746 and 5,683 mgL-1, respectively (Fig. 3b). Excessive VFAs and ammonia nitrogen also causes SS-AD instability. For ammonia nitrogen, values ranging from 4,100 to 5,700 mgL-1 cause toxicity or complete inhibition to methanogenic activity (Xu et al. 2018). The total solids accumulation of the SS-AD reactor at various L-AD effluent recycling rates is shown in Fig. 4. The maximum total solid accumulation of 233 gTSL-1 was achieved in the stage R3. The total solids accumulation of stages R1, R2, R4, and R5 were 86, 124, 130, and 190 gTSL-1 reactor, respectively. The high total solids accumulation (20-30%) could limit mass diffusion in the SS-AD process and cause inhibition of hydrolysis due to accumulated hydrolytic products (Xu et al., 2014). The process performance of the SS-AD reactor with difference liquid effluent recycling rate is shown in Table 3. The highest methane yield of 135 mL-CH4g-1-VS was achieved from stage R2. The methane yields of stage R1, R3, R4, and R5 were 123, 60, 121, and 102 mL-CH4g-1-VS, respectively. The methane yield of stage R3 was significantly lower than that of all other stages (p<0.05). Lin and Li. (2017) found that the SS-AD of yard trimmings with a liquid effluent recycling rate of 1:1 (substrate: inoculum) was favored over 2:1 and 3:1 with significantly higher methane yield and methane

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productivity. A strong linear relationship was observed between the process performance of the SS-AD reactor and L-AD effluent recycling rates (Table 4). The methane yield of EFB was decreased by 55% when using a low L-AD effluent recycling rate of 11.1 mLL-1d-1 in stage R3. The best VS removal efficiency (30.8%) in the SS-AD reactor was obtained from stage R2, while the VS removal efficiency of stage R3 was only 13.7%. The stage R3 was low water content leading to slow mass transfer between microbes and feedstocks, resulting in low methane yield and low VS removal efficiency. Water content in the SS-AD process is critical in facilitating mass transfer (Yang et al. 2015), demonstrating that the liquid effluent of L-AD reactor was readily usable for recycling to the SS-AD reactor as a source of buffer and inoculum. The stage R2 and stage R4 with an L-AD effluent recycling rate of 16.7 mLL-1 reactord-1 could increase the methane production rate, methane yield, VS removal efficiency resulting in more stability in SS-AD reactor. The suitable L-AD effluent recycling rate was able to homogenize the EFB in the SS-AD reactor. However, high L-AD effluent recycling rate could cause washout of nutrients and microorganisms, which could also explain the low methane yield observed by Degueurce et al. (2016).

3.3 Microbial communities in the coupled L-AD and SS-AD processes The bacterial and archaeal communities of sludge taken from coupled L-AD and SS-AD (stage R1-R5) processes were analyzed by 16S rRNA sequencing using MiSeq technology. The composition of the bacterial community is shown in Fig. 5a. Microbial community in the L-AD reactor was dominated by Porphyromonadaceae, Syntrophomonadaceae , Ruminococcaceae, Methanobacteriaceae, and Methanosaetaceae.

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At the family level, the bacterial community was dominated by Porphyromonadaceae (35.0%), Ruminococcaceae (12.7%), and Syntrophomonadaceae (10.9%). In general, Porphyromonadaceae and Ruminococcaceae are the most abundant in AD reactors with wastewater as substrate (Lv et al. 2019). Hahnke et al. (2015) reported the isolation of Porphyromonadaceae from a mesophilic CSTR reactor of maize silage. Genome analysis indicates that Porphyromonadaceae are among the important hydrolytic and acidogenic bacteria in the AD process. The family of Ruminococcaceae is comprised of VFA producing bacteria, including the genera of Ruminococcus sp., Ercella sp. and Eubacterium sp. that were also detected in the L-AD reactor of POME. Many Syntrophomonadaceae, a family of acetate producing bacteria, are known to convert butyrate to acetate during AD (Regueiro et al., 2012). Acetate is a suitable substrate for the acetoclastic methanogens (Yuan et al. 2016). The methanogen community of L-AD reactor of POME was dominated by Methanobacteriaceae (88.3%), Methanosaetaceae (8.09%) and Methanosarcinaceae (2.10%) (Fig 5b). Similarly, Krishnan et al. (2019) observed Methanobacterium sp. to be enriched during AD of POME. Methanobacteriaceae perform hydrogenotrophic methanogenesis that converts formate, hydrogen and CO2 to methane (Eq 5.). In contrast, Methanosaetaceae and Methanosarcinaceae were reported to be dominant in AD reactors fed with hydrogen reactor effluent that showed a high acetate concentration (>1.2 mM) (Eq 6.) (O-Thong et al., 2016). 4𝐻2 + 𝐶𝑂2 ⇄ 𝐶𝐻4 + 2𝐻2𝑂 (∆𝐺 ― 139 𝑘𝐽𝑚𝑜𝑙 ―1)

