Accepted Manuscript

Operation Performance of Up-Flow Anaerobic Sludge Blanket (UASB) Bioreactor

for Biohydrogen Production by Self-Granulated Sludge Using Pre-treated Palm oil
Mill Effluent (POME) as Carbon Source

Safa Senan Mahmod, Azratul Madihah Azahar, Jian Ping Tan, Jamaliah Md Jahim, Peer Mohamed
Abdul, Mohd Shahbudin Mastar Masdar, Nurina Anuar, Mohammed Faisal Mohammed Yunus,
Ahmad Jaril Asis, Shu-Yii Wu

PII: S0960-1481(18)31125-X

DOI: 10.1016/j.renene.2018.09.062

Reference: RENE 10602

To appear in: Renewable Energy

Received Date: 04 April 2018

Accepted Date: 17 September 2018

Please cite this article as: Safa Senan Mahmod, Azratul Madihah Azahar, Jian Ping Tan, Jamaliah
Md Jahim, Peer Mohamed Abdul, Mohd Shahbudin Mastar Masdar, Nurina Anuar, Mohammed
Faisal Mohammed Yunus, Ahmad Jaril Asis, Shu-Yii Wu, Operation Performance of Up-Flow
Anaerobic Sludge Blanket (UASB) Bioreactor for Biohydrogen Production by Self-Granulated
Sludge Using Pre-treated Palm oil Mill Effluent (POME) as Carbon Source, Renewable Energy
(2018), doi: 10.1016/j.renene.2018.09.062

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1 Operation Performance of Up-Flow Anaerobic Sludge Blanket (UASB) Bioreactor

2 for Biohydrogen Production by Self-Granulated Sludge Using Pre-treated Palm oil

3 Mill Effluent (POME) as Carbon Source

6 Safa Senan Mahmod a, Azratul Madihah Azahar a , Jian Ping Tan a, Jamaliah Md Jahim a,b,*, Peer

7 Mohamed Abdul a,b, Mohd Shahbudin Mastar Masdar a,b, Nurina Anuar a,b, Mohammed Faisal

8 Mohammed Yunus c, Ahmad Jaril Asis c, Shu-Yii Wu d

10

11 a Research Centre for Sustainable Process Technology (CESPRO), Faculty of Engineering and

12 Built Environment, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia

13 b Chemical Engineering Programme, Faculty of Engineering and Built Environment, Universiti

14 Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia

15 c Sime Darby Research Sdn Bhd, R&D Centre – Carey Island, Lot 2664, Jalan Pulau Carey,

16 42960 Carey Island, Selangor, Malaysia.

17 d Department of Chemical Engineering, Feng Chia University, Taichung 40724, Taiwan

18

19

20 Correspondence: Jamaliah Md Jahim, Research Centre for Sustainable Process Technology

21 (CESPRO), Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia,

22 43600 UKM Bangi, Selangor, Malaysia

23

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1 Abstract

2 Palm oil mill effluent (POME), an agro-industrial wastewater with high solids content, was

3 subject to hydrolysis by 1% (w/v) nitric acid in order to increase its solubility and the

4 fermentable sugar content from its cellulosic component. POME hydrolysate was then

5 evaluated in an up-flow anaerobic sludge blanket (UASB) bioreactor for the production of

6 biohydrogen gas via mixed culture under thermophilic conditions. The bioreactor was fed

7 with pre-treated POME under varied hydraulic retention time (HRT) between 48-3 h at

8 constant cycle length of 24 h to test the productivity of H2 and the stability of UASB; no

9 washout of biomass occurred at any cycle and the system managed to recover its H2

10 production rate (HPR) after initial fluctuations. In this study, H2-producing granules

11 (HPGs) were formed shortly after the start-up period, and were analysed by FESEM, FTIR,

12 SEM-EDX, and their extracellular polymeric substances (EPS) content. The maximum HY

13 and HPR achieved were 2.45 mol-H2/mol-sugar and 11.75 LH2/LPOME d-1, respectively, at

14 HRT 6 h. Acetic acid was found to be the major by-product at all HRTs, followed by butyric

15 acid, while Clostridium spp. was found to be the most dominant H2-producing bacteria in the

16 system. Results suggest that UASB has a good potential for stable H2 production with high

17 POME digestion rate.

18 Keywords: Biohydrogen; hydraulic retention time; palm oil mill effluent; UASB; H2-

19 producing granules

20 1 Introduction

21 The consumption of palm oil, one of the most popular edible vegetable oils worldwide, is

22 increasing steadily, the production of palm oil is mainly led by Indonesia, Malaysia and

23 Thailand [1]. Despite the massive revenues from palm oil production, a drawback of palm

24 oil milling process is the concomitant generation of palm oil mill effluent (POME) as a

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1 wastewater from the mills [2]. In line with palm oil milling process, approximately 5 to 7

2 tons of water is used to produce 1 ton of crude palm oil (CPO), half of which is turned into

3 POME. The composition of POME includes 0.6–0.7% residual oil and 2–4% of suspended

4 solids, which are mainly the debris of palm fruit mesocarp fiber, while water forms 95-

5 96% of its content [3]. Currently, the global energy demand depends on the fossil fuels’

6 utilization, the impending depletion of which has raised much concern. In addition, the

7 excessive consumption of fossil fuels over the decades has jeopardized the environment,

8 leading to air pollution and severe climate changes [4,5]. In this context, hydrogen has

9 been hailed as a clean and environmentally friendly alternative source of energy since

10 water vapour is the only product of its combustion [4,6]. As compared to hydrocarbon

11 fuels, hydrogen is approximately 2.7 times higher in energy yield [7]. In this regard, POME

12 and its derivatives, being lignocellulosic wastes, have been considered as high potential

13 substrates for the production of biohydrogen because of their abundance, low cost and

14 reliability [8]. Additionally, anaerobic digestion is the preferred treatment for POME due

15 to its numerous merits. Firstly, high COD of POME making anaerobic digestion superior to

16 the aerobic process that requires a balanced nutrient content in order to treat POME [9].

