Bioresource Technology 177 (2015) 17–27

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Simultaneous treatment of raw palm oil mill effluent and biodegradation of palm fiber in a high-rate CSTR Maneerat Khemkhao a, Somkiet Techkarnjanaruk b, Chantaraporn Phalakornkule a,c,d,⇑ a

The Joint Graduate School of Energy and Environment, King Mongkut’s University of Technology Thonburi, Bangkok 10140, Thailand National Center for Genetic Engineering and Biotechnology (BIOTEC), Bangkok 10150, Thailand c Department of Chemical Engineering, King Mongkut’s University of Technology North Bangkok, Bangkok 10800, Thailand d Research and Technology Center for Renewable Products and Energy, King Mongkut’s University of Technology North Bangkok, Bangkok 10800, Thailand b

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 A modified CSTR was used to treat

raw palm oil mill effluent without pretreatment.  Neither physical nor chemical pretreatment of the raw POME was performed.  The deflector installed at the CSTR’s upper section promoted retention of fibers.  Simultaneous degradations of palm fibers and soluble parts in POME were achieved.  Methane production rate was 4.14 l/ l d at a highest OLR 19.0 g COD/l d.

a r t i c l e

i n f o

Article history: Received 31 August 2014 Received in revised form 9 November 2014 Accepted 12 November 2014 Available online 20 November 2014 Keywords: CSTR Palm oil mill effluent Biogas Anaerobic DGGE

Idenficaon based on databases in Gene Bank using BLAST

Sequencing

Community fingerprints

Sequences

DGGE

Bac

Arc

PCR products Nested PCR

Extracon

Nucleic acids

a b s t r a c t A high-rate continuous stirred tank reactor (CSTR) was used to produce biogas from raw palm oil mill effluent (POME) at 55 °C at a highest organic loading rate (OLR) of 19 g COD/l d. Physical and chemical pretreatments were not performed on the raw POME. In order to promote retention of suspended solids, the CSTR was installed with a deflector at its upper section. The average methane yield was 0.27 l/g COD, and the biogas production rate per reactor volume was 6.23 l/l d, and the tCOD removal efficiency was 82%. The hydrolysis rate of cellulose, hemicelluloses and lignin was 6.7, 3.0 and 1.9 g/d, respectively. The results of denaturing gradient gel electrophoresis (DGGE) suggested that the dominant hydrolytic bacteria responsible for the biodegradation of the palm fiber and residual oil were Clostridium sp., while the dominant methanogens were Methanothermobacter sp. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Due to the increased demand for biofuels, there has been a large increase in palm oil production with a corresponding increase in ⇑ Corresponding author at: Department of Chemical Engineering, King Mongkut’s University of Technology North Bangkok, Bangkok 10800, Thailand. Tel.: +66 (0)89 135 3253; fax: +66 (0)2 587 0024. E-mail addresses: [email protected], [email protected] (C. Phalakornkule). http://dx.doi.org/10.1016/j.biortech.2014.11.052 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.

Closest matches of paral 16S rRNA gene sequences

the amount of wastewater generated from its processing. The wastewater from palm oil production, which is commonly called palm oil mill effluent (POME), is mainly produced from three main sources of sterilization condensates, clarification sludge and hydrocyclone drain-off (Borja and Banks, 1994). POME is a viscous brown liquid which is usually discharged at temperatures approximately 73 °C (Mustapha et al., 2003). It typically contains fine suspended solids and is acidic with pH approximately 4.5 (Khemkhao et al., 2011). Raw POME also has a high chemical oxygen demand around

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58 g/l and a high biochemical oxygen demand of approximately 26 g/l (Mustapha et al., 2003). It typically has high concentrations of free fat, oil and grease (FOG), as well as dissolved oil and crude oil solids (Khemkhao et al., 2011). Due to its high organic content, the conversion of the organic matter in POME into methane is a potentially useful method of treatment since methane is an important renewable energy source. This conversion is usually based on anaerobic processes. However, POME usually contains high concentrations of suspended solids (SS) and colloidal components, such as oil and biofibers, which can cause problems in the operations of anaerobic processes. When raw POME was fed to high-rate anaerobic processes without prior removals of biofibers and oil, operational problems such as scum formation, blockage of pipeline, sludge flotation and low methane yields were reported. In early work by Borja and Banks (1994), an upflow anaerobic sludge blanket (UASB) reactor was used to treat raw POME. It was found that at a high organic loading rate (OLR) of 10.6 g COD/l d, an accumulation of SS and FOG in the UASB caused the deterioration of microbial activities and wash-out of active biomass. Excessive surface scum and blockage of pipeline were found in an expanded granular sludge bed (EGSB) reactor used to treat raw POME due to the presence of FOG in the raw POME (Zhang et al., 2008). Furthermore, granular sludge flotation was also encountered in the EGSB because the specific gravity of granular sludge decreased after oil and grease were adsorbed on it. Biofibers were found to settle to the reactor bottom shortly after entering the reactor. As a result, the apparent COD removal was as high as 90%, but the methane yield was relatively low (Zhang et al., 2008). In the work by Fang et al. (2011), both UASB and EGSB were used to comparatively treat raw POME and deoiled POME. Deoiling process removes not only FOG, but also parts of biofibers. When deoiled POME was used as the substrate, both UASB and EGSB gave satisfactory COD reduction, methane production and methane yield. On the other hand, when raw POME was used as the substrate, lower methane composition and methane yields were achieved. It was suggested that raw POME gave lower methane yield compared to deoiled POME because a higher portion of volatile solids in the raw POME was contributed by biofibers. More recent studies about POME treatment therefore employed either physical treatment or chemical treatment to remove fine suspended solids and FOG before processing in high-rate anaerobic reactors such as or anaerobic hybrid reactor (AHR) (Choi et al., 2013; Jeong et al., 2014), UASB (Ahmad et al., 2011; Khemkhao et al., 2012) and upflow anaerobic sludge-fixed film (UASFF) (Zinatizadeh et al., 2007). It is desirable to investigate the possibility of developing anaerobic reactors which can process raw POME without pretreatment. There are at least two advantages of using raw POME without physical or chemical pretreatment. First, the use of chemicals can be reduced resulting in a more environmentally friendly process, while most physical processes such as filtration and screw decanter require regular maintenance and make the overall process more complex. Secondly, suspended solids such as palm fibers and residual oil can be additional carbon sources for methane production. In the study by O-Thong et al. (2012), biodegradability of oil palm empty fruit bunches (EFB) together with raw POME by thermophilic inoculum was found to be possible. Using batch assays under a thermophilic condition, the authors illustrated that the lignocellulosic substrate can be co-digested with raw POME at a mixing ratio up to 0.3:1 on wet weight basis. The co-digestion of EFB with POME enhanced microbial biodegradability and resulted in 25–32% higher methane production. Thermophilic hydrolytic and acidogenic activities of the inoculums were suggested to underlie the successful co-digestion.