Eq.5

𝐶𝐻3𝐶𝑂𝑂 ― + 𝐻2𝑂⇄ 𝐶𝐻4 + 2𝐻𝐶𝑂3― (∆𝐺 ― 28 𝑘𝐽𝑚𝑜𝑙 ―1)

Eq.6

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The analysis of the bacterial community structure in an SS-AD reactor with different L-AD effluent recycling rate (stage R1-R4) demonstrated the dominance of the phylum Firmicutes which represented for 50%–75%, except the bacterial community structure in stage R5 (without recycling L-AD effluent) was dominated by 56% of the phylum Bacteroidetes. The Firmicutes and Bacteroidetes are capable of degrading a wide range of substances such as cellulose, proteins, and pectin (Xu et al., 2018). The main proportion at the family level was affiliated to members of the Caldicoprobacteraceae (33.6-47.3%), Porphyromonadaceae (10.7-22.6%), and Ruminococcaceae (8.1-15.3%) (Fig. 5a). The microbial community in the SS-AD reactor was dominated by Caldicoprobacteraceae, Ruminococcaceae, Porphyromonadaceae, Synergistaceae, Lachnospiraceae, Methanobacteriaceae, and Methanosaetaceae. The relative abundance of Synergistaceae, Caldicoprobacteraceae, and Lachnospiraceae was increased in the SS-AD process, while that of Syntrophomonadaceae was decreased. The relative abundance of Ruminococcaceae and Porphyromonadaceae in the SS-AD process was similar to the LAD process. Synergistaceae can accelerate the transfer of VFAs to acetate (Zou et al., 2018), while Caldicoprobacteraceae, Ruminococcaceae, Porphyromonadaceae, and Lachnospiraceae are responsible for the degradation of complex substrates by producing hydrolytic enzymes (Cheng et al., 2018; Suksong et al., 2019). The dominant bacteria were shifted from Firmicutes to Bacteroidetes during the SS-AD process without L-AD effluent recycling in stage R5 with high VFAs accumulation. Correspondingly, the relative abundance of Caldicoprobacteraceae, Porphyromonadaceae, and Ruminococcaceae in stage R5 were decreased by 96.5, 28.9 and 44.7% when compared with stage R2. This change in the microbiota between R1-R4 and R5 is attributed to the low L-AD effluent

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recycling in the SS-AD reactor leading to inhibition of the microorganisms due to the accumulation of VFAs and an increase in ammonia concentration. However, the methanogen community in R1-R5 stages of the SS-AD reactor was not affected by L-AD effluent recycling rate (Fig. 5b). Similarly, Ruan et al. (2019) found that the community composition of hydrolytic and fermentative bacteria more strongly changed than that of the methanogens in an AD system of sludge hydrolysis by microaeration, which enhanced hydrolysis and acidification of organic matter and biogas production. The methanogen community in R1-R5 stages of SS-AD reactor consisted of 88.3-99.5% Methanobacteriaceae, 0.21-8.09% of Methanosaetaceae and 0.38-2.66% of Methanosarcinaceae. The reason for this resilience in the methanogen community is the high generation time that does not allow a rapid adaptation as in bacteria, which was also found by Walter et al. (2016). The results corroborate those by Suksong et al. (2019), who found that the SS-AD reactor of EFB with L-AD effluent as inoculum was dominated by Methanobacterium (24%) and Methanosaeta (47%) under mesophilic temperature (40ºC). The high abundance of Methanobacteriaceae in the AD reactor is in consistence with the relative high solid accumulation (15-20%) in the reactors, which indicate high hydrogen and carbon dioxide pressure (Stolze et al. 2015).