17 Additionally, in anaerobic fermentation, a valuable product (H2) is obtained from a cheap

18 carbon source (POME) without the need for light and additional nutrients [10]. Thirdly,

19 anaerobic fermentation is associated with high hydrogen production rate (HPR) and

20 simple process controls with a various temperature and pressure conditions [6].

21 Due to the low biohydrogen yield from POME, several pre-treatment methods have been

22 advocated to increase its solubilisation and release the fermentable monomeric sugars

23 that could be converted to H2 through anaerobic fermentation. This is explained by the

24 presence of starch, cellulose, hemicellulose and lignin in the lignocellulosic wastes, which

25 could negatively affect the biodegradability of the substrates [8]. Several pre-treatment

26 methods have been applied on POME, for instance, ultrasonication [2], ozonation [11],

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1 drug fluvastatin and Na2SO4 dosing [12], heating [13], autoclaving [14], and acid or

2 alkaline pre-treatments [13,15]. Out of which, nitric acid has been found to demonstrate a

3 short reaction time and higher efficiency for saccharification [16]. In addition, residual

4 nitric acid may serve as a nitrogen source for fermentation, by forming nitrates when

5 neutralized to replace other common nitrogen sources such as yeast extract ammonium,

6 amino acid, and urea [16,17]. According to our previous study [17], POME pre-treated

7 with 1% (w/v) nitric acid showed 65% improvement in biohydrogen yield as compared to

8 untreated POME, with significant increase in POME’s solubility; sCOD increased from

9 40.05 to 44.93 (g/L), and Total reducing sugars from 15.05 to 22.28 (g/L), with negligible

10 amount of furfural and 5-HMF released.

11 Over the years, studies have reported successful H2 production from POME by mixed

12 cultures in different types of bioreactors; continuous stirred-tank reactor (CSTR) [18–20],

13 ASBR [7,21–23], up-flow anaerobic sludge blanket (UASB) [24,25] and UASB-fixed film

14 (UASB-FF) [3,26]. In contrast to fossil fuels, CO2 produced during the production rather

15 than during its utilization in the downstream stage, this thereby promotes easy capture of

16 CO2 and sequestration. Consequently, biological hydrogen production is carbon-neutral or

17 carbon zero [27]. The application of UASB systems for the conversion of organic waste

18 into anaerobic biohydrogen has been studied extensively [28–31]. Investigation of the

19 formation of granular sludge, although still in its initial stage, have shown significant

20 improvement on the biohydrogen production as compared to suspended sludge systems

21 [32]. As reported by Kongjan et al [30], samples treated by UASB reactors typically

22 consisted of solids concentration in the total range of 1–3%, with high substrate feeding

23 rates and short hydraulic retention times (HRTs) as they can maintain a high biomass

24 concentrations together with a high specific activity by the formation of granular sludge.

25 Among their advantages, UASB bioreactors have recorded higher operational stability

26 than CSTR bioreactors, due to their low sensitivity to fluctuations in environmental

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1 conditions (e.g. acidity and HRT) [33]. In addition, immobilised cells in the UASB

2 bioreactors can risk developing methanogens, since they prevent biomass wash-out, even

3 at high flow rates [30].

4 Raw POME is discharged at high temperatures around 80–90 °C, and is expected to be

5 easily treated under thermophilic anaerobic conditions [9]. Thermophilic mixed cultures

6 not only have a high potential as H2 producers but also are good in utilizing various types

7 of organic wastes. Nitipan et al [10] have listed some of the advantages of using

8 thermophilic mixed cultures for biohydrogen production, which are; sterilization is not

9 required, could easily get adapted to new environments and a mixture of substrates due to

10 the microbial diversity, and the possibility of obtaining a stable and continuous system.

11 Notably, the most common bacterial species used in dark fermentation to produce H2 are

12 Clostridium [34] and Thermoanaerobacterium [21]. On the other hand, although

13 thermophilic temperature yields in higher biohydrogen yield compared to mesophillic

14 temperature, a negative net energy gain has been reported when operating anaerobic

15 fermentation at temperatures above the ambient temperature [35].

16 To this day, literature on the formation mechanisms involved in sludge self-granulation

17 represented as H2-producing granules (HPGs) using POME as a sole carbon source is

18 somewhat limited. Thus, this study aims to evaluate the impact of various HRTs on the

19 biohydrogen producing UASB bioreactor fed with pre-treated POME as a substrate, and to

20 study the formation of HPGs under various HRTs. In order to achieve this goal, HPGs were

21 examined for all HRTs; these granules were examined by using various analytical tools

22 such as the Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy

23 (SEM) and dispersive X-ray spectroscopy (EDX). Additionally, the microbial population

24 was characterized via PCR and DGGE to better elucidate the microbial hydrogen

25 production process.

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1 2 Materials and methods

2 2.1 POME preparation and pre-treatment

3 POME was collected from a local palm oil mill in Selangor, Malaysia. The collected sample

4 was stored in a cold room at 4 °C until further use. Industrial grade nitric acid (69-70%,

5 J.T.Baker) was used to hydrolyse raw POME, whereas KOH (R&M Chemicals) was used to

6 neutralize the pH of the medium. The reaction conditions were adopted from a previous

7 study [17]; hydrolysis reaction time of 5 min, hydrolysis reaction temperature of 45 °C and

8 nitric acid concentrations of 1%(w/v). Table 1 shows the characteristics of raw POME and

9 pre-treated POME; all tests were conducted in triplicates.

