Accepted Manuscript Production of a bioflocculant from Aspergillus niger using palm oil mill effluent as carbon source Ahmad H. Rajab Aljuboori, Yoshimitsu Uemura, Noridah Binti Osman, Suzana Yusup PII: DOI: Reference:
S0960-8524(14)01145-6 http://dx.doi.org/10.1016/j.biortech.2014.08.038 BITE 13801
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
10 June 2014 6 August 2014 7 August 2014
Please cite this article as: Rajab Aljuboori, A.H., Uemura, Y., Osman, N.B., Yusup, S., Production of a bioflocculant from Aspergillus niger using palm oil mill effluent as carbon source, Bioresource Technology (2014), doi: http:// dx.doi.org/10.1016/j.biortech.2014.08.038
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Production of a bioflocculant from Aspergillus niger using palm oil mill effluent as carbon source
Ahmad H. Rajab Aljubooria*, Yoshimitsu Uemuraa, Noridah Binti Osmana , Suzana Yusupb a
Centre for Biofuel and Biochemical Research (CBBR), Universiti Teknologi PETRONAS,
31750 Tronoh, Perak, Malaysia b
Biomass Processing Lab, Universiti Teknologi PETRONAS, 31750 Tronoh, Perak,
Malaysia
*
Corresponding author, Centre for Biofuel and Biochemical Research (CBBR), Universiti
Teknologi PETRONAS. Tel: +60 5 368 7645; Fax: +60 5 368 7649. E-mail address: [email protected] (Ahmad H. Rajab Aljuboori).
Abstract This study evaluated the potential of bioflocculant production from Aspergillus niger using Palm Oil Mill Effluent (POME) as carbon source. The bioflocculant named PM-5 produced by A. niger showed a good flocculating capability and flocculating rate of 76.8% to kaolin suspension could be achieved at 60 hours of culture time. Glutamic acid was the most favorable nitrogen source for A. niger in bioflocculant production at pH 6 and temperature 35 ˚C. The chemical composition of purified PM-5 was mainly carbohydrate and protein with 66.8 % and 31.4 %, respectively. Results showed the novel bioflocculant (PM-5) had high potential to treat river water from colloids and 63 % of turbidity removal with the present of Ca2+ ion. 1
Keywords: Aspergillus niger; Bioflocculant; Biopolymer flocculant; Extracellular biopolymeric substance; Microbial flocculant; Palm oil mill effluent.
1. Introduction
Flocculation process is widely used in various fields of industries, including food production, downstream of fermentation process, water purification and wastewater treatment (Wang et al., 2013a; Wang et al., 2013b). Flocculating agents are generally divided into: inorganic flocculants (aluminium sulfate and polyaluminium chloride), organic synthetic flocculants (polyacrylamide), and naturally occurring flocculants (bioflocculant, chitosan and guar gum) (Aljuboori et al., 2013). Inorganic and organic synthetic flocculants are commonly used in water and wastewater industries (Zhao et al., 2013). However, the excessive use of these chemical flocculants can cause health and environmental problems (Li et al., 2013). Recently, most of bioflocculants have attracted high biotechnological attention due to their outstanding flocculation properties, biodegradability, eco-friendly and free of secondary pollution risk (Guo et al., 2013; Zhang et al., 2013). However, high cost of bioflocculant production related to expensive substrates has limited the practical application and development of bioflocculant (Zhao et al., 2012). Therefore, it is necessary to find low cost substrates such as industrial biological waste to replace conventional media substrates (sucrose, glucose, fructose) and reduce the bioflocculant production cost (Wang et al., 2013b; Zhang et al., 2013; Zhao et al., 2012). For example, Wang et al. (2013b) reported hydrolysate of corn stover was used as carbon source to produce the bioflocculant from Ochrobactrum ciceri W2. Moreover, waste 2
fermenting liquor was successfully used as media substrate to produce bioflocculant from Bacillus subtilis (You et al., 2008). Palm oil mill effluent from palm oil extraction process is one of the most abundant agroindustrial waste containing high organic matters produced in many countries including Malaysia, Indonesia and India (Saidu et al., 2013). Typically 1 tonne of Crude Palm Oil (CPO) production required 5-7.5 m3 of water and more than 50% of the water ends up as POME. Malaysia is the first exporter and second producer of CPO produced around 52.6, 51.4, 49.8, 55.7 and 55.4 million tonnes of POME in 2008, 2009, 2010, 2011 and 2012, respectively (Aljuboori, 2013). Thus, POME could be the most available and sustainable organic source for biological products industries, especially for bioflocculant production. Therefore, the aim of this study are to investigate the bioflocculant production by Aspergillus niger using POME as carbon source, to determine optimal chemical and environmental conditions for bioflocculant production and to investigate the composition and flocculating properties of bioflocculant.