3.4 Feasibility of using coupled L-AD and SS-AD process for biogas production from waste residues in the oil palm industry Over the past decade, the palm oil industry has engaged in method developments for the utilization of the by-products in order to ensure sustainable economic growth (Yoshizaki et al. 2013). A coupled L-AD and SS-AD process of waste residues in the oil

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palm industry was demonstrated technically as a feasible option for stable methane production from both solid and liquid waste. The strengths and benefits of coupled L-AD and SS-AD process are shown in Table 5. This study shows L-AD of POME could produce 330 L of methane from 1 kg-VS or 20.9 L of methane from 1 L-POME. One kg-VS of POME was converted to 0.57 kg of methane plus 0.43 kg of remaining VS in the liquid effluent. The latter was used as inoculum for the SS-AD process. The optimum L-AD effluent recycling rate was 16.7 mLL-1d-1. Under optimum condition, one kg VS of EFB could produce 134 L of methane. Thus 0.31 kg VS of EFB was converted to methane, while the remaining VS after the SS-AD process of EFB was 0.69 kg. Our data support the common practice of SS-AD of EFB, also because the digestate may serve as a valuable fertilizer to be returned to the oil palm plantation area (Yoshizaki et al. 2013). The total energy production of coupled L-AD and SS-AD processes was 16.7 MJ, obtained at an optimum L-AD effluent recycling rate. The synergistic effect of coupled L-AD and SS-AD yielded up to 1.15 MJ of extra energy in the case of an EFB:EFF ratio of 1:1 (Fig. 6). These results indicated that coupled L-AD and SS-AD processes could increase energy production by 29% and 71% compared to single L-AD and SS-AD processes alone, respectively (Fig. 6). The coupled L-AD and SS-AD process could increase energy production and reduce liquid effluent of L-AD reactor treatment by proving a source of methane inoculum for the SS-AD reactor.

4. Conclusions

18

Coupled liquid and solid-AD processes are effective for biogas production from liquid and solid waste residues in the oil palm industry. The liquid effluent of the liquid-AD reactor is suitable for recycling to the SS-AD reactor as a source of buffer and microbial inoculum. The coupled L-AD and SS-AD could increase energy production by 29% and 71% compared to the L-AD and SS-AD alone, respectively, with no outsource SS-AD inoculum required. The coupling of liquid and solid-AD processes acts synergistically in biogas production with reduction of operation cost for the SS-AD reactor and enhanced energy recovery from liquid and solid waste residues.

5. Acknowledgments The authors thank the Research and Development Institute of Thaksin University and Thailand Research Fund through the Royal Golden Jubilee Ph.D. Grant (PHD/0037/2559), TRF Mid-Career Research Grant (Grant No. RSA6180048), TRF Senior Research Scholar (Grant No. RTA6280001) for the financial support.

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List of figures Fig 1. Schematic diagram of liquid effluent recycling systems for biogas production by coupled liquid and solid-state anaerobic digestion process. Fig 2. Methane production rate (A), alkalinity, and volatile fatty acids (B) from palm oil mill effluent by liquid-state anaerobic digestion reactor. Fig 3. Variation of methane production rate (A), alkalinity, volatile fatty acids, and ammonia nitrogen (B) from EFB via solid-state anaerobic digestion operation at different liquid effluent recycling rates. Fig. 4. Total solids accumulation during the SS-AD process of EFB with various liquid effluent recycling rates. Fig. 5 Bacterial (A) and archaeal (B) community of liquid AD sludge (L-AD) and solidstate AD sludge (SS-AD) of coupling liquid and solid-state anaerobic digestion for biogas production from POME and EFB at families level. Fig. 6. Volatile solids (VS) balance and energy production of coupling liquid and solidstate anaerobic digestion for biogas production from POME and EFB.