10 2.2 Seed inoculum

11 The seed sludge used in this study was collected from a digested sludge that originated

12 from a thermophilic sequential batch reactor treating raw POME. The sludge was pre-

13 heated at 80 °C for 60 min in a reciprocating water bath (Julabo, model SW22, Germany) in

14 order to inactivate the CH4-producing and other non-H2 producing bacteria and used as

15 the inoculum for biohydrogen fermentation. Characterization of the seed sludge revealed

16 the following: pH 4.98; 53.19 g/L for total COD, 23.30 g/L for volatile suspended solid

17 (VSS); and 30 g/L for total suspended solid (TSS) [17].

18 2.3 Experimental set-up and operation

19 A laboratory-scale, 20 L UASB bioreactor (Fig. 1) was used in this study with 15 L working

20 volume. The stainless steel UASB bioreactor column was designed with an external

21 diameter of 25 cm and a height of 120 cm. An inverted funnel was placed at the top of the

22 bioreactor as a gas-liquid separator to reduce the amount of water vapour passing through

23 the gas outlet. The temperature was controlled electrically and was operated under

24 thermophilic conditions (55 °C). The pre-treatment of the POME was performed

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1 separately before feeding into the UASB bioreactor, and was pumped via a peristaltic

2 pump (Shinshin, Taiwan). For a uniform influent distribution, a liquid distributor was

3 fixed at the bottom of the column bioreactor. Mixing was performed by a circulation pump

4 (air-operated double diaphragm pump, Blagdon, Irland). The effluent was collected from

5 the 1 L overflow unit in order for the HPGs to settle at the bottom of the overflow unit and

6 recycled back to the bioreactor. The biogas produced was monitored by a gas flow meter

7 (Ritter, Germany). The system was considered to be in a steady-state when the fluctuation

8 in the biogas production reading was not more than 5 L. Biohydrogen productivity in

9 UASB was measured on daily basis, the hydrogen production rate (HPR) was measured in

10 Eqn (1).

H2 (L) 1 24 (ℎ)
11 HPR (LH2/LPOME .d-1) = Working volume(L) × HRT (h) × 𝑑 (1)

12 The hydrogen yield was calculated by dividing the moles of biohydrogen produced per day

13 over the total carbohydrate consumed with glucose equivalent, as shown in Eqn (2),
H2 (mol)
14 HY = TC (2)
𝑖 ‒ TC𝑓 (mol)

15 The biomass wash-out was measured based on the solid retention time (SRT), which is

16 calculated in Eqn (3) based on the VSS analysis at the react and settle phase [36],

VSS reactor
17 SRT (h) = VSS × HRT (3)
effluent

18

19 2.4 Analytical methods

20 The composition of the biogas produced was monitored daily, and liquid samples (from

21 the effluent port) were analysed daily for their volatile fatty acids (VFAs), ethanol, total

22 carbohydrates, TSS and VSS. H2 and CO2 were determined by gas chromatography (GC,

23 model SRI 8600C, USA) equipped with thermal conductivity detector (TCD) and helium

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1 ionization detector (HID). The carrier gas used for the GC was helium gas (99.99% purity)

2 at a flow rate of 25 mL/min. The initial oven temperature of the GC was set at 40 °C and

3 2.7 psi. The compositions of sugar and of ethanol were measured using HPLC (Agilent

4 1200, California, USA) with an RI detector and REZEX ROA column. The HPLC was

5 operated at 60 °C and the mobile phase comprised of HPLC grade 0.005 N H2SO4 with a

6 flow rate of 0.6 mL/min. The VFAs were analysed by a UV detector HPLC (Agilent 1100,

7 California, USA) in ROA column with a mobile phase of 0.005 N H2SO4 running at a flow

8 rate of 0.6 mL/min.

9 2.5 Net energy gain

10 The theoretical net energy gain En (kJ/g COD) was calculated according to Perera et al [37],

11 that is defined as the total energy produced equivalent to the hydrogen volume generated,

12 minus the heat energy required to raise the reactor contents from ambient temperature to

13 the fermentation temperature, as shown in Eqn (4),

𝐺𝜌𝐻 (𝐿𝐻𝑉) ‒ 𝑉𝜌𝑤𝐶𝜌(𝑇𝑓 ‒ 𝑇𝑎)

2
14 𝐸𝑛 = 𝑉𝐶 (4)

15 Where, G is the biohydrogen volume (L); 𝜌𝐻 is H2 gas density (8.9×10-5 kg/L); LHV is the
2

16 lower heating value of H2 (120,000 kJ/kg); V is the working volume of the bioreactor (L);

17 𝜌𝑤 is the water density (1 kg/L); 𝐶𝜌 is the water specific heat (4.2 kJ/kg K); 𝑇𝑓 is the

18 temperature at which the reaction took place (K); 𝑇𝑎id the ambient temperature (K); and C

19 is the feed’s COD concentration (g COD/L).

20

21 2.6 HPGs characterization and imaging

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1 The H2-producing granules (HPGs) developed in the UASB were viewed under field

2 emission scanning electron microscopy FESEM (Supra 550VP, Germany). Gluteraldehyde

3 (2% (w/w)) was used for the fixation of the self-granulated granules overnight at 4 °C. The

4 fixed granules were washed thrice with 0.1 M phosphate buffer saline (PBS) solution for

5 10 min in each wash. Dehydration was carried out at 30% to 90% for 10 min each time,

6 and proceeds to three times in 100% (w/w) ethanol. The dehydrated HPGs were placed

7 into a Critical Point Dryer (Leica Microsystems EM CPD 300, Germany) for 1.5 h. For

8 FESEM viewing, dried HPGs were sputter-coated with platinum.

9 The chemical composition of the freeze dried HPGs was verified using FTIR spectroscopy

10 (Thermo Scientific Nicolet 6700, USA) by the attenuated total reflectance (ATR) method.