2. Materials and methods
2.1 Substrate Preparation Raw POME collected from Felcra Nasaruddin, Felcra Oil Mill, Bota, Perak, Malaysia, was used as substrate (carbon source) with characteristics as follows: Total Carbon (TC) 11,891 mg L-1; Total Organic Carbon (TOC) 11,794 mg L-1 ; Total Nitrogen (TN) 290.4 mg L-1 and pH 4.6 (this sample was used for optimization of PM-5 production).
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Additionally, two samples of POME collected from different palm oil mills were used to study the effect of POME chemical content on PM-5 production. The first sample was collected from Felcra Berhad Bidor mill (B-POME), Perak, Malaysia, with characteristics as follows: Total Carbon (TC) 12,720 mg L-1 ; Total Organic Carbon (TOC) 12,710 mg L-1 ; Total Nitrogen (TN) 570.7 mg L-1 and pH 4.5. The second sample was collected from Felcra Berhad Seberang mill (S-POME), Perak, Malaysia, with characteristics as follows: Total Carbon (TC) 9,722.5 mg L-1 ; Total Organic Carbon (TOC) 9,715 mg L-1 ; Total Nitrogen (TN) 402.1 mg L-1 and pH 4.75. Samples were analyzed in triplicate. The collected POME samples were stored at temperature of 4 ˚C.
2.2 Microorganism and growth conditions A. niger, isolated by the Department of Biotechnology and preserved at the Microbial Culture Collection Unit (UNiCC), Laboratory of Industrial Biotechnology, Institute of Bioscience, University Putra Malaysia (Selangor, Malaysia), was maintained on slant media at 4˚C and sub-cultured every 30–40 days. The medium for slant and subculture consisted of (g L-1): potato extract, 4; glucose, 20 and agar, 15. Meanwhile the initial pH was adjusted to 5.6 ± 0.2.
2.3 Production of bioflocculant The production medium consisted of (g L-1): POME, 10 (TOC); glutamic acid, 7.92; MgSO4·7H2O, 0.5; KCl, 0.5; FeSO4, 0.01; K2HPO4, 1.0; and its initial pH was adjusted to 6.0. The fungus was cultured in 100-mL Erlenmeyer flasks containing 50 ml of medium and incubated in a shaker at 150 rpm for 3 days at 32 ˚C. Samples were taken at different 4
time intervals to determine flocculation rate and fungal biomass weight. The biomass samples were filtered and dried at 105 ˚C in an oven for 2 hours. Distilled water was used to prepare all medium solutions and the media were sterilized at 121 ˚C for 20 minutes.
2.4 Optimization of culture conditions of A. niger for bioflocculant production Six factors inclusive nitrogen source, C/N ratio, initial pH, culture temperature, metal ions and culture time were investigated. To determine the effect of nitrogen sources on bioflocculant production, glutamic acid was replaced with (NH4)2SO4, NH4NO3, NaNO3, urea and yeast extract (1 g L-1 nitrogen source). For the C/N ratio, different concentrations of POME (TOC) were used in order to get the different C/N ratio of 0/1 to 40/1. The initial pH of production media were adjusted at 3 to10. Temperature of production media were adjusted at 25 to 40 ˚C. To study the effect of metal ions on bioflocculant production, KCl was replaced with NaCl, CaCl2, MgCl2, MnCl2 and FeCl3 at the same concentration. The effect of time course of bioflocculant production was investigated between 0 and 96 hours. All experiments were conducted in duplicate.
