Improvement of Biohydrogen Production through Combined Reuses of

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Improvement of biohydrogen production through combined reuses of palm oil mill effluent together with pulp and paper mill effluent in photofermentation

Pretty Mori Budiman1, Ta Yeong Wu1*, Ramakrishnan Nagasundara Ramanan1, Jamaliah Md. Jahim2

1

Chemical Engineering Discipline, School of Engineering, Monash University, Jalan Lagoon

Selatan, 47500 Bandar Sunway, Selangor Darul Ehsan, Malaysia 2

Department of Chemical and Process Engineering, Faculty of Engineering and Built

Environment, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul Ehsan, Malaysia

*E-mail: [email protected]; [email protected] Tel: +60 3 55146258 Fax: +60 3 55146207

Keywords: Biohydrogen; Palm Oil Mill Effluent; Pulp and Paper Mill Effluent; Photofermentation; Rhodobacter sphaeroides; Waste Reuse

Notes: The authors declare no competing financial interest

ACS Paragon Plus Environment

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ABSTRACT

Rhodobacter sphaeroides NCIMB8253 and palm oil mill effluent (POME) were applied as the purple

non-sulfur

bacteria

and

substrate,

respectively

to

produce

biohydrogen

in

photofermentation process. Due to the dark color of POME, pulp and paper mill effluent (PPME) was used as a diluting agent to reduce the turbidity of substrate and thus, improving light penetration. Anaerobic batch experiments were performed by varying the concentration of POME from 12.5 to 100% (v/v) with 10% (v/v) inoculum in a total of 100 ml substrate. The highest biohydrogen yield of 4.670 ml H2/ml medium was obtained using NS4 treatment containing 25 and 75 % (v/v) of POME and PPME, respectively. A maximum production rate of 0.496 ml H2/ml medium/h and light efficiency of 2.40% were also achieved in NS4. Furthermore, a simultaneous 28.8% CODtotal removal was obtained after 3 days of photofermentation. Additional increase of POME concentration (>25%, v/v) did not support higher production of biohydrogen due to the increase of turbidity (>16,450 NTU) which resulted in hindrance of light penetration. This study showed that the potential of reusing and combining two different effluents together, in which case one had lower turbidity than the other wastewater, for improving light penetration and thus, photo-biohydrogen production.


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1.

INTRODUCTION

Depletion of fossil fuels and increasing environmental problems associated with fossil fuels consumption have amplified the needs to discover an alternative energy resource. During the past decade, hydrogen has been considered by many countries as a potential substitute to conventional fossil fuels.1 Meanwhile, the application of biohydrogen as an energy source has gained more attention as one of the most suitable answers to this energy shortage due to its renewability and environmental-friendly features. Among the existing production methods, photofermentation by purple nonsulfur (PNS) bacteria has been suggested as one of the most promising ways to produce biohydrogen. Ability to reuse waste material, high theoretical biohydrogen yield, lack of oxygen-evolving activity and ability to utilize wide spectrum of light have been regarded as the main advantages of photofermentative biohydrogen production.2 Photofermentative biohydrogen production is strongly coupled with the photosynthesis electron transport system, through which the PNS bacteria, such as Rhodobacter sphaeroides, obtain energy.3 Under nitrogen-deficient and photo-anaerobic environment, PNS bacteria are able to convert organic acids into biohydrogen and carbon dioxide via nitrogenase activity.1 PNS bacteria, such as Rhodobacter sp.,4-6 Rhodopsuedomonas palustris 7-8 and mixed culture bacteria9 have been reported to have high biohydrogen-producing potential. Furthermore, a utilization of wastewater as a production substrate is expected to produce biohydrogen in a sustainable way and at the same time, reducing the contamination level of the effluent before it is discharged into the watercourses. Under photoaerobic condition, Kornochalert et al.10 reported that 91% COD reduction was achieved in latex rubber sheet wastewater treatment using fermented pineapple extract inoculated indigenous PNS bacteria. Wastewaters, such as olive mill, tofu, dairy, sugar


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refinery, and brewery, have been reported to be potential photofermentation substrates due to its high organic matter content.2,11 The development of palm oil industries in Malaysia has resulted in the upsurge of palm oil mill effluent (POME) productions annually.12 In Malaysia, crude palm oil extraction generates about 50 million tonnes of POME annually and the discharge of POME is projected to escalate in the future.13 The discharge of untreated POME has been seen as a severe pollution due to its high BOD loading and low pH, together with the high colloidal nature of the suspended solids.14 Recently several methods were introduced to treat POME such as using natural coagulation,15-16 vermitechnology,17 adsorption18 and the others but high level of organic acids, carbohydrate, lipids, minerals, protein, and nitrogen available in the POME was also reported to support the growth of several microorganisms which helped to transform the POME into valueadded products.12 Besides, Budiman et al.19 reported that a concentration of 25% (v/v) POME was the best to be reused as a growth medium for R. sphaeroides due to the presence of adequate nutrients and light penetration. However, Budiman et al.19 did not further investigate if the diluted POME could be reused as a substrate in biohydrogen production through photofermentation process. During photofermentation, natural or artificial illumination is supplied to support the bacterial growth and initiate biohydrogen production. Androga et al.20 suggested that the incident light energy supplied during photofermentation was required to meet the high energy demand of photosynthetic apparatus to trigger the photo-biohydrogen production mechanisms. Since the efficiency of light penetration plays an important role in photofermentation, the use of POME without any dilution or pre-treatment may lead to the reduction of light penetration, due to the dark color and high turbidity of this wastewater. Previous study done by Eroğlu et al.21 reported that the highest biohydrogen yield of 14 L H2/L medium was obtained when 2% (v/v) olive mill