CSTR have been studied as a more suitable type of reactor than a UASB reactor for treatment of substrate with high SS and FOG content. It is well known that CSTR provides a good contact between wastewater and biomass (Choorit and Wisarnwan, 2007). In Choorit and Wisarnwan (2007), a CSTR was operated successfully at a maximum OLR of 17 g COD/l d and obtained a biogas production rate of 4.7 l/l d with 70% of methane content. However, only 70% COD reduction was achieved, which in turn suggested relatively low degradation of POME fed to the CSTR. In this study, a modified CSTR with a deflector installed at its upper section to promote the retention of suspended solids in the reactor was used to treat raw POME. Neither physical pretreatment nor chemical pretreatment was performed to remove biofibers and FOG. The reactor performance was analyzed in terms of stability and efficiency of the process in removing COD and in producing biogas. Biodegradation of palm fiber and residual oil were investigated in the high-rate CSTR. The rates of palm fiber hydrolysis and fiber accumulation in the reactor were determined and the compositions of palm fibers at the influent, the effluent and accumulated in the CSTR were compared. DGGE was used to tentatively identify the working microorganisms that may be responsible for the biodegradation of the palm fiber and residual oil. 2. Methods 2.1. Reactor setup Two configurations of CSTR were used in this study, a typical configuration CSTR (e.g. Choorit and Wisarnwan, 2007) and a modified CSTR similar to that used in Show et al. (2007). The CSTR vessel used was a glass cylinder of 14 cm in diameter, 40 cm in height, and a working volume of 5.8 l and with a water jacket for heating. The lid of the reactor was fitted with ports for a temperature probe, an agitator and a gas outlet. The agitator was composed of two flat disc impellers, each having 6 flat blades, located at 10 mm and 60 mm above the reactor bottom. There were four sampling ports located along the reactor at heights of 4 cm, 14 cm, 24 cm and 37 cm from the bottom. The top sampling port served as the effluent outlet, while the other three ports were used for periodic collection of mixed liquor suspended solids (MLSS) from the reactor vessel. The MLSS values reported in this study were the average values of the data from these three ports. Raw POME was fed in at the bottom of the reactor, and the treated effluent flowed out through the top sampling port, and biogas flowed out at the gas outlet located on the reactor’s lid. In the modified CSTR configuration, a prism-shaped deflector was connected to the inner wall of the reactor vessel at its upper section to promote the retention of suspended solids (Fig. 1). The typical CSTR configuration was the same except that a deflector was not installed at the upper section. 2.2. Wastewater Raw POME was obtained from Sooksomboon Palm Oil Co., Ltd. (Chonburi province, Thailand). The characteristics of POME are reported in Table 1. The values are mean ± standard deviation of triplicates. Determinations of total COD (tCOD), soluble COD (sCOD), total solid (TS), volatile solid (VS), suspended solid (SS), volatile suspended solid (VSS), total Kjeldahl nitrogen (TKN), ammonia nitrogen (NH3-N), fat, oil and grease (FOG), alkalinity, and total volatile acid (TVA) were performed according to the Standard Methods (APHA, 2005). The raw POME was used as the substrate for biogas production in the CSTR. Before feeding the POME into the reactor, the pH was adjusted to 7 with 6 N NaOH and then diluted with tap water to obtain suitable organic loading rates for this study.

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Fig. 1. Schematic diagram of (a) the typical CSTR, (b) the modified CSTR with a deflector and (c) the experimental setup. 1: Feeding tank, 2: CSTR, 3: Gas counter, 4: Effluent storage tank, 5: Thermometer, 6: Hot water circulation bath. Table 1 Characteristics of POME.

The hydraulic retention time (HRT) was controlled at 80 h. The effluent was collected and was analyzed for the TS and fiber content.

Parameters

Range

Values

Total chemical oxygen demand (tCOD) Soluble chemical oxygen demand (sCOD) Biochemical oxygen demand (BOD) Total solids (TS) Suspended solids (SS) Volatile solids (VS) Volatile suspended solids (VSS) Alkalinity Total volatile acids (TVA) pH Total Kjeldahl nitrogen (TKN) Ammonia nitrogen (NH4-N) Fat, oil and grease (FOG)

69,500–88,150 38,500–64,700 33,500–36,400 51,560–52,180 23,180–29,100 43,100–43,360 20,800–25,680 1319–1389 2640–2652 4.69–4.75 1003–1053 138–194 6830–7610

70,500 ± 917 39,067 ± 1159 34,950 ± 1450 51,880 ± 310 26,547 ± 3043 43,260 ± 140 23,513 ± 2486 1344 ± 39 2647 ± 6 4.73 ± 0.03 1020 ± 29 176 ± 32 7150 ± 410

Notes: all parameters are in units of mg/l except pH; values are mean ± standard deviation of triplicates.

2.3. Preliminary experiments for comparing the retention of total solids and palm fibers in a typical CSTR and a modified CSTR with installed deflector In order to investigate the effectiveness of the deflector in retaining solids especially fibers in the CSTR, the same POME samples were fed to the typical CSTR and the modified CSTR. The reactors were not inoculated. POME diluted with tap water at the ratios of 1:4 2:3, 3:2 and 4:1 POME:water (v/v) were used as the feed.