2.5 Purification of the bioflocculant Two volumes of cold ethanol (at 4˚C) were added to 1 L culture broth (supernatant). The precipitate was dissolved in 100 ml deionized water and 50 mL of 2% cetylpyridinium chloride solution (CPC) was added to the solution and thoroughly mixed. After three hours, the precipitate was collected and dissolved in 100 mL of 0.5 M NaCl. Two volumes of cold ethanol were added and the precipitate was washed with ethanol, dissolved in 5 mL of deionized water and vacuum-dried (Aljuboori et al., 2013; Deng et al., 2005). 5
2.6 Analysis of bioflocculant The total sugar content of bioflocculant was determined according to the phenol sulfuric acid method using glucose as standard (Dubois et al., 1956; Krishnaveni et al., 1984). The total protein content was determined by the Bradford method with bovine serum albumin as standard (Bradford, 1976). The functional groups of bioflocculant were determined with a Spectrum One FT-IR spectrometer (PerkinElmer, USA). Elemental analysis of PM-5 was carried out with a PerkinElmer Series II CHNS/O 2400 elemental analyzer (USA).
2.7 Determination of flocculating rate of bioflocculant A kaolin suspension was used to determine the flocculating rate of the bioflocculant in culture broth. 2 grams of Kaolin clay (Merck, Germany) was suspended in 1 L of deionized water. One ml of culture broth and 0.5 ml of CaCl2 (10 mmol/L) were added to 198.5 ml of kaolin suspension in a 500-ml beaker and the pH value was adjusted to 7.0 using 1 M NaOH or HCl. The mixture was stirred at 200 rpm for 1 min, slowly stirred at 60 rpm for 5 min, and allowed to stand for 10 min using jar tester (FC6S, VELP SCIENTIFICA, Italy). The optical density (OD) of the supernatant was measured with a spectrophotometer (DR5000 UN-VIS, HACH, USA) at 550 nm. In the control experiment, 1 ml of culture broth was replaced with 1 ml of fresh culture medium. The flocculating rate was calculated according to the following equation: Flocculating rate (%) = (A550 - B550)/A550 X100
(1)
where A550 and B550 were the OD550 (optical density at 550 nm) of control and sample supernatant, respectively.
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2.8 Purification of river water Different concentration (1 to 50 mg L-1) of PM-5 bioflocculant and CaCl2 (5 mmol/L) were added into 200 ml real river water obtained from Perak river, Perak, Malaysia, mixed at 200 rpm for 1 min, and then at 60 rpm for another 5 min, then allowed to stand for 10 min using 6-breaker jar tester (FC6S, VELP SCIENTIFICA, Italy), and the supernatant was taken for analysis. Turbidity was measured according to the procedure of turbidity meter (2100Q, HACH, USA). The pH value of water was measured by a pH meter (OAKTON, EUTECH INSTRUMENTS, SINGAPORE). The Total suspended solids (TSS) of river water was determined by 2540 D (APHA, 2005). The turbidity removal was calculated as follows: Turbidity removal efficiency (%) = (Ca- Cb)/Ca X100
(2)
where Ca is the initial turbidity value and Cb is the turbidity value after treatment.