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wastewater was used as a photofermentation substrate. The visibility of olive mill wastewater was enhanced by diluting the wastewater with distilled water. According to Wu et al., 22 pretreatment of certain wastewaters, typically by dilution, was necessary prior to photo-biohydrogen production due to the dark color of the wastewater. In this study, the dark color of POME could be reduced by reusing pulp and paper mill effluent (PPME) as an alternative diluting agent instead of using distilled water. PPME was selected as a diluting agent in this study because it is a lighter-in-color wastewater than the POME. Furthermore, recent study showed that R. sphaeroides NCIMB8253 were able to grow adequately in POME which was diluted with PPME.19 Thus, an addition of PPME was predicted to reduce the turbidity of POME so that the combined wastewater between POME and PPME could be reused as one single substrate in photofermentation to produce biohydrogen. The main aim of this study was to investigate the effect of different POME concentration, which was diluted by PPME, on bacterial growth and biohydrogen production through photofermentation using R. sphaeroides. Besides, COD (CODtotal and CODsoluble) removals from the combined wastewater would also be evaluated. Kinetic analysis on photo-biohydrogen production would be conducted to describe the cumulative biohydrogen production by fitting the results into modified Gompertz model.

2.

MATERIALS AND METHODS

2.1. Wastewater collection POME and PPME were obtained from Seri Ulu Langat Palm Oil Mill Sdn. Bhd. and Muda Paper Mills Sdn. Bhd., respectively. Both raw effluents were stored and refrigerated at 4°C to reduce


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biological degradation. The physical and chemical characteristics of both effluents are summarized in Table 1.

2.2. PNS bacteria R. sphaeroides NCIMB8253 used in this study were acquired from Faculty of Engineering and Built Environment, Universiti Kebangsaan Malaysia. The bacteria were grown on agar slants and incubated for 24 h at 30oC under 4 klux illumination (WalkLAB Digital Lux meter (Trans Instruments (S) Pte. Ltd., Singapore)). Then, the bacteria were stored and preserved at 4oC.

2.3. Inoculum preparation Before photofermentation experiment, R. sphaeroides NCIMB8253 were cultivated on modified liquid medium consisted of (in 1 liter): K2HPO4,1 g; KH2PO4, 0.5 g; NaCl, 0.4g; MgSO4•7H2O, 0.5g; sodium glutamate, 1.8 g; DL-malic acid, 1.0 g; CaCl2•2H2O, 0.05 g; yeast extract, 10 g; ferric citrate (0.1 w/v%), 5 ml; vitamins solution, 1 ml; trace elements solution, 1 ml; and HCl (37%), 0.68 ml. Trace elements solution (in 100 ml) contained: H3BO3, 0.06 g; CoCl2•2H2O, 0.2 g; ZnCl2, 0.07 g; Na2MoO4•2H2O, 0.04 g; MnCl2•4H2O, 0.1 g; NiCl2•6H2O, 0.02 g; and CuCl2•2H2O, 0.02 g. Vitamin solutions (in 1 liter) contained: thiamin, 500 µg; niacin, 500 µg; and biotin, 15 µg. Cultivation was done under 4 klux illumination and anaerobic condition for 24 h. Further inoculation of R. sphaeroides NCIMB8253 was done in 48 h by following the method described by Budiman et al.19 before the bacteria were transferred into photo-biohydrogen production medium.


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2.4. Media preparation Untreated POME was subjected to various dilutions with PPME at proper concentrations and pH adjustment to 7 (Mettler Toledo FE20, Australia) by an addition of 5 mol/L NaOH. In all experimental sets, production medium was purged with pure argon gas for 10 min to create an anaerobic environment. Finally, liquid inoculum (~82.9 × 108 CFU/ml) of pre-activated bacteria from the growth medium was transferred into Schott bottle containing production medium (10%, v/v inoculum) with various POME concentrations. The characteristics of the production medium were summarized in Table 2.

2.5. Photofermentative biohydrogen production Photofermentative hydrogen production was conducted in a 100 ml Schott bottle. External illumination was provided by fluorescent lamps and kept at an average of 4 klux on the outer surface of Schott bottle. The intensity of the light was measured as an average of the light intensity on the surface of the Schott bottle from the front and back side using a WalkLAB Digital lux meter (Trans Instruments (S) Pte. Ltd., Singapore). The temperature of the broth was maintained at 30oC using water bath and hotplate heater. An agitation of 250 rpm was applied to provide better heat and mass transfer in the broth. Frequent temperature measurement and adjustment were done throughout the experiment due to the possible heating from the light source. The produced biogas was collected using water displacement method. All experiments were done in triplicate.

2.5. Analytical methods Content of produced biohydrogen and carbon dioxide produced in the collected biogas was measured with gas chromatography equipment (Agilent 7890A, U.S.) which was equipped with


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a thermal conductivity detector (TCD). The loss of organic matters through photofermentation was calculated by measuring total (CODtotal) and soluble (CODsoluble) COD values. COD values were analyzed using Reactor Digestion Method (HACH Method 8000) and measured spectrophotometrically using HACH DR2800. CODtotal was measured from the sample without undergoing filtration prior to analysis, while CODsoluble was referred to the supernatant of the sample after centrifugation at 13,500 rpm for 15 min. Bacterial cell concentration was determined by colony-forming-unit (CFU) method. Collected samples were diluted two times by 2,000 dilution factor each. Diluted samples were then spread onto petri dish agar near fire source and left under illumination (4,000 lux) for 24 h inside the growth chamber (PROTECH GC-1050, U.S.). Petri dish agar consisted of (in 1 liter): yeast, 2.5 g; peptone, 2.5 g; and bacteriological agar, 15 g. After 24 h, the number of bacterial colonies was calculated and used as the indication of bacterial growth. Total carbohydrate concentrations were measured spectrophotometrically (Thermo Spectronic GENESYS 10 VIS, U.S.) at 490 nm through phenol-sulfuric acid method.23 Experimental data were generated as the averages of the values obtained from three replications of each experimental set. ANOVA test would be applied on the cumulative biohydrogen production results to identify the significance of the experimental data sets.