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28

POME (Inlet) Biogas outlet

Liquid state anaerobic digestion (L-AD)

L-AD reactor operation • Palm oil mill effluent (POME) as substrate • Hydraulic retention time 30 days • Organic loading rate 1.66-2.00 gVS∙L-1 reactor∙d-1 • Temperature 40 ˚C

M

Gas counter EFB Liquid effluent

SS-AD reactor operation • Oil palm empty fruit bunches (EFB) as substrate • Hydraulic retention time 30 days • Organic loading rate 6.03 gVS∙L-1 reactor∙d-1 • Temperature 40 ˚C • Liquid effluent recycling rates • Stage R-1 (22.2 mL∙L-1 reactor∙d-1) (EFB 0.5:1EFF) • Stage R-2 (16.7 mL∙L-1 reactor∙d-1) (EFB 1:1EFF) • Stage R-3 (11.1 mL∙L-1 reactor∙d-1) (EFB 2:1EFF) • Stage R-4 Confirm teste (16.7 mL∙L-1 reactor∙d-1) (EFB 1:1EFF) • Stage R-5 Without liquid effluent recycling

Liquid effluent mixed with EFB (Inlet)

Biogas outlet

Solid state anaerobic digestion (SS-AD) M

10 L. Working volume 5 L.

Fig 1. Schematic diagram of liquid effluent recycling systems for biogas production by coupled liquid and solid-state anaerobic digestion process.

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(A) Methane production rates

1.0

Methane production rates (L-CH4 L-1 reactor d-1)

Start-up

Organic loading rate 1.66 gVS L-1 reactor d-1

0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0

10

20

30

40

50

60

70

80

90

100 110 120 130 140 150 160 170 180

Operation times (Day) Volatile fatty acids

Alkalinity

18000 Start-up

(B)

16000

1400

14000

1200

12000 1000 10000 800 8000 600 6000

Volatile fatty acids (mg L-1)

Alkalinity (mg-CaCO3 L-1)

1600

Organic loading rate 1.66 gVS L-1 reactor d-1

400

4000

200

2000

0

0 0

10

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30

40

50

60

70

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100 110 120 130 140 150 160 170 180

Operation times (Day)

Fig 2. Methane production rate (A), alkalinity, and volatile fatty acids (B) from palm oil mill effluent by liquid-state anaerobic digestion reactor.

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(A) Effluent recycling rate (mL-EFF L-1 reactor d-1)

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Stage R-1 EFB0.5:1EFF

Start-up

Methane production rate

Stage R-2 EFB1:1EFF

Stage R-3 EFB2:1EFF

Stage R-4 EFB1:1EFF

Stage R-5 Without-recycling

1.0

20

0.8

15

0.6

10

0.4

5

0.2

0

Methane production rate (L-CH4 L-1 reactor d-1)

Effluent recycling rate

0.0 0

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Operation times (Day) Alkalinity

15000

Stage R-3 EFB2:1EFF

Stage R-2 EFB1:1EFF

Stage R-1 EFB0.5:1EFF

Start-up

Volatile fatty acids

Ammonia nitrogen Stage R-4 EFB1:1EFF

Stage R-5 Without-recycling

(B) 20000

13500

18000

12000

16000

10500

14000

9000

12000

7500

10000

6000

8000

4500

6000

3000

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Volatile fatty acids (mg L-1)

Alkalinity (mg-CaCO3 L-1)

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4000

0

0 0

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20

30

40

50

60

70

80

90

2000

0

100 110 120 130 140 150 160 170 180

Operation times (Day)

Fig 3. Variation of methane production rate (A), alkalinity, volatile fatty acids, and ammonia nitrogen (B) from EFB via solid-state anaerobic digestion operation at different liquid effluent recycling rates.