11 The FTIR spectra were obtained within the range of 4000–400 cm-1 at a transmission

12 mode resolution of 2 cm-1.

13 Extracellular polymeric substances (EPS) was extracted using a modified method by Lutpi

14 et al [38] in which one HPG was immersed in 10 mL of ultrapure water. The sample was

15 treated for 1 h by formaldehyde (37% (w/w)) at 4 °C. Then, it was treated with NaOH (1

16 M) for 3 h. To obtain the soluble EPS, the mixtures were centrifuged at 10000 g for 15 min.

17 The carbohydrate content of the extracted EPS was calculated using the phenol–sulphuric

18 acid method [39]. The protein content was evaluated by the use of the Bradford method

19 with bovine serum albumin (BSA) as the standard [40]. Herein, the quantity of volatile

20 suspended solids (VSS) of the HPGs represents the total of carbohydrates and proteins

21 which is measured using the standard method of American Public Health Association

22 APHA [41].

23 2.7 Microbial community analysis

24 a) Sample collection, sample storage, DNA extraction, and PCR

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1 The sample collected from UASB bioreactor was stored in a freezer at -20 ℃. The modified

2 DNA extraction was carried out using FavorPrep Soil DNA Isolation Mini Kit (Favorgen,

3 USA). The extracted DNA sample was PCR-amplified using the bacterial 16S rRNA gene

4 primer set were: 357F, 5’-GAC TCC TAC GGG AGG CAG CAG-3’ [42] and 518R, 5’-ATT ACC

5 GCG GCT GCT GG-3’ [43]. The GC-clamp (CGC CCG CCG CGC CCC GCG CCC GGC CCG CCG CCC

6 CCG CCC CCC) [43] was attached at the 5’ of the forward primer. The PCR component (PCR

7 Master Mix, Promega, USA) was performed according to manufacturer instructions. The

8 PCR program comprised of an initial denaturation for 3 min at 94 ℃, followed by repeated

9 denaturation for 40 cycles at 94 ℃ for 30 s, annealing at 55 ℃ for 1 min, extension at 72 ℃

10 for 1.5 min, and ended with a final extension for 10 min at 72 ℃. The amplifications of

11 samples were conducted using Eppendorf Mastercycler (Eppendorf AG, Germany).

12 b) DGGE and DNA sequencing

13 The PCR products were separated using VS20WAVE -DGGE (Cleaver Scientific, UK) on a

14 vertical gel of 8% (w/v) acrylamide with a denaturant concentration of 30% (top) to 60%

15 (bottom) of the polyacrylamide gel. After loading the samples, electrophoresis was carried

16 out in 1x TAE buffer at 150 V for 4 h at 60 ℃. Then, the polyacrylamide gel was stained

17 with SYBRR Green Nucleic Acid Gel Stain for 40 min then visualized and sliced out on Gel

18 Imaging (FireReader V10, Uvitec, United Kingdom). The PCR program and DNA

19 sequencing for the eluted DGGE bands were conducted as prescribed by Yasin et al [44].

20 The closest match for the partial 16S rRNA gene sequence was identified by the ribosomal

21 database project [45]. The evolutionary relationship of taxa for the phylogenetic tree was

22 inferred using the UPGMA method [46] which were conducted in MEGA6 [47].

23 3 Results and discussion

24 3.1 Influence of variation in HRT on H2 production in UASB reactor

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1 To date, the UASB system has gained popularity for biohydrogen generation via anaerobic

2 digestion due to its operation at desirably short HRT, high concentrations of self-

3 granulated biomass, and more stable operation under extreme conditions [48]. As shown

4 in Fig 2, the UASB bioreactor reached steady state after ten days and, subsequently,

5 operated steadily for 18 days when fed with pre-treated POME under HRT of 48 h. Upon

6 the attainment of a stable volume of the biogas production for 4 continuous days in the

7 range of 26-28 L/d, the HRT was reduced to 24 h. The H2 production was gradually rising,

8 as might be attributed to the microorganisms being adapted to the new growth conditions

9 of increased substrate feeding rate. The reactor was considered at steady-state at HRT 24

10 h when the biogas production ranged between 48-49 L/d, and then HRT was reduced to

11 12 h. During 12 h HRT, the reactor was fed 4 times a day, and the HPR reached the steady

12 state in the last three days of this period. In general, the increase in HPR and H2 content in

13 the biogas produced has been noted to be associated with lowering the HRT; this can, in

14 turn, be attributed to the provision of more carbon sources for the microorganisms

15 secondary to the elevated substrate feeding rate, which enhanced the microbial activity to

16 produce H2 more abundantly [24]. The maximum H2 production rate of 11.75 ± 0.15

17 LH2/LPOME d-1 and H2 yield of 2.45 ± 0.24 mol-H2/mol-sugar was achieved at HRT of 6 hours.

18 These results suggested that the H2 production using the UASB bioreactor was more

19 favourable for a low HRT (6 h). The bacterial mixed culture had the ability to adapt to a

20 higher substrate availability because of their active substrate metabolism, which in turn

21 caused an early and high H2 production [24]. A drastic decline in the biogas production

22 was observed with the reduction in the HRT to 3 h, during which the feeding was done

23 every 1.5 h. Such a decline could have resulted from a low mixing and poor contact of the

24 substrate with the microorganisms [49]. After the drastic decline that resulted in a low H2

25 production, the system was recovered by increasing the HRT back to 6 h and afterwards to

26 12 h. The HPR had a similar trend as the H2 content under the HRTs of 6 and 12 h. Notably,

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1 the biogas produced from the UASB bioreactor consisted exclusively of H2 and CO2, and no

2 CH4 could be detected over the 70 days of operation which is indicating that the initial heat

3 treatment of the sludge and the slightly acidic conditions (pH 5.5-5.8) in the fermentation

4 medium had successfully inhibited the activity of CH4-producing bacteria [50].