2.4. Seed sludge Seed sludge was obtained from Ngaung-Khaem water quality control plant (Bangkok, Thailand). This plant is a domestic wastewater treatment plant which is operated at a mesophilic condition. The sludge had 54,170 ± 440 mg TS/l, 18,800 ± 30 mg VS/l, 53,290 ± 290 mg SS/l, 18,670 ± 90 mg VSS/l, a VSS/SS ratio of 0.35, a sludge volume index (SVI) of 17.08 ml/g SS and a settling velocity of 10.24 cm/min. 2.5. Start-up period and experimental run The CSTR was seeded with 10 g VSS/l of the anaerobic sludge. The reactor was initially fed with diluted POME corresponding to an OLR of 2 g COD/l d. The feed flowrate was controlled at 870 ml/d, giving an HRT of 160 h. The reactor was operated at 25 °C overnight before a single step increase of temperature to 55 °C (Boušková et al., 2005; Poh and Chong, 2010). The reactor temperature was maintained by circulating hot water through the reactor jacket. The reactor was operated until stable COD removal and biogas production were achieved. After the startup period, an experimental run was started with an OLR of 2 g COD/l d and an HRT of 80 h (3.3 days). The process was then operated at this OLR until a

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tCOD removal of 80% was achieved. Then, the OLR was increased by a step of 2.1 ± 0.9 g COD/l d by increasing the COD concentration in the POME feed. The HRT of 3.3 days was maintained throughout the experimental run. The stepwise increase in OLR was repeated until signs of reactor instability were observed. 2.6. Analytical methods The gas produced was measured using a gas counter. Gas composition was determined by a gas chromatograph (GC) equipped with a thermal conductivity detector (SHIMADZU, GC-2014, Japan), a stainless steel packed column and a helium carrier gas with a flow rate of 50 ml/min. Liquid effluent samples from the reactor were also routinely sampled. The following parameters were analyzed according to the Standard Methods (APHA, 2005): TS, SS, VSS, tCOD, sCOD, FOG, alkalinity and TVA. The fiber residues, which were mainly hemicelluloses, cellulose, and lignin, were determined using an automated fiber analyzer (Fibretherm FT12, Germany) based on the acid and neutral detergent fibers method (Van Soest et al., 1991). Compositions of long chain fatty acids (LCFA) were determined by a GC with a flame ionization detector (SHIMADZU, GC-2010, Japan), a capillary column (Agilent Technologies, DB-Wax, USA) and a nitrogen carrier gas with an average linear velocity of 36 cm/s. All measurements were repeated three times and the standard errors were all within 10% of the mean value. A test of significant difference based on the paired t-statistic was performed using the Excel Solver Add-in. The difference was regarded as not significant if the paired t-statistic showed probability; P > 0.05 and significant if P < 0 .05. 2.7. DGGE analysis Microbial DNA was extracted using DNA extraction kits (Omega, USA). A nested Polymerase Chain Reaction (PCR) technique was used to increase the sensitivity of PCR. The bacterial 16S rRNA gene was amplified by PCR with the bacterial primer EUB8F/U1492R in the first round and the specific primer set 338GC-F/518R in the second round. The archaeal 16S rRNA genes were amplified by PCR with the primer A20F/U1492R in the first round and the specific primer set ARC344F/ARC522R in the second round. A 40 bp GC clamp (50 -CGCCCGCCGCGCGCGGCGGGCGGGGC GGG-GGCACGGGGGG-30 ) was added at the 5 end of BAC338F and ARC344F. The nested PCR products were analyzed by DGGE on the DGGE-2000 system apparatus (CBS Scientific Co. Inc., USA). The PCR products were loaded onto 8% polyacrylamide gel in 1 TAE (Tris-acetate-EDTA) buffer. The gradients were created by the addition of 0–80% denaturant (5.6 M urea and 40% v/v formamide) into polyacrylamide. After electrophoresis, the DGGE gel was stained with SYBR Gold nucleic acid gel (Invitrogen, USA) and visualized under UV light. Most of the bands were excised from the gel and re-amplified with the M13F (20) primer for sequencing reaction. The 16S rRNA gene sequences were compared with those in the database of the National Center for Biotechnology Information (NCBI) using the BLASTn program (Altschul et al., 1990).

3. Results and discussion 3.1. Comparative retentions of total solids and palm fibers in a typical CSTR and the modified CSTR with installed deflector Table 2 shows the TS and the amounts of fibers in the feed and in the effluent of both reactor configurations. The fibers accounted for approximately 20% of the TS in the POME feed. The TS in the effluents of the typical CSTR were reduced by 21%, 47%, 38% and 36%, respectively, for POME feeds at dilutions of 1:4, 2:3, 3:2 and 4:1. The TS in the effluents of the modified CSTR were reduced by 39%, 63%, 59% and 57%, respectively, for POME at dilutions of 1:4, 2:3, 3:2 and 4:1. From these results, it is clear that the modified CSTR can retain TS more effectively than the typical CSTR, resulting in lower concentrations of TS in the effluent. The fiber content in the TS effluent from the typical CSTR was approximately the same as the fiber content in the TS POME feed. In contrast, the fiber concentrations in the effluent from the modified CSTR ranged between 0.23 and 0.35 g/l, which was markedly lower than in the feed and also in the effluent of the typical CSTR. The data indicated that the deflector in the CSTR can effectively retain the fibers inside the CSTR. The retention times of TS and fibers in the reactors were calculated using a similar method to that used for estimating sludge retention times (see, e.g., Wongnoi et al., 2007). The retention time was obtained by dividing the total mass of solids in the reactor by the concentration of solids removed in the unit of time.

RTi ¼ Mi =ðQ e Xe Þ

ð1Þ

where RTi is the retention of component i in the reactor (h); Mi is the total mass of component i in the reactor (g); Qe is the flow rate of effluent (l/h) and Xe is the concentration of component i in the reactor effluent (g/l). The retention times of fibers in the reactors were calculated to be 1.5 ± 0.6 d for the typical CSTR and 23 ± 9 d for the modified CSTR. A paired t-test analysis showed that the SRT of fibers in each reactor were significantly different (P < 0.05). 3.2. Reactor performances In the experimental runs, the OLR was increased in nine steps. The average OLRs in the nine steps were 2.0, 3.3, 6.0, 7.5, 10.0, 11.4, 13.7, 17.6 and 19.0 g COD/l d. The highest OLR of 19.0 g COD/l d was obtained by feeding the POME diluted at a ratio of 7.5:2.5 (v/v) raw POME:water with an HRT of 3.3 days. For each input OLR value, the pH of the POME feed into the reactor was adjusted to 7 by adding NaOH. Fig. 2 shows plots of the effluent pH, alkalinity and ratios between total volatile acids and total alkalinity (TVA/T-ALK ratio) against operating time. Throughout an experimental run, the pH values of the effluent varied in the range of 7.0–8.0 which is suitable for methane production. The pH of POME was adjusted at the beginning of the operation only, and no further external pH adjustment was required as the pH remained in the desired range. As shown in Fig. 2, the buffering capacity as measured by the TVA/T-ALK ratio was approximately 0.5. The TVA/T-ALK ratio is slightly higher than 0.4, the value which