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Total solids accumulation (g L-1-reactor)

300 R-1 (R20.997) R-2 (R20.999) R-3 (R20.998) R-4 (R20.992) R-5 (R20.991)

250

200

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32

Operation times (Day)

Fig. 4. Total solids accumulation during the SS-AD process of EFB with various liquid effluent recycling rates.

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(A) 100

unidentified_Atribacteria Thermoanaerobacteraceae Syntrophomonadaceae Synergistaceae ST-12K33 Ruminococcaceae Rikenellaceae Prevotellaceae Porphyromonadaceae Peptococcaceae Others Marinilabiaceae Lachnospiraceae Heliobacteriaceae Family_XIII Family_XI Eubacteriaceae Erysipelotrichaceae Enterococcaceae Draconibacteriaceae Clostridiaceae_1 Christensenellaceae Carnobacteriaceae Caldicoprobacteraceae Bacteroidales_UCG-001 Anaerolineaceae Aerococcaceae

Relative abundance distribution (%)

80

60

40

20

0

L-AD

SS-AD (Stage-R1)

SS-AD (Stage-R2)

SS-AD (Stage-R3)

SS-AD (Stage-R4)

SS-AD (Stage-R5)

100

Halobacteriaceae ARC26 unidentified_Thermoplasmatales unidentified_Bathyarchaeota Methanosarcinaceae Methanosaetaceae Methanobacteriaceae

Relative abundance distribution (%)

80

60

40

20

0

(B)

L-AD

SS-AD (Stage-R1)

SS-AD (Stage-R2)

SS-AD (Stage-R3)

SS-AD (Stage-R4)

SS-AD (Stage-R5)

33

Fig. 5 Bacterial (A) and archaeal (B) community of liquid AD sludge (L-AD) and solidstate AD sludge (SS-AD) of coupling liquid and solid-state anaerobic digestion for biogas production from POME and EFB at families level.

34

POME

Waste residues of palm oil extraction process

1 kg of VS

L-AD Reactor

EFB 1 kg of VS

HRT 30 day Temp. 40 ˚C

EFB 0.5:1 EFF 1 kg of VS

Effluent (EFF)

SS-AD reactor HRT 30 day Temp. 40 ˚C

EFB 1:1 EFF

0.19 kg VS

(B)

Energy production of coupling L-AD and SS-AD reactor

Synergistic effect (Energy production)

Methane 123 L (0.28 kg of VS) Digested 0.72 kg of VS Total energy 4.43 MJ

(A) + (B) Total energy 16.3 MJ

0.76 MJ

Methane 134 L (0.31 kg of VS) Digested 0.69 kg of VS Total energy 4.82 MJ

(A) + (C) Total energy 16.7 MJ

1.15 MJ

Methane 60 L (0.14 kg of VS) Digested 0.86 kg of VS Total energy 2.16 MJ

(A) + (D) Total energy 14.0 MJ

-1.51 MJ

(C)

1 kg of VS Mixing tank EFB 2:1 EFF

(D)

1 kg of VS (A)

Methane 330 L (0.81 kg of VS) Total energy 11.9 MJ

EFB 1 kg of VS Without EFF recycling

(E)

Methane 102 L (0.23 kg of VS) Digested 0.77 kg of VS Total energy 3.67 MJ

Fig. 6. Volatile solids (VS) balance and energy production of coupling liquid and solidstate anaerobic digestion for biogas production from POME and EFB.

35

Table 1 Characteristics of palm oil mill effluent and empty fruit bunch. Characteristics Moisture content Total solids Volatile solids VS/TS ratio Nitrogen content Chemical oxygen demand C:N ratio Cellulose Hemicellulose Lignin Lipids content pH TVFA

Palm oil mill effluent (POME) 4.98 4.26 0.86 0.06 63925 27.0 0.62 4.42 1273

Empty fruit bunch (EFB) 61.6 38.4 36.2 0.95 0.13 64.0 44.1 26.1 29.8 0.43 7.02 182

Unit %wet weight %wet weight %wet weight %wet weight mgL-1 % of TS % of TS % of TS %wet weight mgL-1