3.

RESULTS AND DISCUSSION

3.1. Effect of POME concentration on biohydrogen production In this study, six different concentrations of POME (12.5, 20, 25, 30, 40 and 100 %, v/v) were applied as photo-biohydrogen production substrates with PPME as a diluting agent. The efficiencies of biohydrogen production for all media were measured by calculating three


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parameters, namely biohydrogen yield (Y), maximum biohydrogen production rate (Rmax), and light conversion efficiency (ɳ). Y is defined as the amount of total volume of biohydrogen obtained over the amount of production medium applied, with the unit of ml H2/ml medium. Rmax is calculated by dividing the amount of biohydrogen produced over the amount of production medium and the time period of gas collection (ml H2/ml medium·h). Meanwhile, light conversion efficiency, ɳ (%) is calculated using Equation 1 as shown:

η=

H 2 energy content × H 2 output 33.6 ρ H 2VH 2 × 100 = × 100 light energy input IAt

Equation 1

where ߩுଶ is the density of hydrogen (g/L), ܸுଶ is the volume of hydrogen produced (L), I is the light intensity (W/m2, 1 lux = 0.0161028 W/m2), t is the duration of hydrogen production (h), and A is the irradiated area. From this study, it was observed that the amount of POME concentration in photofermentative biohydrogen production medium affected the amount of biohydrogen yield mainly due to the turbidity and the availability of carbon source, which was measured as a total carbohydrate concentration in the medium. An increase of POME concentration in the production medium resulted in increases of both turbidity and total carbohydrates in the combined wastewater (Table 2). Figure 1 displays that at lower POME concentration range (NS4) contributed to the higher carbohydrate concentration (Table 2), the measured biohydrogen yields decreased exponentially as turbidity increased higher that its optimum (>16,450 NTU), which could be observed in Figure 1. These results showed that photofermentative biohydrogen production was highly influenced by the turbidity of the


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production medium. Hence, an introduction of PPME as a diluting agent in this study was necessary to promote photo-biohydrogen production by increasing the visibility of substrate and improving the light penetration into the system. Budiman et al.19 also reported that a combination of POME and PPME as single substrate could be applied as a suitable growth medium for R. sphaeroides because of the enhanced light penetration and faster adaptation in the diluted media.19 Furthermore, the introduction of PPME (a low nitrogen source) helped increase the C/N ratio of photofermentation substrate (Table 1). It was reported that high C/N ratio promoted the production of biohydrogen by allowing PNS bacteria to dispose their excess energy and reducing power through production of hydrogen.22

3.1.1. Effect of POME at lower concentrations (NS1-NS3) As POME concentration was increased from 0 to 20% (v/v), an improvement of biohydrogen production was obtained (Figure 2). Among these three treatments, 100% PPME treatment (NS1) yielded the lowest amount of cumulative biohydrogen. An inhibition of biohydrogen production might occur due to the lack of carbon source (Table 2) which led to the lowest bacterial growth as observed in Figure 3. Although biohydrogen production rate in NS2 and NS3 were faster during the first 12 h of photofermentation, cumulative biohydrogen amount at the end of the experiment for these two treatments were lower than NS4. Faster biohydrogen production might occur due to lower turbidity of these media (Table 2) and thus, promoting early biohydrogen release. However, as time proceeded, biohydrogen productions in NS2 and NS3 were limited by the availability of nutrients which resulted in the suppression of bacterial growth (Figure 3). This suppression of bacterial growth in NS2 and NS3 might have led to the inhibition of biohydrogen production at the later stage of photofermentation. In general, production of biohydrogen is positively influenced by the growth of PNS bacteria.2 Sasikala et al.24 stated that at higher cell


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concentration (1.6-1.8 mg dry weight/ml), production of biohydrogen was increased up to 100% as compared to using 0.2 mg dry weight/ml during photofermentation. Furthermore, biohydrogen production by R. sphaeroides OU001 was also observed to be highly dependent on the cell growth rate, especially at the later part of photofermentation process.25

3.1.2. Effect of POME at recommended concentration (NS4) Cumulative photo-biohydrogen production data presented in Figure 2 shows that increases of POME concentration until 25% (v/v) could lead to an increase of cumulative photo-biohydrogen amount up to 3.5 times. NS4 (25% POME, v/v) achieved the highest cumulative photobiohydrogen production of 467 ml H2 in 100 ml of medium. From Table 2, a similar trend of results as the cumulative biohydrogen production was observed for Y and ɳ. An increase of POME volumetric concentration up to 25% enhanced the biohydrogen yield and light conversion efficiency. Further increase beyond 25% did not result in improvement of biohydrogen production, but inhibitions of both biohydrogen production (Figure 2) and biomass (Figure 3) were observed. In comparison with raw POME (NS7), a significant improvement of photobiohydrogen production yield (almost 16 times higher) was observed when NS4 was applied as a production medium. Moreover, NS4 had 16 times higher ɳ value as compared to NS7 (Table 2) because dilution decreased the color intensity and thus, enhancing the light penetration needed during photofermentation process. Similar findings were reported by various authors. Eroğlu et al.26 studied the effect of various pre-treatment methods to enhance light absorption during photofermentation process of olive mill wastewater. 100% improvement of biohydrogen production was obtained due to the application of dilution and clay pre-treatment with the highest ɳ of 0.23%. Their results showed that a reduction of color in the production medium facilitated the enhancement of light absorption and biohydrogen production.