Table 2 Comparative amounts of total solids and fiber in the effluents of the typical and the modified CSTR. POME at various dilutions (POME:water)

1:4 2:3 3:2 4:1

Effluent from the control CSTR

Effluent from the modified CSTR

TS (g/l)

Influent Fiber (g/l)

TS (g/l)

Fiber (g/l)

TS (g/l)

Fiber (g/l)

5.68 13.03 18.80 25.31

1.13 2.58 3.41 4.69

4.50 6.89 11.71 16.24

0.78 1.54 2.66 3.57

3.48 4.82 7.66 10.90

0.23 0.33 0.33 0.35

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Alkalinity (mg/l)

3000 2500 2000

Alkalinity

18

Eff. pH

16

TVA/T-ALK ratio

14

OLR

12 10

1500

8

1000

6

pH TVA/T-ALK ratio OLR (gCOD/l.d)

20

3500

4 500

2 0

0 0

25

50

75 100 Operation day

125

150

Fig. 2. Effluent pH, alkalinity and ratios between total volatile acids and total alkalinity (TVA/T-ALK) against operating time.

75

20 16 14

45 30

Biogas

12

Methane

10

% methane

8

OLR

6

15

OLR (gCOD/l.d)

Production rate (l/d) Methane content (%)

18 60

4 2

0

0 0

25

50

75 100 Operation day

125

150

Fig. 3. Biogas and methane production rates.

has been regarded as indicating that the buffering capacity is adequate (Zinatizadeh et al., 2007). Biogas and methane production rates are shown in Fig. 3. The biogas production increased with the stepwise increases in OLR from 2.0 to 3.3, 6.0, 7.5, 10.0, 11.4 and 13.7 g COD/l d, while the methane content in the biogas varied between 60–72% and was 68% on average. The biogas and methane production rates responded to the shift in OLR relatively fast; i.e., within the working HRT. For the two highest ORLs of 17.6 and 19.0 g COD/l d, the biogas production did not increase with the increased OLR. However, the methane content in the biogas remained high at around 70% for these two OLR values. The fact that the biogas and methane production did not increase for these two highest OLR values suggested that the rates of methanogenesis reactions also did not increase and that the overall process then became limited by slow methanogenic growth rates. It has been reported in the literature that methane formation is associated with cell growth of Methanosarcina and that nearly equimolar methane was produced from acetate during the methanogenic growth (Yang and Okos, 1987). Fig. 4 illustrates that the sCOD removal was relatively constant throughout an experimental run, with >90% sCOD removal efficiency on average. In contrast, the tCOD removal efficiency was around 93% for the first three OLRs and slowly decreased for the last six OLRs to an efficiency of 82% at the highest OLR of 19.0 g COD/l d. The increasing OLR was found to have a strong effect on the efficiencies of the tCOD removal, but a much weaker effect on the efficiencies of the sCOD removal. The tCOD in the effluent increased from a value of 2000 mg/l for the ORLs of 2.0–7.5 g

COD/l d, to 5000 mg/l for the OLRs of 10.0–11.4 g COD/l d and finally to 12,000–13,000 g COD/l d for the OLRs of 13.7, 17.6 and 19.0 g COD/l d. Similar to the biogas and methane production rates, the tCOD in the effluent responded to the shift in OLR relatively fast; i.e., within the working HRT. The tCOD values in the effluent suggested that a further treatment would be required before the effluent is discharged to the environment. The sCOD is a parameter that indicates the oxygen demand for soluble compounds and tCOD is a parameter that indicates the oxygen demand for the degradation of both soluble and insoluble compounds. Therefore, the decrease in the tCOD removal efficiency with relatively constant sCOD removal efficiency as the OLR is increased suggests that it is the biodegradation of the insoluble components that is a limiting factor in the COD removal efficiency. The results of an investigation on hydrolysis of fibers, the major insoluble component in raw POME, will be given in the next section. A comparison of the performance of the modified CSTR with other reactors is shown in Table 3, which gives a summary of the operating OLR, COD removal efficiencies, biogas production rates and methane yields. Anaerobic upflow reactors are usually sensitive to loading of suspended solids. When UASB and EGSB were used to treat raw POME, relatively low methane yield and granular sludge flotation were reported (Borja and Banks, 1994; Zhang et al., 2008; Fang et al., 2011). AHR contain a granular sludge bed in a lower zone, which acts like a conventional UASB, and an upper zone that contains filter media which prevent the washout of granules from the lower zone.

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100 90 80

50000

70 40000

60 50

30000

40 20000

30 20

10000

Removal efficiency (%)

COD concentration (mg/l)

60000

Inf. tCOD Eff. tCOD Inf. sCOD Eff. sCOD % tCOD removal % sCOD removal

10 0

0 0

25

50

75 100 Operation day

125

150

Fig. 4. COD removals against operating time.