5 Overall, the substrate consumption presented as total carbohydrate (TC) was generally in

6 the range of 55-75.2 %, with the exemption TC consumption of 24-34 % under the HRT of

7 3 h, as illustrated in Fig. 2. This drop could be related to the presence of non-H2 producing

8 bacteria that consumed the carbon substrate and generated CO2 with less H2 production,

9 high substrate feeding rate, the insufficient retention time for the complete substrate

10 conversion, or a high food to microorganism ratio that exceeded the maximum capacity of

11 the H2 producers [51].

12

13 3.2 Evaluation of UASB effluent

14 Based on the results reported in Fig. 3, the UASB system demonstrated excellent stability

15 throughout all the HRTs after the start-up period. Fig. 3(a) shows that the dominant

16 soluble metabolites produced from the UASB bioreactor were acetic acid (HAc) and

17 butyric acid (HBu), with minor production of propionic acid (HPr) and ethanol (EtOH).

18 The acidogenic microorganisms were responsible for the high H2 yield by accepting extra

19 electrons during the HAc and HBu production, as shown in Eqn (5) and (6) [52] [53].

20 Based on the microbial metabolic pathways, HAc and HBu are produced along with the

21 production of H2, whereas the formation of HPr is accompanied by the consumption of H2,

22 shown in Eqn (7) [54]. In theory, 4 mol of H2 is produced from 1 mol of glucose in the

23 fermentation of HAc (C2H4O2) and 2 mol H2 is produced in the fermentation of HBu

24 (C4H8O2):

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1 C6H12O6 +2H2O → 4H2 + 2CO2 + 2C2H4O2 (acetic acid) (5)

2 C6H12O6 → 2H2 +2CO2 + C4H8O2 (butyric acid) (6)

3 Whereas no H2 formation is associated with the production of HPr (C3H6O2), instead, 2 mol

4 of H2 are consumed:

5 C6H12O6 + 2H2 → 2H2O + 2C3H6O2 (propionic acid) (7)

6 During the start-up period, HPr was copiously produced especially on Days 9 and 10, due

7 to a failure in circulation pump that also lead to an increase the pH value to 8. The system

8 was recovered by adding fresh seed to the reactor, and the H2 recorded stable

9 productivity. Sivagurunathan et al [55] have listed various reasons for the HPr

10 accumulation, such as; the overloading of feed during the start-up period, the higher

11 hydrogen partial pressure, or a shift in the dominant acidogenic species. However, the

12 level of HPr was consistently low following the reduction in the HRTs. Moreover, the

13 production of HAc recorded a drastic decline when EtOH was produced, for instance, on

14 Days 26 and 35. EtOH is known to be an unfavourable pathway in the H2 production

15 process by shifting the pathway towards solvent formation [36], as illustrated in Eqn (8),

16 whereby 1 mol of ethanol consumes 1 mol of H2 under anaerobic conditions [24].

17 C6H12O6 2CO2 +2C2H5OH (Ethanol) (8)

18 In this study, any variations in the production pathway that lead to the accumulation of

19 EtOH or HPr in the system were recovered shortly and returned to HAc and HBu

20 pathways. The concentrations of HPr and EtOH did not exceed 100 mM under normal

21 conditions. Hence, no toxicity was caused from accumulation of VFAs in this UASB system.

22 In this regard, Khemkhao et al. (2012) have suggested that the relatively high VFAs

23 concentrations resulted in, or from, the breakup of HPGs in a UASB-H2 producing

24 bioreactor.

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1 In Fig. 3(b), the greatest fluctuations were noted in the concentrations of the TSS and VSS

2 in the effluent during the HRT of 48 h, which reflect the low stability of the reactor. With

3 the reduction in the HRT, the UASB shows that the biomass was well maintained even

4 when the HPR diminished under the HRT of 3 h, indicating that UASB has less risk of

5 biomass washout [30]. Under the HRT of 12 h, the biomass (VSS) ranged between 14 and

6 18 g/L, whereas, under the HRT of 6 h, it ranged between 17 and 20 g/L.

7 The solid retention time (SRT) of the system recorded higher values than HRT throughout

8 all different HRT runs, as shown in Table 2. The aforementioned indicates that no

9 washout of biomass cells have occurred, lending further evidence to the good stability of

10 UASB bioreactors. Furthermore, this finding is in agreement with Kim et al [57] who

11 suggested that, for complex substrates, herein POME, a higher SRT might be more

12 favourable due to the slow degradation of the organic compounds. In contrast to a study

13 conducted by Oh et al [58], that stated that during biohydrohen production using mixed

14 cultures, the increase in total biomass (SRT) might be accompanied with the increase in

15 non-H2 producing bacteria due to some changes in the microbial population, this could be

16 due to the drastic increase in SRT.

17

18 3.3 HPGs formation and characterization

19 The HPGs in the UASB bioreactor were produced shortly after the start-up period in

20 different shapes and sizes. Fig. 4 reveals the morphology of the HPGs to be of a non-

21 spherical, irregular and uneven shape with different sizes. Despite the different sizes of

22 HPGs produced, the biggest granules were produced under the HRT of 12 h.

23 As depicted on Fig. 5(a: i&ii) the FESEM performed on a HPG showed dominant rod-

24 shaped bacteria on the surface alongside POME residues. The cross-sectioned image of the

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1 HPG in Fig. 5(a:iii) did not show clear image of bacterial community therein. Hence, it was

2 deemed necessary to carry out both SEM-EDX and FTIR analysis in order to determine the

3 content of HPGs that could not be determined by visual imaging.