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Although NS4 treatment exhibited the highest yield, a comparison of Rmax among treatments (Table 2) showed that the highest production rate was obtained in NS3 (1.533 ml H2/ml medium·h), which occurred during the first hour of biohydrogen production. Higher production rate was also observed in NS2 (1.077 ml H2/ml medium·h). These findings occurred as a result of faster biohydrogen production during the early stage of photofermentation in both NS2 and NS3 treatments. It could be seen in Figure 2 that during the first 12 h of photofermentation, NS2 and NS3 produced higher amount of biohydrogen as compared to NS4. However, both treatments experienced a drop in biohydrogen production rate after 12 h of photofermentation, whereas NS4 treatment displayed a steady and higher production rate (Figure 2). Biohydrogen production rate in NS4 started to slow down after 48 h. Koku et al.27 indicated that a reduction of biohydrogen production with time might occur due to the decline of electron carrier activity of PNS bacteria. At the end of photofermentation, NS4 yielded the highest amount of cumulative biohydrogen production. Additionally, ANOVA results also showed that NS4 yielded the highest cumulative biohydrogen production with the most significant results as compared to the other treatments (Figure 2).

3.1.3. Effect of POME at higher concentrations (NS5-NS7) Further increase of POME concentration (> 25%, v/v) resulted in the reduction of cumulative photo-biohydrogen production significantly. At higher POME concentration, an increase in turbidity of production medium (Table 2) started to hinder the efficiency of light penetration. As shown in Table 2, ɳ started to decrease significantly at higher POME concentrations, which justified the inadequate illumination of photofermentation system in NS5, NS6 and NS7. Furthermore, lower biohydrogen accumulation in these treatments could also be related to the suppression of bacterial growth and the presence of inhibitory compounds. An inhibition of


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bacterial growth in NS5-NS7 was observed in Figure 3. In general, photofermentative biohydrogen production rate is proportional to the bacterial growth rate.2 Besides, photoheterotrophy growth has been highlighted by Koku et al.28 as a preferred growth environment of PNS bacteria to produce biohydrogen. Sevinç et al.29 observed that the intensity of light supply affected the rate of bacterial growth during photofermentation by R. capsulatus. At 3 klux illumination, bacterial growth rate was reported to be 141% higher than 1.5 klux photofermentation at 20oC. This result showed that during photofermentation, a reduction of light supply suppressed the bacterial growth, which was not favorable for biohydrogen production. Similar restrictions were also reported by Eroğlu et al.21 and Seifert et al. 5. Seifert et al.5 reported that an increase of biomass concentration was obtained when higher intensity and efficiency of light penetration was supplied. Therefore, Seifert et al.5 limited the amount of dairy effluent at 40% (v/v) to increase light distribution and substrates accessibility by PNS bacteria. Also, Eroğlu et al.21 found that no biohydrogen could be produced in higher concentration of olive mill wastewater (> 4%, v/v) due to excessive presence of inhibitory compounds and low visibility. In the case of continuous photofermentation, Tawfik et al.9 also reported that biohydrogen production potential was limited by the amount of organic loading rate (OLR). The use of effluent as a substrate at higher OLR (> 6.4 g COD/L) resulted in lower biohydrogen production due to the inhibition of biohydrogen-producing enzyme activity as a result of reduced light penetration. At the highest POME concentration (100%, v/v) or NS7, photo-biohydrogen production was almost completely inhibited with only 27.7 ml of biohydrogen collected after 72 h of photofermentation. The reasons of this inhibition was due to suppression of bacterial growth (Figure 3) as a result of the presence of inhibitory compounds and the dark color of NS7,


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indicated by the highest turbidity value (Table 2). Dark color of the medium reduced the light penetration efficiency, which led to lower biohydrogen production.21 Furthermore, Wu et al.12 mentioned that the use of concentrated POME as a fermentation substrate might inhibit the growth of microorganisms due to the presence of water-soluble antioxidants, phenolic acids, and flavonoids in high amounts.

3.2. Kinetics and efficiency of biohydrogen production in NS4 treatment A kinetic analysis on cumulative photo-biohydrogen production was conducted by fitting the experimental results of NS4 into modified Gompertz model,30 as shown in Equation 2:  R e   H = H m exp − exp  m (λ − t ) + 1    Hm  

Equation 2

where H, Hm, Rm, λ and e represent the cumulative volume of biohydrogen production (mL), biohydrogen production potential (mL), maximum biohydrogen production rate (mL/h), lag phase time (h) and 2.718, respectively. Figure 4 and Table 3 show the results of kinetic analysis of photo-biohydrogen production in NS4. The shape of fitted curve displayed a good correlation using modified Gompertz model with R2 = 0.990. Sevinç et al.29 also reported a well-fitted cumulative photobiohydrogen production by R. capsulatus (R2 >0.994) at different temperature and light intensity using modified Gompertz model. However, small differences in total amount of biohydrogen and lag phase between experimental and predicted values were observed (Table 3). Experimental results showed that the total amount of biohydrogen produced was 467 ml of H2 with a lag phase of 0.5 h, whereas the total amount of biohydrogen produced was predicted to be 445.8 ml of H2 with a lag phase of 0.44 h by using modified Gompertz model. Error (%) between these values were calculated based on Equation 3.


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Error =

exp erimental − prediction × 100 prediction

Equation 3

Using Equation 3, 4.76 and 13.6% errors were calculated for the total amount of biohydrogen and lag phase, respectively. Modified Gompertz model also showed the predicted maximum biohydrogen production rate (Rm) in NS4 was 35.76 ml H2/h as compared to 49.60 ml H2/h obtained from the experimental results. The efficiency of photo-biohydrogen production in NS4 was then compared to other previous studies.4,5,7,31 In comparison with other results using wastewater as a photofermentation substrate, NS4 contained 25% (v/v) POME and 75% (v/v) PPME generated a comparable biohydrogen production performance, with high photo-biohydrogen production yield (4.670 ml H2/ml medium or 713 ml H2/g CODtotal-consumed), high production rate (0.496 ml H2/ml medium·h) and short lag phase (0.5 h). Photo-biohydrogen yield obtained was higher than 1.9 ml H2/ml medium biohydrogen yield reported by Zhu et al.31, where tofu wastewater was diluted with water in the ratio of 4 to 6. However, in comparison with 4% (v/v) olive mill wastewater4 and 40 % (v/v) dairy wastewater5 as photo-biohydrogen production substrates, the biohydrogen production yield obtained in NS4 was relatively lower. In the case of photofermentation using 40 % (v/v) dairy wastewater as a substrate, higher amount of inoculum (30%, v/v) was added into the production medium.5 Besides, the dairy wastewater underwent filtration process and was combined with a defined medium prior to photofermentation.5 Meanwhile, olive mill wastewater was pre-filtered before it was diluted with distilled water.4 On the other hand, NS4 used in this study did not undergo any pre-treatment and was only diluted with lighter color wastewater, namely PPME.