The combined features of a UASB and an anaerobic filter enable the AHR to process POME at higher OLRs than a UASB (Ahmad et al., 2011; Khemkhao et al., 2012). However, high concentrations of SS and residual oil in raw POME cause scum and clogging problems in the AHR. The results for AHR in Table 3 (Choi et al., 2013; Jeong et al., 2014) were obtained by using POME pretreated by a screw decanter as the substrate for the AHR. UASFF reactors have been successfully used to treat raw POME (Najafpour et al., 2006). A UASFF reactor is a combination of UASB and upflow fixed film (UFF) reactors. The bottom part of the UASFF reactor is the UASB reactor, the middle part serves as a fixed film reactor, and the top part serves as a gas–liquid–solid separator. High COD removal of 97% at HRT of 3 d and methane yields of 0.346 l CH4/g COD removed were achieved at an OLR of 11.6 g COD/l d. However, when the HRT was reduced from 3 d to 1.5 d, corresponding to an OLR of 23 g COD/l d, excessive built-up of sludge in the reactor, sludge wash-out and turbid effluent were observed. Najafpour et al. (2006) suggested that there are two main factors controlling the stability of the UASFF reactor at high OLRs. The first factor is internal packing that effectively provides capture of solids in the reactor. The second factor is a high ratio of effluent recycling that helps create internal dilution for the elimination of effects of high organic loading. Therefore, Najafpour et al. (2006) used raw POME mixed with recycled effluent in a ratio of 1:11.25. A stirred tank reactor is considered to be more suitable than an anaerobic upflow reactor for treating raw POME with high SS and FOG. In early work by Borja-Padilla and Banks (1993), a semicontinuous anaerobic reactor was successfully operated under thermophilic conditions at a maximum OLR of 15 g COD/l d. Raw POME was added to the reactor every 3 h, and effluent was simultaneously withdrawn. The feed was mixed for 10 min by a stirrer, and the system was allowed to settle for a 3-h period. It was suggested that the accumulation of a high biomass concentration in the reactor and efficient degradation of the POME were achieved by keeping the mixing cycle short, and the settling time relatively much longer. However, the reactor was not operated at an OLR higher than 15 g COD/l d because high suspended solid concentrations were observed in the reactor. Such high suspended solid concentrations can lead to sludge flotation. In a later study by Choorit and Wisarnwan (2007), a CSTR was successfully operated at 55 °C at a maximum OLR of 17 g COD/l d, and a HRT of 5 d with a COD removal of 70% and a biogas production rate of 4.66 l/l d with 70% of methane content. The COD removal of 70% was relatively low compared to other studies reported in Table 3. A possible explanation for the relatively low COD removal is that the CSTR had no component part to capture biofibers before they left the reactor with the effluent. In Poh and Chong (2010), a CSTR was

used to cultivate a thermophilic mixed culture for treatment of raw POME. However, the CSTR operation was investigated at relatively low OLRs, i.e., 3.5–6.3 g COD/l d. The modified CSTR in this study was operated successfully with raw POME at the highest OLR of 19 g COD/l d. As shown in Table 3, the methane production rate of 4.14 l/l d is higher than that of all other rectors, while the COD removal, CH4 content and methane yield are comparable with other reactors. The modified CSTR does not require an effluent recycle stream, and therefore it is not surprising that the methane production rate per reactor volume is higher than that reported for the UASFF by Najafpour et al. (2006). 3.3. Biodegradation of fiber and oil Fiber accumulation in the reactor and rates of fiber hydrolysis were analyzed for the OLRs of 7.5, 10.0, 11.4, 13.7, 17.6 and 19.0 g COD/l d. Fig. 5(a) shows the fiber mass inflow and outflow rates, the rate of fiber accumulation and the hydrolysis as a function of OLR. It can be seen that the fiber inflow rate was proportional to OLR and that the palm fiber accumulation in the reactor remained at the low rates of 0.32–0.75 g/d for all OLR values. The fiber outflow rates increased from 0.93 g/d at OLRs of 7.5–3.04 g/d at OLR of 11.4 g COD/l d, and to 4.25 g/d at 13.7 g COD/l d, but then decreased to 3.27 g/d at 17.6 g COD/l d and to 2.24 g/d at 19.0 g COD/l d. The increasing/decreasing rates of the fiber outflow were found to correspond with the increasing/decreasing concentrations of tCOD in the effluent for the same OLR values (Fig. 4). The data therefore indicate that fiber was a major component contributing to the increasing tCOD values in the effluent, and that the tCOD removal efficiencies depended upon the degradation of fibers. Palm fibers can be biodegraded by hydrolysis reactions of cellulose, hemicelluloses and lignin. The operating temperature of 55 °C has been reported to be beneficial for the hydrolysis of the fibers. The thermophilic temperature not only increases thermal degradation, but also, based on PCR-based DGGE analysis, enriches a number of working hydrolytic, acidogenic and acetogenic bacteria (Khemkhao et al., 2012). It has been reported in the literature that the well-known anaerobic cellulolytic bacteria, Clostridium, requires high temperatures both for its growth and for cellulose degradation (Pérez et al., 2002). In this study, hydrolysis rates of palm fibers were calculated by mass balance in a rate form. The hydrolysis rate was calculated as:

Hydrolysis rate ¼ fiber mass inflow rate  fiber mass outflow rate  fiber accumulation rate:

Table 3 Comparative anaerobic digestion performances for POME treatment. System

Pretreatment of raw POME

Operating conditions

Fiber feed rate (g/l d)

SS feed rate (g/l d)

Parameters

References CH4 content (%)

Biogas production rate (l/l d)

CH4 production rate (l/l d)

Methane yield (l CH4/g COD removed)

4–4.5

93.5

65

2.8

1.64

0.27

Choi et al. (2013)

n/a

0.6

95

72

3.0

2.16

0.11

Jeong et al. (2014)

12.3

n/a

6.6

71

71

3.7

2.65

0.30

2 3

17.5 5.3

n/a n/a

10.8 n/a

91 87

70 69

n/a 2.1

n/a 1.37

0.18 0.33

No pretreatment

4

10.6

n/a

3.5

97

54

n/a

n/a

n/a

UASB UASFF

Filtration No pretreatment

4 3

12.5 11.6

n/a n/a

n/a 2.3

82 97

48 72

3.2 5

1.51 3.65

0.32 0.32

UASFF

Sedimentation

2.2

12.9

n/a

2.6

95

n/a

n/a

n/a

0.33

Choorit and Wisarnwan (2007) Zhang et al. (2008) Faisal and Unno (2001) Borja and Banks (1994) Ahmad et al. (2011) Najafpour et al. (2006) Zinatizadeh et al. (2007)

UASFF

Cationic and anionic polyacrylamide

1.5

9.3

n/a

0.01

93

n/a

n/a

n/a

0.31

Screw decanter

6.2

15

n/a

0.6

95

72

4.4

3.17

0.24

Jeong et al. (2014)