4 To identify the composition of the surface and the inner layers of the HPGs produced,

5 energy dispersive X-ray spectroscopy (EDX) was used, the results were shown in Fig. 5 (b:

6 i&ii). Carbon and oxygen were the most dominant elements on the outer and inner layers

7 of the HPGs, highlighting that the HPGs consisted of organic materials (biomass). The pH of

8 the medium affected not only the extent of metal accumulation on microbial surfaces, but

9 also that of salt precipitation within the HPGs. In fact, metallic salts are precipitated within

10 the HPGs, this could be attributed to the higher pH therein as compared to the bulk liquid

11 in the UASB reactor [59]. The presence of calcium in POME had an added advantage to the

12 formation of HPGs. Being a positive ion, calcium can neutralize the negatively charged

13 surfaces of bacteria by adsorption, leading to a decrease in the electrical repulsion

14 between bacteria in a significant way creating a cell-to-cell interaction, which is a decisive

15 step towards granulation. This agrees with the DLVO (Derjaguin, Landau, Verwey, and

16 Overbee) theory of microbial granulation, which considers the overall Gibbs free energy as

17 a function of the sum of attractive and repulsive forces over a distance between the cells

18 [28]. Furthermore, the multi-valence positive ion (Ca+2) may promote sludge granulation

19 by bonding with extracellular polymer substance (EPS). This in turn implies that Ca+2 may

20 bridge EPS-to-EPS and cells-to-EPS together to form an initial 3D structure of microbial

21 community, wherein bacteria can further develop [60]. Unfortunately, nitrogen could not

22 be detected by EDX. Therefore, for accurate quantification of the microbial content, the

23 bacterial extracellular polymeric substance (EPS) was measured, and the protein-to-

24 carbohydrate (P/C) ratio was found to be 1.69. In this regard, a similar ratio has been

25 obtained by Lutpi et al [38] for an attached biofilm on granular activated carbon in a H2

26 bioreactor under HRT of 24 h. Wang et al [61] have reported that a lower protein-to-

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1 polysacchride ratio in the EPS of an activated sludge could reduce the cell surface

2 hydrophobicity, hence compromising the flocculating ability of the sludge and its settle-

3 ability. Likewise, Chen et al [62] reported that a low P/C ratio in the EPS could lead to a

4 decline in the sludge settle-ability.

5 Furthermore, the FTIR spectrum (Fig. 5(c)) revealed the functional groups available on

6 the HPG surface to be –OH, –COOH, C(N)=O, C–N and N–H groups. A broad absorption

7 band at 3337.3 cm-1 denoted the presence of –OH groups [38] and the same band could be

8 representing the hydrogen vibrations of alcoholic groups (OH), phenol or carboxyl groups

9 (COOH) that are attributed to N-H vibrations of the amide groups [59,63]. At 700 cm-1, the

10 band reflected the existence of unsaturated bonds. Collectively, these functional groups

11 suggested the presence of the EPS in the samples. Similar data have been represented by

12 Lutpi et al [38] and Kumar et al [64]. The two sharp bands at 2919.6 cm-1 and 2851.9 cm-1

13 are attributed to C–H stretching vibrations [63,65] these bands were also found in samples

14 originated from different types of wastes and were assigned for the fat and lipid

15 components [66]. This may refer to the oil that usually leads to the formation of scum but,

16 due to the mixing mechanism in the UASB system, it led to the formation of HPGs

17 alongside the solids of POME and the microbes. The peaks around the 1500-1600 cm-1

18 region can indicated the presence of the aromatic rings of lignin [66]. The bands in the

19 region of 1700 to 1500 cm-1 can be assigned to the proteins [67]. According to Shi et al

20 [63], peptidic bond of protein (amide I) were represented by the peaks of C–O and C–N at

21 1655 cm-1, while the peptidic bond (amide II) of protein stretching vibration of C–N and

22 deformation vibration of N–H was assigned at 1577.9 cm-1, Fig. 5(c). These bands confirm

23 the proteinaceous content of HPGs which may be attributed to the microbial population on

24 the surface; this finding is further corroborated by the FESEM imaging in Fig. 5(a).

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1 The formation of HPGs shortly after the start-up period could be attributed to the

2 presence of solids in POME that acted as a support to the bacteria and facilitated the

3 agglomeration process; HPGs produced in this study are larger in size as compared to

4 other substrates, Table 3. This is the first report that shows sludge granulation at long

5 HRTs.

6 Table 4 summarises the maximal biohydrogen yield by the different microorganisms

7 operating in different bioreactors using POME as the sole carbon source. To our

8 knowledge, the current study appears to be one of the pioneering studies, demonstrating

9 the highest biohydrogen production from POME under the HRT of 6 h with 2.45 mol-

10 H2/mol-sugar consumed, as compared to other studies. In addition to the effect of the

11 POME pre-treatment on HY [17], the resultant HPGs succeeded in maintaining the stability

12 of the UASB bioreactor for shorter HRTs.

13 The system’s net energy gain EN (-1.91 kJ/d) was calculated based on Eqn (4), noting that

14 the resulting improvement in HY under thermophilic system is not high enough to

15 compensate for the energy required for heating the reactor contents from ambient

16 temperatures of ~25 ͦC to mesophilic or thermophilic conditions [35]. However as

17 mentioned earlier, POME is discharged from the mill at 90-80 ͦC, hence, measuring the

18 energy gain would be according to the discharge temperature not the ambient

19 temperature as conducted in the laboratory scale. Therefore, when same reaction

20 conditions are conducted at the pilot plant, EN is calculated to be 3.60 kJ/d.