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3.3. Organic matter removals during biohydrogen production During photofermentation, a simultaneous decrease of COD in all treatments occurred due to the consumption of organic matters for promoting bacterial growth and biohydrogen production. Figure 5 displays the COD removal and the initial and final values of both COD levels in all treatments. Moderate reductions of both CODtotal (Figure 5a) and CODsoluble (Figure 5b) were obtained in all treatments within three days of photofermentation process. NS4 achieved the highest removal of CODtotal up to 28.8%, while CODsoluble removal was 10.5%. The present study showed that NS4 yielded the highest photo-biohydrogen production (Table 2). Hence, further COD removal analysis was conducted to measure the environmental benefit of reusing NS4 in photo-biohydrogen production. Figure 6 summarizes the time course changes in CODtotal and CODsoluble throughout 72 h of photofermentation process. As R. sphaeroides NCIMB8253 grew and photo-biohydrogen was produced, organic matter in NS4 was also consumed, indicated by the reductions of both CODtotal and CODsoluble (Figure 6). In the beginning of photofermentation, significant reductions of both COD values were observed due to the occurrence of not only photo-biohydrogen production but also the exponential phase of bacterial growth. However, towards the mid of photofermentation (> 24h), both COD contents decreased in a slower rate as stationary phase of bacterial growth and biohydrogen production started to take place. Xie et al.32 estimated that bacterial cell growth was responsible for 37% of substrate consumption during photo-biohydrogen production by PNS bacteria. Meanwhile, only 16% of acetic acid feed was utilized for biohydrogen production. These findings indicated that a significant amount of substrate was consumed for cell growth instead of biohydrogen production. Overall, this study indicated that COD removals from the wastewaters were mediocre because the predominant PNS bacteria belonging to Rhodospirillum and Rhodoferax are more well-known for their key role in carbon and nitrogen metabolism than Rhodobacter.33 In


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comparison with other literature studies using mainly R. sphaeroides, it was found that CODtotal removal from NS4 were comparable to other photofermentation process that utilized other wastewater as a substrate. In this study, 28.8 and 20.5% of CODtotal and CODsoluble removals from NS4 respectively were achieved during 72 h of photofermentation by using R. sphaeroides NCIMB8253. On the other hand, other PNS bacteria, such as R. sphaeroides O.U.001, achieved 30 and 21% of CODtotal removals using olive mill wastewater4 and dairy wastewater,5 respectively. However, longer periods of photofermentation (155 h in 4% (v/v) olive mill wastewater and 120 h in 40 % (v/v) dairy wastewater) were required to achieve these removals. This comparison showed that 25% (v/v) POME diluted with PPME could be reused by PNS bacteria as a photo-biohydrogen production substrate, while removing moderate level of COD contents from the combined wastewaters.

3.4. Growth of PNS bacteria during photofermentative biohydrogen production Figure 3 shows the time course of the CFU number change as an indication of R. sphaeroides NCIMB8253 growth. Typically, standard bacterial growth curve consists of four distinctive phases such as lag, exponential, stationary and death phase.1 However, as opposed to the standard bacterial growth curve, lag phase was not observed for all treatments, except in NS1. In Figure 3, the lag phase of NS1 lasted for 6 h. During the lag phase, bacterial growth was suppressed due to the adaptation of PNS bacteria to the new environment or as a result of nutrient deficiency. Meanwhile, for other treatments, increases of CFU number were observed in the early stage of bacterial growth, indicating a significantly shorter amount of adaptation period. It is important to highlight that NS1 contained raw PPME with low nitrogen content (Table 1) which was not favorable to support the growth of R. sphaeroides.


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Page 18 of 35

Figure 3 also shows that as concentration of POME increased up to 25%, the growth of R. sphaeroides was positively influenced. This increase of growth was due to the increase of food contents inside the medium as POME concentration increased (Table 2). The highest bacterial growth was obtained in NS4 (25% POME, v/v) with a maximum CFU number of 196 × 108 CFU/ml. On the contrary, concentrations of POME higher than 25% (v/v) (NS5-NS7) inhibited the growth of R. sphaeroides (Figure 3). The inhibition trend represented a suppression of bacterial growth in these treatments, possibly due to the higher turbidity of wastewater (Table 2) and the presence of inhibitory compounds. Budiman et al.19 reported that the photo-anaerobic growth of R. sphaeroides could be promoted by enhancing light penetration through an introduction of proper dilution rate into POME medium. Similar trend was reported by Eroğlu et al.21, in which the growths of R. sphaeroides OU001 in concentrated olive mill wastewater media (5%, 10% and 20% olive mill wastewater, v/v) were significantly inhibited by the dark color of the wastewater and inhibitory substances such as phenolic compounds. On the other hand, Hay et al.34 reported an aerobic growth of R. sphaeroides OU001 was only significantly affected by pH, agitation and temperature, but the presence of light did not play a significant role in the bacterial growth. Analysis of pH changes showed that both NS5 and NS6 experienced slight decreases in pH after 12 h of fermentation (Figure 7). Meanwhile, pH of NS7 decreased considerably from 024 h and remained almost constant afterwards (Figure 7). Budiman et al.19 suggested that the evolution of bacterial growth could be observed with an increase of pH during the growth period. Meanwhile, a decrease of pH in raw POME (NS7) could be interpreted as an indication of dark fermentation. During dark fermentation, an accumulation of volatile fatty acids, such as acetic acid and butyric acid could result in the reduction of pH medium.2 The present results were concurrent with the results obtained by Budiman et al.19, in which a decrease in pH (Figure 7)


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was observed for the treatment with the highest turbidity (NS7), possibly due to a substantial organic acid production (not reported in the present study). On the contrary, no decrease of pH (Figure 7) was observed for the treatments with lower turbidity (Table 2) whereby the pH values were maintained almost at neutral level mainly due to the consumption of organic acid.19

4.