No pretreatment

5

17.0

n/a

9.2

70

70

4.7

3.24

0.28

Modified CSTR with a deflector EGSB EGSB UAF

No pretreatment

3.3

19.0

4.5

6.6

82

68

6.2

4.14

0.27

Choorit and Wisarnwan (2007) This study

No pretreatment Deoiling process No pretreatment

5 5 5

9.7 19.4 10.9

n/a n/a n/a

4.1 8.2 4.6

92 92 88

60 73 n/a

4.2 1.98 4.9

2.55 1.44 n/a

0.29 0.08 n/a

UASB UASB UASB

No pretreatment Deoiling process Aluminum sulfate

5 5 2.4

9.7 19.4 9.5

n/a n/a n/a

4.1 8.2 2.1

93 94 81

61 70 76

4.2 2.1 3.0

2.56 1.56 2.40

0.28 0.09 0.32

OLR (g COD/l d)

Three-phase screw decanter Screw decanter

0.7– 2.4 5.8

19–23

n/a

15

No pretreatment

7

EGSB MABR

No pretreatment n/a

UASB

35–38 °C AHR followed by ABF and ADF AHR followed by ABF CSTR

55–57 °C AHR followed by ABF CSTR

Fang et al. (2011) Fang et al. (2011) Mustapha et al. (2003) Fang et al. (2011) Fang et al. (2011) Khemkhao et al. (2012)

M. Khemkhao et al. / Bioresource Technology 177 (2015) 17–27

COD removal (%)

HRT (d)

Notes: n/a refers to not available; AHR anaerobic hybrid reactor; ABF anaerobic baffled filter reactor; anaerobic downflow filter reactor; EGSB expanded granular sludge bed; MABR modified anaerobic baffled bioreactor; UAF upflow anaerobic filter; UASFF upflow anaerobic sludge-fixed film.

23

24

M. Khemkhao et al. / Bioresource Technology 177 (2015) 17–27

(a) 30 25

Rate (g/d)

20

Fiber mass inflow

15

Fiber mass outflow Rate of fiber accumulation

10

Hydrolysis rate 5 0 7.5

10

11.4

13.7

17.6

19

OLR (g COD/l.d)

100

5.0

90

4.5

80

4.0

70

3.5

60

3.0

50

2.5

40

2.0 1.5

30

1.0

20

0.5

10

0.0

FOG removal efficiency (%)

FOG concentration (g/l)

(b) 5.5

Inf FOG Eff FOG %FOG removal

0 7.5

10

11.4

13.7

17.6

19

OLR (g COD/l.d) Fig. 5. Biopolymer degradations as a function of OLR (a) palm fiber and (b) FOG.

Fig. 5a illustrates the hydrolysis rates of fibers in the CSTR. It can be seen that the hydrolysis rates are increasing with the inflow rates of fiber, indicating that the hydrolytic bacteria can adjust to the increasing amounts of fiber in the reactor. The concentrations of cellulose, hemicelluloses and lignin in the wastewater feed were measured as 4.27 g/l, 2.48 g/l and 2.28 g/l, respectively, corresponding to a percentage composition of 47% cellulose, 28% hemicelluloses and 25% lignin. The concentrations of cellulose, hemicelluloses and lignin in the effluent were measured as 0.41 g/l, 0.72 g/l and 0.92 g/l, respectively, corresponding to percentage compositions of 20% cellulose, 35% hemicelluloses and 45% lignin. The concentrations of cellulose, hemicelluloses and lignin accumulated in the reactor were 1.67 g/l, 0.86 g/l and 10.87 g/l, respectively, corresponding to percentage compositions of 13% cellulose, 6% hemicelluloses and 81% lignin. The hydrolysis rates of each component were calculated from the mass balance formula given above and the results are shown in Table 4. The hydrolysis rate of cellulose was calculated to be 6.65 g/d, which is approximately double the hydrolysis rate of the hemicelluloses and more than 3 times the hydrolysis rate of lignin (approximately 2 g/d). The slow rate of lignin hydrolysis resulted in a high rate of lignin accumulation in the reactor of approximately 0.5 g/d and

Table 4 Mass balance of fiber component.

Mass inflow Mass outflow Accumulation rate Hydrolysis rate

Cellulose (g/d)

Hemicelluloses (g/d)

Lignin (g/d)

7.43 0.71 0.07 6.65

4.31 1.26 0.04 3.02

3.96 1.60 0.46 1.91

Note: The samples were taken on day 138.

therefore lignin became the major component accumulated in the reactor. The biodegradation of cellulose and hemicelluloses proceeded at desirable rates in the CSTR, but because of the lignin accumulation it may be necessary to periodically drain the solids from the reactor. The FOG concentrations in the influent and the effluent are shown in Fig. 5b. As the OLR was consecutively increased from 7.5 to 10.2, 11.4, 13.7, 17.6 and 19.0 g COD/l d, the FOG concentrations in the influent increased approximately linearly, whereas the FOG concentrations in the effluent remained at low levels of 0.01–0.22 g/l. The FOG removal based on the difference in FOG concentrations in the influent and the effluent was between 92–99.6% and was 97% on an average. In addition, scum, which is typically formed at high FOG concentrations, was not observed in the reactor. Therefore, the CSTR operation consistently yielded high FOG removals even though oil in wastewater is normally difficult to degrade by microorganisms. For example, Hanaki et al. (1981) reported that LCFAs are a cause of operational problems in anaerobic systems because of their low solubility and potential inhibition of anaerobic microorganisms. The operating temperature of 55 °C used in the present study was thought to be beneficial to the hydrolysis of oils. The thermophilic temperature can enhance the solubility of long-chain fatty acids, which is rather limited at moderate temperatures (Hanaki et al., 1981). Table 5 shows the compositions of LCFAs in the original POME and in the treated POME at OLR of 17.6 g COD/l d and at OLR of 19.0 g COD/l d. In contrast to palm oil, which is known to contain palmitic acid and oleic acid as the major components (Jairurob et al., 2013), the POME used in this study was found to contain myristoleic acid (43%) and heptadecanoic acid (39%) as the major components and 10-heptadecenoic acid (12%) and palmitoleic acid (6%) as minor components. The two major components in the original POME were also found to be the major components of the