21 3.4 Microbial community analysis

22 The DGGE analysis of the retrieved sample from the UASB bioreactor, as illustrated in Fig

23 6, a total of nine bands were obtained and three different species where identified;

24 Clostridium celerecrescens, Clostridium sp., and Proteobacteria. The most dominant

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1 bacteria present in the biohydrogen system is Clostridium sp. which is a Gram-positive

2 bacteria from the phylum Firmicutes and related to Clostridiaceae family that dominated

3 the biohydrogen system [68] and has been found to be one of the dominant biohydrogen

4 producers present in biohydrogen systems with different substrates such as POME [34],

5 food waste [44], EFB, corn stover, oil palm frond, oil palm trunk [69]. Clostridium sp. is a

6 cellulolytic bacteria known for its ability to hydrolyse a variety of celluloses, lignin and

7 hemicelluloses; polysaccharides in the plant cell wall and used it as energy and carbon

8 sources [70]. Clostridium sp. uses lactose and glucose to produce acetate, butyrate and

9 hydrogen [69]. Also, the high hydrogen yield obtained in this study could be attributed to

10 the activity of this bacterium under strictly anaerobic conditions [44,71]. Bacteria from

11 phylum Proteobacteria was also present in the system, Proteobacteria is one of the

12 dominant bacteria present in the anaerobic bioreactors [72]. As reported earlier by

13 Sivagurunathan et al [73], the bacteria within the phylum Firmicutes and Proteobacteria

14 such as Bacilli, Clostridium, Klebsiella, and Enterobacter are considerably important

15 populations that are involved in stable and efficient biohydrogen production.

16 One of the major concerns in using mixed cultures is the H2-consuming pathway by

17 homoacetogenesis and methanogenesis. The inoculum pre-treatment, thermal pre-

18 treatment in this study, was reported to be one of the ways to create extreme conditions

19 where spore-forming H2-producers will survive, whereas non-spore-forming

20 methanogens are killed or inhibited. However, Kraemer and Bagley [74] reported that H2

21 consumption was of minor concern in continuous fermentative biohydrogen systems,

22 because it could only account for 2–11% of the HY decrease, and this could be further

23 avoided by sparging N2 to the continuous bioreactor. A study conducted by Luo et al [75]

24 revealed that methanogenesis and homoacetogenesis depend highly on the fermentation

25 conditions. Methanogenic activity is inhibited at pH lower than 6, both under mesophilic

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1 and thermophilic conditions, while homoacetogenesis require the combination of

2 thermophilic condition (55 ͦC) and lower initial pH 5.5 to be inhibited.

4 4. Conclusion

5 In conclusion, the operational performance of biohydrogen production from POME was

6 demonstrated in this study in a UASB bioreactor under different HRTs for 70 days. The

7 maximum HRT of 6 h provided the optimal H2 production rate due to the Clostridium

8 population responsible for the H2 production, which constituted of 52% of the biogas

9 produced. The UASB system has been demonstrated to have the ability to initiate

10 microbial self-granulation of sludge shortly after the start-up period, within a HRT of 48 h.

11 Factors such as dilute nitric acid pre-treatment, thermophilic conditions and HPGs

12 formation had positively affected the HPR and HY of 11.75 LH2/LPOME.d-1 and 2.45 mol-

13 H2/mol-sugar, respectively, achieved in this study. Additionally, based on the SRT values

14 obtained in this study, no biomass washout occurred during all HRT cycles, and the decline

15 in the HPR after running the UASB in a short HRT was recovered successfully, due to the

16 presence of the HPGs. These findings indicate that the UASB bioreactor is suitable for

17 POME treatment and could be superior in terms of biohydrogen production.

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Fig. 1 Schematic diagram of 20 L UASB bioreactor experimental apparatus

Fig. 2 H2 production profile in 20L UASB operated at HRT 48-3 hour

Fig. 3 Effluent of UASB bioreactor operated at HRT 48-3 hour (a) soluble metabolic products, and (b) TSS

and VSS

Fig. 4 HPGs produced at different HRTs (48, 24, 12, 6 and 3 hours)

Fig. 5 (a) FESEM image of (i) HPG surface (magnification 5000x) &(ii) HPG surface (magnification 10000x)

and (iii) HPG cross sectioned (magnification 1000x), (b) SEM-EDX of (i) HPG surface and (ii) HPG cross

sectioned, and (c) FTIR of freeze dried HPG

Fig. 6 Microbial community analysis results for the mixed culture from biohydrogen systems. (a) DGGE
profile of 16S rRNA gene fragments. Numbers indicate DNA bands excised and sequenced for identification
of microbes. (b) 16S rDNA phylogenetic tree of the mixed culture. This phyogenetic tree is based on the
UPGMA method and was contructed using a Maximum Composite Likelihood algorithm. The scale bar
represents 0.1 nucleotide divergence.
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Reactor volume= 20 L
Working volume = 15 L
pH controller= 5.2 (low
value)- 6.0 (high value)
Temperature= 55-60 ˚C

Overflow
unit

Fig. 1
 H2 produced (L) Sugar consumed (%) HPR (L-H2/L-POME .d-1)
80 14
HRT 48 h HRT 24 h HRT 12 h HRT 6 h HRT HRT 6 h HRT 12 h
3h
70 12

HPR (LH2/LPOME. d-1)

Sugars consumed (%)
H2 produced (L)

60
10
50
8
40
6
30 start-up
4
20

10 2

0 0
0 10 20 30 40 50 60 70
Days

Days

Fig. 2
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A 1000 60
900

HRT (hr)
50
mM

800
700
40
600
500 30
400
20
300
200
10
100
Days
0 0
0 10 20 30 40 50 60 70
Days

Acetic acid Butyric acid Propanoic acid EtOH HRT

50 50
b45 45
TSS & VSS (g/L)

40 40

HRT (h)
35 35
30 30
25 25
20 20
15 15
10 10
5 5
0 0
0 10 20 30 40 50 60 70

Days

TSS (g/L) VSS (g/L) HRT (hr)

Fig. 3
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Fig. 4
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A
i ii iii

B
i ii

Fig. 5
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(a) (b)

Fig. 6
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Highlights:

 Raw POME was pre-treated using dilute nitric acid.