CONCLUSIONS

In conclusion, the reuse of PPME as a diluting agent helped reduce the dark color of POME so that photo-biohydrogen production could be improved. From this study, NS4 containing 25% (v/v) POME and 75% (v/v) PPME achieved the highest biohydrogen yield of 4.670 ml H2/ml medium (713 ml H2/g CODtotal-consumed) with a maximum production rate of 0.496 ml H2/ml medium/h and light efficiency of 2.40%. A simultaneous 28.8% CODtotal removal was also achieved at the end of photofermentation. On the contrary, POME concentration which was higher than 25% resulted in higher turbidity, thus causing the hindrance of light penetration and inhibition of biohydrogen production.

ACKNOWLEDGEMENTS The authors would like to thank Monash University Malaysia for providing P.M. Budiman with a PhD scholarship.


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REFERENCES (1) Xie, G.J.; Liu, B.F.; Xing, D.F.; Ding, J.; Nan, J.; Ren, H.Y.; Guo, W.Q.; Ren, N.Q. The kinetic characterization of photofermentative bacterium Rhodopseudomonas faecalis RLD-35 and its application for enhancing continuous hydrogen production. Int. J. Hydrog. Energy 2012, 37, 13718-13724. (2) Hay, J.X.W.; Wu, T.Y.; Juan, J.C.; Jahim, J.M. Biohydrogen production through photo fermentation or dark fermentation using waste as a substrate: Overview, economics, and future prospects of hydrogen usage. Biofuels Bioprod. Biorefining 2013, 7, 334-352. (3) Mudhoo, A.; Kumar, S. Effects of heavy metals as stress factors on anaerobic digestion processes and biogas production from biomass. Int. J. Environ. Sci. Technol. 2013, 10 (6), 1383-1398. (4) Eroğlu, E.; Eroğlu, I.; Gündüz, U.; Yücel, M. Effect of clay pretreatment on photofermentation hydrogen production from olive mill wastewater. Bioresour. Technol.

2008, 99, 6799-6808. (5) Seifert, K.; Waligorska, M.; Laniecki, M. Hydrogen generation in photobiological process from dairy wastewater. Int. J. Hydrog. Energy 2010, 35, 9624-9629. (6) Assawamongkholsiri, T.; Reungsang, A. Photo-fermentational hydrogen production of Rhodobacter sp. KKU-PS1 isolated from an UASB reactor. Electron. J. Biotechnol 2015, 18, 221-230. (7) Jamil, Z.; Mohamad Annuar, M.S.; Ibrahim, S.; Vikineswary, S. Optimization of phototrophic hydrogen production by Rhodopseudomonas palustris PBUM001 via statistical experimental design. Int. J. Hydrog. Energy 2009, 34, 7502-7512.


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(8) Pott, R.W.M.; Howe, C.J.; Dennis, J.S. Photofermentation of crude glycerol from biodiesel using Rhodopseudomonas palustris: Comparison with organic acids and the identification of inhibitory compounds. Bioresour. Technol. 2013, 130, 725-730. (9) Tawfik, A.; El-Bery, H.; Kumari, S.; Bux, F. Use of mixed culture bacteria for photofermentative hydrogen of dark fermentation effluent. Bioresour. Technol. 2014, 168, 119-126. (10) Kornochalert, N.; Kantachote, D.; Chaiprapat, S.; Techkarnjanaruk, S. Bioaugmentation of later rubber sheet wastewater treatment with stimulated indigenous purple nonsulfur bacteria by fermented pineapple extract. Electron. J. Biotechnol. 2014, 17, 174-182. (11) Wu, T.Y.; Mohammad, A.W.; Lim, S.L.; Lim, P.N.; Hay, J.X.W. Recent advances in the reuse of wastewaters for promoting sustainable development. In Wastewater Reuse and Management; Sharma, S.K., Sanghi, R., Eds.; Springer: Netherlands, 2013; pp 47-103. DOI: 10.1007/978-94-007-4942-9_3. (12) Wu, T.Y.; Mohammad, A.W.; Jahim, J.M.; Anuar, N. Holistic approach to managing palm oil mill effluent (POME): Biotechnological advances in the sustainable reuse of POME. Biotechnol. Adv. 2009, 27, 40-52 (13) Teh, C.Y.; Wu, T.Y.; Juan, J.C. Optimization of agro-industrial wastewater treatment using unmodified rice starch as a natural coagulant. Ind. Crops Prod. 2014, 56, 17-26. (14) Wu, T.Y.; Mohammad, A.W.; Jahim, J.M.; Anuar, N. Pollution control technologies for the treatment of palm oil mill effluent (POME) through end-of-pipe processes. J. Environ. Manage. 2010, 91 (7), 467-1490. (15) Shak, K.P.Y.; Wu, T.Y. Coagulation-flocculation treatment of high-strength agroindustrial wastewater using natural Cassia obtusifolia seed gum: Treatment efficiencies and flocs characterization. Chem. Eng. J. 2014, 256, 293-305.