25

M. Khemkhao et al. / Bioresource Technology 177 (2015) 17–27 Table 5 Long-chain fatty acids contents. Samples

Long-chain fatty acids

Raw POME (mg/l) Effluent sample from OLR of 17.6 g COD/l d (mg/l) Effluent sample from OLR of 19.0 g COD/l d (mg/l) Raw POME (mg/g FOG) Effluent sample from OLR of 17.6 g COD/l d (mg/g FOG) Effluent sample from OLR of 19.0 g COD/l d (mg/g FOG) Raw POME (%) Effluent sample from OLR of 17.6 g COD/l d (%) Effluent sample from OLR of 19.0 g COD/l d (%)

Myristoleic acid (14:1)

Palmitoleic acid (16:1)

Heptadecanoic acid (17:0)

10-Heptadecenoic acid (17:1)

3.37 0.20 0.23 0.47 4.00 2.56 42.93 48.78 63.89

0.44 0.07 0 0.06 1.40 0 5.61 17.07 0

3.07 0.14 0.13 0.43 2.80 1.44 39.11 34.15 36.11

0.97 0 0 0.14 0 0 12.36 0 0

(a) Uncultured Firmicutes bacterium gene for 16S rRNA, paral sequence Pseudomonas sp. jg7 16S rRNA (97%) (JX624259.2) Chryseobacterium sp. THG-EP9 16S rRNA (88%) (KF532126.1) Lactobacillus manihovorans strain TCP017 16S rRNA gene (100%) ( KF312682.1) Bacillus sublis strain NB10 16S rRNA (88%) (JX489616.1) Uncultured Firmicutes bacterium gene for 16S rRNA, paral sequence Acinetobacter sp. MU02 16S rRNA gene (92%) (KF261600.1) Clostridium popule strain 743A 16S rRNA (95%) (NR026103.1) Clostridium indolis strain 7 16S rRNA (98%) (NR026493.1) Clostridium caenicola strain EBR596 16S rRNA (92%) (NR041311.1) Clostridium cadaveris strain JCM 1392 16S rRNA (100%) ( NR104695.1) Clostridium histolycum strain ATCC 19401 16S rRNA (94%) (NR104889.1) Exiguobacterium sp. strain AT1b 16S rRNA (94%) (NR074970.1) Thermotoga lengae strain TMO 16S rRNA (98%) (NR074951.1) Natranaerobius thermophilus strain JW/NM-WN-LF 16S riRNA (86%) (NR074181.1) Acetomicrobium flavidum strain DSM 20664 16S rRNA (100%) ( NR104752.1) Pelobacter seleniigenes strain KM 16S rRNA (91%) (NR044032.1) Rhodothermus marinus strain DSM 4252 16S rRNA (87%) ( NR074728.1)

(b) Acidianus brierleyi strain IFO 15269 (DSM 1651) 16S rRNA (74%) (NR043409.1)

Uncultured Methanolinea sp. gene for 16S rRNA (87%) (AB479395.1)

Uncultured Methanothermobacter sp. clone H6 16S rRNA (87%) (HQ271186.1) Uncultured Desulfurococcales archaeon clone slmarc120 16S rRNA (88%) (HQ700670.1) Uncultured Methanolinea sp. 16S rRNA (83%) (AB434764.1) Uncultured Methanolinea sp. 16S rRNA (87%) (AB602620.1) Methanothermobacter defluvii ADZ 16S rRNA (90%) (NR028248.1) Uncultured Methanosaeta sp. clone CINBIN-32R27 (100%) (HQ392652.1) Uncultured Methanothermobacter sp. clone 420DA-27 16S rRNA (86%) (JQ772456.1) Methanothermobacter marburgensis str. Marburg 16S rRNA (95%) (NR102881.1) Methanothermobacter thermoformicicum Z-245 16S rRNA (87%) (X68712.1) Methanogenium cariaci strain JR1 16S ribosomal RNA (93%) (NR104730.1) Uncultured Methanothermobacter sp. clone B51-45 16S rRNA (93%) (JF754557.1) Methanothermobacter thermautotrophicus CaT2 DNA (97%) (AP011952.1) Methanosaeta thermophila PT 16S ribosomal RNA (99%) (NR074214.1 ) Fig. 6. DGGE profiles of (a) the bacterial and (b) the archaeal communities on day 161.

26

M. Khemkhao et al. / Bioresource Technology 177 (2015) 17–27

residual oil in the reactor effluent. The results shown in Table 5 suggest that myristoleic acid and heptadecanoic acid may be more difficult to degrade biologically than palmitoleic acid and 10-heptadecenoic acid which were found to be degraded in the CSTR. Other LCFAs originally present in palm oil may have been biodegraded by natural microorganisms during the collection process, while palmitoleic acid and 10-heptadecenoic were found to be degraded in the CSTR.

with either H2/CO2 or formate as substrates (Schink, 1997). Methanolinae sp. can use H2 or formate for growth and methane production (Imachi et al., 2008). Methanosaeta sp. and hydrogenotrophic methanogen have been detected in an anaerobic methane production from crude glycerol. Methanosaeta sp. is known to utilize only acetate for methane production (Smith and Ingram-Smith, 2007).