3. Results and discussion
3.1 Bioflocculant production
3.1.1 Effect of POME, nitrogen sources and C/N ratio on bioflocculant production As shown in Fig. 1(a and b), POME was a good carbon source for bioflocculant production by A. niger with the flocculating rates up to 80%. In order to maximize the bioflocculant production, organic and inorganic nitrogen sources were used to find the most favorable nitrogen source for A. niger. Glutamic acid (organic nitrogen) was efficiently used in the production of PM-5 with the flocculating rate up to 81%. Although yeast extract and urea 7
were favorable nitrogen sources for biomass growth, the flocculating rate showed not higher than 65%. Similarly, Ugbenyen et al. (2012), reported yeast extract and urea used for bioflocculant production were poorly utilized by Cobetia sp. Moreover, the low productions of PM-5 and biomass growth were observed when (NH4)2SO4, NH4NO3 and NaNO3 were used as inorganic nitrogen sources. This result is in agreement with Liu et al. (2010) finding, where inorganic nitrogen sources ((NH4)2SO4, NH4NO3 and NaNO3) led to poor production of bioflocculant and cell growth of Chryseobacterium daeguense W6. Glutamic acid was the most favorable nitrogen source for PM-5 production and used in the following experiments. The effect of C/N ratio on bioflocculant production was investigated in this study. Results showed the production of bioflocculant gradually increased as the C/N ratio increased up to 20/1 with flocculating rate of 79% (Fig.1b), but further increased in C/N ratio slightly decreased the PM-5 production. Thus, 20/1 ratio was chosen for the next experiments.
3.1.2 Effect of the initial pH, temperature and metal ions on PM-5 production For most microorganisms the microbial product such as bioflocculant is regularly increase between minimum and the optimum pH, and a corresponding regularly decrease in microbial product between the optimum and the maximum pH. This reflects the effect of [H+] change on enzymatic reaction rates and nutrient absorption. Fig. 2 shows the effect of initial pH of culture medium on PM-5 production. The production of PM-5 dramatically increased as the pH increased from 3 to 6, and about 77% of highest flocculating rate was recorded at pH 6. While, culture medium with initial pH between 7 to 10 showed low bioflocculant production. Similarly, Mabinya et al. (2012), reported that bioflocculant 8
produced by Arthrobacter sp. at acidic pH range was higher than basic pH range. Thus, pH 6 was selected as the initial pH in the following experiments. Physical conditions affect the microbial growth and productivities, as enzymatic activity of microorganism depends on temperature of culture medium. The production of bioflocculant (PM-5) rapidly increased as the temperature of culture medium increased from 25 to 30 ˚C, and then slightly increased as the temperature reached 35˚C (Fig. 3). The highest flocculating rate was 76.3% at 35˚C, further increased in culture medium temperature (40˚C) significantly affected the PM-5 production. This may be due to the enzymes of bioflocculant production are deactivated at 40˚C (Xia et al., 2008). The optimal temperature range for bioflocculant-producing microorganisms was reported to be between 25 to 37˚C (Nam et al., 1996; Salehizadeh and Shojaosadati, 2001; Wu and Ye, 2007). However, the A. niger growth gradually decreased as the temperature increased from 25 to 40˚C. As shown, the optimum temperature for PM-5 production was 35 °C, this temperature was chosen for the following experiments. In addition, PM-5 production by A. niger was stimulated in the presence of different metal ions Mn2+, Ca2+, K+, Mg2+ and Na+ in culture medium with the flocculating rate of 74.7, 70.6, 69, 66.4 and 56.2 %, respectively. In contrast, Fe3+ poorly stimulates the PM-5 production with the flocculating rate of 26.1% (Fig. 4). Ugbenyen and Okoh (2013) also reported, metal ions such as Ca2+, Mn2+, K+, Mg2+ and Na+ were significantly stimulated the bioflocculant production by Bacillus sp. Although Mn2+ was the most favorable metal ion for PM-5 production, A. niger growth was the lowest in comparison with other metal ions
9
and control (no metal ion added) culture medium. Hence, as Mn2+ was the most favorable metal ion and used in culture medium for the following experiments.