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(16) Teh, C.Y.; Wu, T.Y.; Juan, J.C. Potential use of rice starch in coagulation-flocculation process

of

agro-industrial

wastewater:

Treatment

performance

and

flocs

characterization. Ecol. Eng. 2014, 71, 509-519. (17) Lim, S.L.; Wu, T.Y.; Clarke, C. Treatment and biotransformation of highly polluted agroindustrial wastewater from a palm oil mill into vermicompost using earthworms. J. Agric. Food Chem. 2014, 62, 691-698. (18) Alkhatib, M.F.; Mamun, A.A.; Akbar, I. Application of response surface methodology (RSM) for optimization of color removal from POME by granular activated carbon. Int. J. Environ. Sci. Technol. 2015, 12, 1295-1302. (19) Budiman, P.M.; Wu, T.Y.; Ramanan, R.N.; Hay, J.X.W. Treatment and reuse of effluents from palm oil, pulp, and paper mills as a combined substrate by using purple nonsulfur bacteria. Ind. Eng. Chem. Res. 2014, 53, 14921-14931. (20) Androga, D.D.; Sevinç, P.; Koku, H.; Yücel, M.; Gündüz, U.; Eroglu, I. Optimization of temperature and light intensity for improved photofermentative hydrogen production using Rhodobacter capsulatus DSM1710. Int. J. Hydrog. Energy 2014, 39, 2472-2480. (21) Eroğlu, E.; Gündüz, U.; Yücel, M.; Türker, L.; Eroğlu, I. Photobiological hydrogen production by using olive mill wastewater as a sole substrate source. Int. J. Hydrog. Energy 2004, 29, 163-171. (22) Wu, T.Y.; Hay, J.X.W.; Kong, L.B.; Juan, J.C.; Jahim, J.M. Recent advances in reuse of waste material as substrate to produce biohydrogen by purple non-sulfur (PNS) bacteria. Renew. Sust. Energ. Rev. 2012, 16, 3117-3122. (23) Dubois, M.; Gilles, K.A.; Hamilton, J.K.; Rebers, P.A.; Smith, F. Colorimetric method for determination of sugars and related substances. Anal. Chem. 1956, 28, 350-356.


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(24) Sasikala, K.; Ramana, C.V.; Rao, P.R. Environmental regulation for optimal biomass yield and photoproduction of hydrogen by Rhodobacter sphaeroides O.U.001. Int. J. Hydrog. Energy 1991, 16, 597-601. (25) Basak, N.; Das, D. Photofermentative hydrogen production using purple non-sulfur bacteria Rhodobacter sphaeroides O.U.001 in an annular photobioreactor: A case study. Biomass Bioenerg. 2009, 33, 911-919. (26) Eroğlu, E.; Eroğlu, I.; Gündüz, U.; Yücel, M. Treatment of olive mill wastewater by different physicochemical methods and utilization of their liquid effluents for biological hydrogen production. Biomass Bioenerg. 2009, 33, 701-705. (27) Koku, H.; Eroğlu, I.; Gündüz, U.; Yücel, M.; Türker, L. Kinetics of biological hydrogen production by the photosynthetic medium Rhodobacter sphaeroides O.U. 001. Int. J. Hydrog. Energy 2003, 28, 381-388. (28) Koku, H.; Eroğlu, I.; Gündüz, U.; Yücel, M.; Türker, L. Aspects of the metabolism of hydrogen production by Rhodobacter sphaeroides. Int. J. Hydrog. Energy 2002, 27, 1315-1329. (29) Sevinç, P.; Gündüz, U.; Eroglu, I.; Yücel, M. Kinetic analysis of photosynthetic growth, hydrogen production and dual substrate utilization by Rhodobacter capsulatus. Int. J. Hydrog. Energy 2012, 37, 16430-16436. (30) Han, H.; Liu, B.; Yang, H.; Shen, J. Effect of carbon sources on the photobiological production of hydrogen using Rhodobacter sphaeroides RV. Int. J. Hydrog. Energy 2012, 37, 12167-12174. (31) Zhu, H.G.; Suzuki, T.; Tsygankov, A.A.; Asada, Y.; Miyake, J. Hydrogen production from tofu wastewater by Rhodobacter sphaeroides immobilized in agar gels. Int. J. Hydrog. Energy 1999, 24, 305–310.


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(32) Xie, G.J.; Liu, B.F.; Ren, H.Y.; Xing, D.F.; Nan, J.; Ren, N.Q. Material flow analysis of feedstock for enhancing its conversion efficiency during continuous photo-hydrogen production. Glob. Change Biol. Bioenergy 2014, 6, 621-628. (33) Belila, A.; Fazaa, I.; Hassen, A; Ghrabi, A. Anoxygenic phototrophic bacterial diversity within wastewater stabilization plant during 'red water' phenomenon. Int. J. Environ. Sci. Technol. 2013, 10 (4), 837-846. (34) Hay, J.X.W.; Wu, T.Y.; Teh, C.Y.; Jahim, J.M. Optimized growth of Rhodobacter sphaeroides O.U.001 using response surface methodology (RSM). J. Sci. Ind. Res. 2012, 71, 149-154.


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List of Tables:

Table 1. Properties of POME and PPME

Parameter

POME

PPME

NS4a

pH

4.3 ± 0.3

6.15 ± 1.3

7.00 ± 0.25

turbidity

67,500 ± 1,910 NTU

4,700 ± 141 NTU

16,450 ± 636 NTU

COD

84,450 ± 19,500 mg/l

2,716 ± 125 mg/l

22,900 ± 849 mg/l

total suspended solids

19,610 ± 7,900 mg/l

841 ± 878 mg/l

5,559 ± 100 mg/l

total organic carbon

4,251 ± 112.70 mg/l

473.3 ± 18.56 mg/l

1433.5 ± 6.36 mg/l

total nitrogen

650 ± 300.00 mg/l

3.70 ± 3.65 mg/l

170.5 ± 2.12 mg/l

C/N ratio

6.54 ± 3.43

128 ± 22

8.41 ± 0.16

malic acid

17.76 ± 0.28 g/l

0.290 ± 0.08 g/l

4.655 ± 0.18 g/l

sodium glutamate

10.87 ± 0.02 g/l

0.515 ± 0.13 g/l

3.04 ± 0.12 g/l

total phenol

n.d.b mg/l

5.25±0.35 mg/l

3.89 ± 0.36 mg/l

heavy metal content

a

-

Cu

n.d.

n.d.

n.d.