 H2-producing granules from POME were produced in UASB.
 Optimum biohydrogen yield achieved was 2.45 molH2/molsugar.
 Bacterial community in H2 producing sludge was dominated by Clostridium spp.
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Table 1: Physiochemical characteristics and chemical composition of raw POME and pre-treated
POME hydrolysate

Raw POME HNO3 Pre-treated POME

Color Brown Yellowish-brown
pH 4.1-4.7 3.42
tCOD (g/L) 44.07 ± 12.25 41.25 ± 3.21
sCOD (g/L) 22.33 ± 1.15 24.93 ± 7.09
Total carbohydrates (TC) (g/L) 18.37 ± 0.21 22.15 ± 0.32
Total reducing sugars (DNS) (g/L) 15.17 ± 0.20 19.25 ± 1.41
TSS (g/L) 23.85 ± 0.17 18.45 ± 0.14
VSS (g/L) 17.90 ± 0.16 15.44 ± 0.04
Glucose (g/L) 5.92 ± 0.12 6.05 ± 0.15
Xylose (g/L) 5.19 ± 0.10 5.83 ± 0.22
Arabinose (g/L) 0.70 ± 0.05 0.77 ± 0.08
Furfural (mg/L) 1.44 ± 0.05 1.5 ± 0.13
5-HMF (mg/L) 0.25 ± 0.02 0.7 ± 0.15
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Table 2: UASB bioreactor performance at different HRTs of H2 production

HRT (d) SRT* (d) HPR ** HY*** (mol H2/mol TC consumed)

LH2/LPOME . d-1 mmol H2/LPOME .h-1
2.00 7.18 0.27 ± 0.07 0.50 ± 0.13 0.38 ± 0.11
1.00 3.32 1.10 ± 0.35 2.04 ± 0.66 1.12 ± 0.30
0.50 1.82 3.88 ± 0.26 7.22 ± 0.49 1.69 ± 0.16
0.25 0.77 11.75 ± 0.15 21.86 ± 0.28 2.45 ± 0.24
0.13 0.4 5.75 ± 0.28 10.70 ± 0.53 1.46 ± 0.51
 Table 3: Comparison of HPGs’ sizes produced in different substrates

Samples/substrate H2 System Temperature HRT (h) Diameter of HPGs Dominant Microbe H2 Production (mol-H2/mol- Ref.
(℃) sugar)
Glucose ASBR 28 6.7 h 1.7 ± 0.2 mm Clostridium sp. 1.36 (Liang et al., 2010)

Galactose UASB 37 2h N.Aa Class Bacilli, 2.25 (Sivagurunathan et

Clostridia and al., 2016)
Micrococcales
Coffee drink manufacturing UASB 35 6h 0.5mm Clostridium sp. 1.29 (Jung et al., 2010)
wastewater
Glucose CSTR 37 0.5 h ~1.52 mm, After 114h Clostridium 1.81 (Zhang et al., 2007)
pasteurianum
Sucrose UASB 35 8h 1.4 mm - 2.9 (Chang and Lin,
2007)
Sucrose wastewater CSTR 35 12 h 0.5 mm after 10 days - 4.3 L/L.d (Salem et al., 2017)
POME UASB 38 12 h >1mm, within 22 days - 0.514 L H2/g VSS d (Mohammadi et al.,
2014)
Pre-treated POME UASB 55 48 h < 0.4 cm, after 10 days Clostridium sp. 0.38 This study
24 h <0.5 cm 1.12
12 h <2 cm 1.69
6h <1.1 cm 2.45
3h < 0.6 cm 1.46
Table 4: Comparison of biohydrogen production from POME with different bioreactors

Bioreactor Operation Inoculum HRT (h) HPR HY* Ref.

Type temperature (˚C) LH2/LPOME.d-1 (mol-H2/mol-sugar)
ASBR 60 Mixed culture 48 9.10 2.10 (Nitipan et al., 2014)
ASBR 55 Thermoanaerobacterium 48 9.70 1.72 (Mamimin et al., 2016)
thermosaccharolyticum PSU-2
ASBR 37 Mixed culture 72 6.70 2.92 (Badiei et al., 2011)
ASBR- 2 stages 55 & 37 Mixed culture 12 5.56 2.52 (Maaroff et al., 2018)
CSTR 37 Clostridium butyricum LS2 Batch - 0.78 ml H2/ml POME (Mishra et al., 2016)
CSTR 37 Bacillus anthracis Batch - 2.02 (Mishra et al., 2017)
PUNAJAN 1 – isolated from POME
CSTR 55 Mixed culture 48 1.1 0.52 (O-thong et al., 2016)
UASB 37 Clostridium LS2 immobilized on 16 8.76 3.26 (Singh et al., 2013)
Polyethylene glycol (PEG) gel
UASB 55 Mixed culture 48 1.92 1.85 (Krishnan et al., 2016a)
UASB 55 Mixed culture 12 2.5 0.29 (Krishnan et al., 2017)
UASB 55 Mixed culture 9 2.1 0.42 (Krishnan et al., 2016b)
UASB-FF 38 Mixed culture 36 - 2.66 (Mohammadi et al., 2017)
UASB 55 Mixed culture 6 11.75 2.45 This study
ASBR= Anaerobic sequencing batch reactor; CSTR= continuous stirred-tank reactor; UASB-FF= up-flow anaerobic sludge blanket-fixed film

*The total sugar consumed as glucose equivalent