36

Table 2. Process performance of liquid anaerobic digestion reactor for biogas production from palm oil mill effluent. Parameters Hydraulic retention time Organic loading rate (based on COD) Organic loading rate (based on VS) Flow rate Working volume Temperature Initial pH Final pH Methane content Methane production Methane production rate Methane yield (based on COD) Methane yield (based on VS) Final alkalinity Final volatile fatty acids Final ammonium COD removal efficiency VS removal efficiency

L-AD reactor 30 2.14 1.66 33.3 5 40 7.50 8.63 61.8 20.9 0.42 293.1 329.5 8166 109.3 800 83.7 81.8

Unit Day g-CODL-1d-1 g-VSL-1d-1 mLL-1d-1 L ˚C %(v/v) m3-CH4m3 POME L-CH4L-1-reactord-1 mL-CH4g-1-COD mL-CH4∙g-1-VS mg-CaCO3L-1 mgL-1 mg-NH4+L-1 % %

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Table 3. Process performance of solid-state anaerobic digestion reactor for biogas production from an empty fruit bunch at various L-AD effluent recycling rates. Parameters Hydraulic retention time Organic loading rate Flow rate Effluent recycling rates Working volume Temperature Initial pH Final pH Methane content Methane production Methane production rate Methane yield Final alkalinity Final volatile fatty acids Final ammonium VS degradation efficiency

R1 30 6.03 33.3 22.2 5 40 7.62 7.80 53.6 44.3 0.50 123.3 5025 178 1113 28.2

R2 30 6.03 33.3 16.7 5 40 7.13 7.83 57.2 49.0 0.81 134.9 4800 370 1580 30.8

R3 30 6.03 33.3 11.1 5 40 6.80 7.46 53.4 22.2 0.48 60.0 6331 7576 5746 13.7

R4 30 6.03 33.3 16.7 5 40 7.18 7.86 54.1 47.1 0.73 121.4 5280 337 1438 27.7

R5 30 6.03 33.3 0 5 40 6.51 7.42 54.8 36.9 0.61 101.5 6299 2200 5683 23.2

Unit Day g-VSL-1d-1 mLL-1d-1 mLL-1d-1 L ˚C %(v/v) m3-CH4tonne-1 EFB L-CH4L-1-reactord-1 mL-CH4g-1-VS mg-CaCO3L-1 mgL-1 mg-NH4+L-1 %

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Table 4. Correlation between process performance of solid-state anaerobic digestion reactor and liquid effluent recycling rates.

Factor (x) VFAs (mgL-1) Ammonia nitrogen (mgL-1) Alkalinity (mgL-1) Methane yield (mL-CH4g-1 VS) VS removal (%)

L-AD effluent recycling rate (y) (mLL-1 reactord-1) Equation R2 y = 0.0002x + 0.611 0.9578 y = 0.0003x + 0.3488 0.9426 y = 0.0008x - 3.2705 0.7949 y = -0.017x + 2.9672 0.7936 y = -0.0739x + 2.9646 0.7705

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Table 5 Strengths and benefits of oil palm industrial waste utilization technology. Strengths/Benefits

L-AD

Waste utilization

Liquid waste

Solid waste

Product

Biogas

Biogas and Compost 30

Compost 120

Coupled L-AD and SS-AD Liquid and solid waste Biogas and compost 30

6.0

-

7.6

67.3

-

120

23.2

-

57.3

Hydraulic 30 retention time (days) Organic loading 1.7 rate (kgVSm3reactord-1) Biogas 33.8 productivity (m3 tonne-1 waste) Overall energy 83.7 recovery a Chiew and Shimada (2013)

SS-AD

Aerobic composting a Solid waste

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Declaration of interests

☒ 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.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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Highlights 

Coupling liquid and solid anaerobic digestion (AD) process were investigated.



Optimum liquid AD effluent recycling rate was 16.7 mLL-1 reactord-1.



Coupling L-AD and SS-AD process could increase 27-71% of biogas production.



Coupling L-AD and SS-AD were suitable for treating both solid and liquid waste.