-

Fe

70.7±1.65 mg/l

0.50±0.01 mg/l

18.35 ± 0.64 mg/l

-

Zn

7.53±1.07 mg/l

0.12±0.01 mg/l

2.01 ± 0.21 mg/l

-

Mn

6.47±1.43 mg/l

0.09±0.01 mg/l

1.72 ± 0.09 mg/l

-

Mg

1,144±7.00 mg/l

3.28±1.08 mg/l

289.5 ± 2.12 mg/l

-

Al

334±22.65 mg/l

33.43±1.10 mg/l

109 ± 4.24 mg/l

NS4 was a combined POME (25%, v/v) and PPME (75%, v/v) substrate.

b

n.d., not detectable


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Table 2. Summary of biohydrogen production media characteristics and experimental kinetic parameters of cumulative biohydrogen production in different POME concentration treatment

Mediuma POME concentration

Turbidity

Total

(NTU)

carbohydrate

(%, v/v)

Kinetic parameters

Y

Rmax

ɳ

λ

(ml

(ml H2/ml

(%)

(h)

H2/ml

medium·h)

concentration (g/l)

medium) NS1b

0

4,700 ± 141

0.385 ± 0.04

1.343

0.217

0.69 2

NS2

12.5

11,150±919

6.565 ± 0.36

3.734

1.077

1.92 0.5

NS3

20

16,300±1,273

9.745 ± 0.18

4.038

1.533

2.07 0.5

NS4

25

16,450 ± 636

12.33 ± 6.56

4.670

0.496

2.40 0.5

NS5

30

23,000±566

16.30 ± 0.62

1.816

0.280

0.93 0.5

NS6

40

28,300±424

17.86 ± 0.65

1.609

0.244

0.83 0.5

NS7

100

67,500±1,910

32.35 ± 1.20

0.298

0.012

0.14 4

a

No additional nutrients were added into any medium.

b

NS1 was 100% raw PPME.


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27

Table 3. Comparison of experimental and predicted kinetics parameters of cumulative biohydrogen production in NS4

Parameters

Hm (ml H2)

Rm (ml H2/h)

ߣ (h)

experimental

467.0

49.60

0.5

prediction (by modified Gompertz 445.8

35.76

0.44

38.7

13.6

Model) error difference (%)

4.76


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28 Figure captions:

Figure 1. Correlation between biohydrogen yield and turbidity value for low POME concentration (NS1-NS4; ▬) and high POME concentration (NS4-NS7; ▬) range.

Figure 2. Effect of POME concentration on cumulative biohydrogen production by R. sphaeroides NCIMB8253. Different letter assigned to each treatment represents the significant differences of experimental results (one-way ANOVA, Tukey’s test; P < 0.05)

Figure 3. Effect of POME concentration on photo-anaerobic growth of R. sphaeroides NCIMB8253

Figure 4. Kinetic fitting of cumulative biohydrogen production in NS4 into Modified Gompertz Model (▬)

Figure 5. CODtotal (a) and CODsoluble (b) reductions in different POME concentration treatment after biohydrogen production by R. sphaeroides NCIMB8253

Figure 6. Changes in CODtotal (−▲−) and CODsoluble (−×−) during R. sphaeroides NCIMB8253 growth (−■−) and biohydrogen production (−●−) in NS4

Figure 7. Changes of pH in different POME concentration treatment during biohydrogen production by R. sphaeroides NCIMB8253


Page 29 of 35

29 List of Figures:

Biohydrogen yield (ml H2/ml medium)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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5 4.5 4 3.5 3 2.5 2 1.5 1

y = 7.2904e-0.00005x R² = 0.9429

y = -2×10-8x2 + 0.0007x - 1.4795 R² = 0.9687

0.5 0 0

10000

20000

30000 40000 50000 Turbidity (NTU)

Figure 1


60000

70000

Energy & Fuels

30

Cumulative Biohydrogen Production (ml H2)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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600 500 d c c

400 300

b b

200

b

100

a

0 0

12

NS1

24

NS2

36 48 Time (h) NS3

NS4

NS5

Figure 2


60

NS6

72

NS7

Page 31 of 35

31 250

CFU • 10-8 (CFU/ml)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Energy & Fuels

200 150 100 50 0 0

12

NS1

24

NS2

36 48 Time (h) NS3

NS4

NS5

Figure 3


60

NS6

72

NS7

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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32

Figure 4


Page 33 of 35

33 90

35

80

30

70

40

15

30

COD Removal (%)

CODtotal (g/l)

20

50

Initial

COD Removal (%)

25 60

Initial

Final Removal

10 20 5

10 0

0 NS1

NS2

NS3

NS4 Medium

NS5

NS6

NS7

Figure 5 (a)

80

35

70

30

60 CODsoluble (g/l)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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25

50 20 40 15 30 10

20

5

10

0

0 NS1

NS2

NS3

NS4 Medium

NS5

NS6

Figure 5 (b)


NS7

Final Removal

Energy & Fuels

34

500

26

450 24

400 350

22

300 250

20

200 18

150 100

16

50 0

14 0

12

24

36 Time (h)

48

Figure 6


60

72

COD (g/l)

Cumulative biohydrogen production (ml H2) CFU number • 10-8 (CFU/ml)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Page 35 of 35

35

7.3 7.1 6.9 6.7 pH

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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6.5 6.3 6.1 5.9 5.7 0

12

NS1

24

NS2

36 48 Time (h) NS3

NS4

NS5

Figure 7


60

NS6

72

NS7

Improvement of Biohydrogen Production through Combined Reuses of

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