3.4. Identification of the working microorganisms

4. Conclusion

The working microorganisms in the CSTR operated at OLR 19.0 g COD/l d were determined by DGGE analysis. The MLSSs were collected on day 161, when the steady-state operation at OLR 19.0 g COD/l d was assumed. Fig. 6a shows the results of the DGGE analysis for the bacterial community and Fig. 6b shows the results for the archaeal community. Fig. 6a shows that for the bacterial community, the majority of the bacteria belong to the phylum Firmicutes with Clostridium, Bacillus, Lactobacillus, Natranaerobius and Exiguobacterium being members of this phylum. Other bacteria present include Acetomicrobium, Chryseobacterium and Rhodothermus which belong to the phylum Bacteroides, Pseudomonas and Acinetobacter which are members of the phylum Gammaproteobacteria, and Acetobacter, Pelobacter and Thermotoga which belong to the phylum Alphaproteobacteria, Deltaproteobacteria and Thermotogae, respectively. In the archaeal community, members of hydrogenotrophic and aceticlastic methanogens were observed. The DGGE in Fig. 6b indicates that Methanothermobacter, a member of hydrogenotrophic, was dominant among the methanogens followed by the hydrogenotrophic Methanolinea. In addition, aceticlastic Methanoseata was detected. In anaerobic processes, proteins, carbohydrates, cellulosic materials and fats are normally hydrolyzed by extracellular enzymes. The known activities of the microorganisms detected in the CSTR are as follows. Clostridium have been classified as cellulolytic bacteria, which produce cellulase and xylanase for degradation of cellulose and hemicelluloses and for acetate, lactate, ethanol and H2 production (Sasaki et al., 2012). Pseudomonas can produce cellulase and xylanase (Cheng and Chang, 2011). Bacillus is able to degrade lignocellulosic biomass for H2 production (Song et al., 2013). In addition, Rhodothermus marinus can produce cellulase, xylanase, b-mannase, a-L-arabinofuranosidase and a-galactosidase (Gomes and Steiner, 1998). Pseudomonas, Bacillus and Clostridium have been classified as acidifying bacteria. These bacteria are known to utilize watersoluble chemical substrates to produce short-chain organic acids, alcohols, aldehydes, CO2 and H2. Pseudomonas ferments glucose to methanol, CO2 and H2. Bacillus and Lactobacillus produce organic acids, especially lactic acids, from hydrolytic products. Thermotoga, a thermophilic methanol-degrading bacterium, is able to ferment methanol to CO2 and H2 in syntrophic culture with Methanothermobacter thermautotrophicus. In addition, Acetomicrobium flavidum, a thermophilic anaerobic bacterium, produces acetate, CO2 and H2 from glucose (Soutschek et al., 1984). Natranaerobius thermophilus utilizes fructose, cellobiose, ribose, sucrose, trehalose, trimethylamine, pyruvate, casamino acids, acetate, xylose, and peptone as carbon and energy sources. During growth on sucrose, N. thermophilus produces acetate and formate as major fermentation products (Mesbah et al., 2007). Members of the order Methanobacterials, particularly those belonging to the genera Methanobacterium, use a CO2 reduction pathway with H2 as an electron donor for methanogenesis. Methanothermobacter thermautotrophicus can drive methanogenesis by utilizing the CO2 reduction pathway with H2 and/or formate as an electron donor. In the order Methanomicrobiales, Methanolinea and Methanogenium are able to utilize the CO2 reduction pathway

A greater amount of palm fibers can be captured in the modified CSTR with a deflector compared with that captured in a typical CSTR. The modified CSTR can be operated with raw POME at the highest OLR of 19.0 g COD/l d. Both cellulose and hemicelluloses were degraded at significant rates in the modified CSTR. The results of DGGE suggested that the dominant hydrolytic bacteria responsible for the biodegradation of palm fiber was Clostridium sp. The methane productivity (rate of methane production per reactor volume) was found to be the highest among the productivities reported in the literature. Acknowledgements The authors are grateful to the research funding from the Commission of Higher Education Thailand. The Royal Golden Jubilee scholarship granted to M. Khemkhao from Thailand Research Fund and the Joint Graduate School of Energy and Environment (JGSEE) are acknowledged. The authors also would like to thank Ngaung-Khaem water quality control plant for providing sludge and Suksomboon Palm Oil Co., Ltd. for POME samples. Special thanks to Dr. Elvin Moore for his critical reading of the manuscript. References Ahmad, A., Ghufran, R., Wahid, Z.A., 2011. Role of calcium oxide in sludge granulation and methanogenesis for the treatment of palm oil mill effluent using UASB reactor. J. Hazard. Mater. 198, 40–48. Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215 (3), 403–410. American Public Health Association (APHA), 2005. Standard methods for the examination of water and wastewater, 21st ed. Washington, DC. Borja, R., Banks, C.J., 1994. Anaerobic digestion of palm oil mill effluent using an upflow anaerobic sludge blanket reactor. Biomass Bioenergy 6 (5), 381–389. Borja-Padilla, R., Banks, C.J., 1993. Thermophilic semi-continuous anaerobic treatment of palm oil mill effluent. Biotechnol. Lett. 15 (7), 761–766. Boušková, A., Dohányos, M., Schmidt, J.E., Angelidaki, I., 2005. Strategies for changing temperature from mesophilic to thermophilic conditions in anaerobic CSTR reactors treating sewage sludge. Water Res. 39, 1481–1488. Cheng, C.L., Chang, J.S., 2011. Hydrolysis of lignocellulosic feedstock by novel cellulases originating from Pseudomonas sp. CL3 for fermentative hydrogen production. Bioresour. Technol. 102 (18), 8628–8634. Choi, W.-H., Shin, C.-H., Son, S.-M., Ghorpade, P.A., Kim, J.-J., Park, J.-Y., 2013. Anaerobic treatment of palm oil mill effluent using combined high-rate anaerobic reactors. Bioresour. Technol. 141, 138–144. Choorit, W., Wisarnwan, P., 2007. Effect of temperature on the anaerobic digestion of palm oil mill effluent. Electron. J. Biotechnol. 10, 376–385. Faisal, M., Unno, H., 2001. Kinetic analysis of palm oil mill wastewater treatment by a modified anaerobic baffled reactor. Biochem. Eng. J. 9, 25–31. Fang, C., O-Thong, S., Boe, K., Angelidaki, I., 2011. Comparison of UASB and EGSB reactors performance, for treatment of raw and deoiled palm oil mill effluent (POME). J. Hazard. Mater. 189, 229–234. Gomes, J., Steiner, W., 1998. Production of a high activity of an extremely thermostable b-mannanase by the thermophilic eubacterium Rhodothermus marinus, grown on locust bean gum. Biotechnol. Lett. 20 (8), 729–733. Hanaki, K., Matsuo, T., Nagase, M., 1981. Mechanism of inhibition caused by longchain fatty acids in anaerobic digestion process. Biotechnol. Bioeng. 23, 1591– 1610. Imachi, H., Sakai, S., Sekiguchi, Y., Hanada, S., Kamagata, Y., Ohashi, A., Harada, H., 2008. Methanolinea tarda gen. nov., sp. nov., a methane-producing archaeon isolated from a methanogenic digester sludge. Int. J. Syst. Evol. Microbiol. 58, 294–301. Jairurob, P., Phalakornkule, C., Na-udom, A., Petiraksakul, A., 2013. Reactive extraction of after-stripping sterilized palm fruit to biodiesel. Fuel 107, 282– 289.

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