3.1.3 Time course of PM-5 production Fig. 5 shows the time courses of flocculating rate, growth and pH of A. niger culture broth. In general, the flocculating rate of the culture broth increased in parallel with cell growth. The flocculating rate increased dramatically after 24 hours and reached maximum value of 77% at early stationary phase (60 h) and decreased gradually thereafter. This may be due to enzymatic activity and cell lysis (Aljuboori et al., 2013; Xiong et al., 2010). This phenomenon showing the PM-5 produced by biosynthesis during A. niger growth (Okaiyeto et al., 2013). Studies similarly reported that bioflocculants from Rhodococcus R3 (Guo et al., 2013), Serratia ficaria (Gong et al., 2008) and Ochrobactium ciceri W2 (Wang et al., 2013b) were produced by biosynthesis during cell growth. The biomass growth was rapidly increased from 12 to 24 h of culture time then slowly increased and reached stationary phase with maximum biomass growth of 3.82 g L-1. Referring to pH, the pH profile was relatively changed during PM-5 production. Results showed as the flocculating rate increased the pH level decreased from 5.89 to 4.4 within 72 h, which might be due to the presence of organic acid components of the bioflocculant (Lu et al., 2005; Xiong et al., 2010). About 2.6 g L-1 of purified PM-5 was obtained from culture broth when the flocculating rate reached the maximum level at 60 h. Moreover, this study investigated the effect of POME chemical content on PM-5 production. B-POME and S-POME are two different samples with different chemical content were used to produce PM-5 by A. niger at optimum conditions. This study found 10
that B-POME produced 2.73 g L-1 of purified PM-5 with average flocculating rate of 77.2%. While S-POME produced 2.21 g L-1 of purified PM-5 with average flocculating rate of 62.6%. These results shows that POME with high TOC concentration produced high concentration of PM-5 by A. niger.
3.2 Chemical analysis of PM-5 The phenol-sulfuric acid method showed that purified PM-5 bioflocculant contain 66.8 % of carbohydrate and Bradford method determined about 31.4 % of total protein in PM-5. The elemental analysis of purified PM-5 showed that the mass proportion of C, H, O and N, were 25.2%, 4.6%, 69.1% and 1.1% (w/w), respectively. The IR spectrum of purified PM-5 showed a broad stretching peak at 3430 cm-1 which is an indication of −OH stretching from hydroxyl group. A weak peak at 2927 cm-1 indicated C-H asymmetrical stretching vibration and known to be typical of carbohydrate derivatives and the peak at 1631 cm-1 displayed a carboxylate anion group. Another peak at 1413 cm-1 could be attributed to the symmetric stretching of the ̶COO−group. A weak peak at 1324 cm-1 showed carboxyl group. The behavior of the wide peaks in 1078 - 1047 cm-1 range, represents the stretch vibration of CO in the carbohydrate region. The presence of hydroxyl and carboxyl groups in purified PM-5 bioflocculant are preferable functional groups for the bioflocculation process and induce high binding capacity (Guo et al., 2014). Metal ions are stimulating agents for the flocculating activity of cation-dependent bioflocculant. Therefore, Ca2+ ion induced the flocculating activity of PM-5 on kaolin particles by neutralizing and stabilizing the negative charge of those functional groups and thereby bridging mechanism occurs when the kaolin particles adsorbed onto the bioflocculant chain (Gao et al., 2006). 11
3.3 Purification of river water Real river water with initial turbidity of 73.2 NTU, Total Suspended Solids of 117 mg L-1 and pH 6.9 was used to investigate the effect of PM-5 on turbidity removal. Results showed PM-5 bioflocculant produced by A. niger could effectively use to reduce the turbidity of river water. The turbidity removal was increased dramatically as the PM-5 concentration increased from 20 to 35 mg L-1 and recorded the highest removal of turbidity of 63%. This is due to high concentration of PM-5 in the presence of Ca2+ ion that provided more bridging sites to colloids and particles (Doherty et al., 2003). Thereafter the turbidity removal was sharply decreased as the PM-5 concentration increased beyond 35 mg L-1 (overdose), due to restabilization of colloid complex (Renault et al., 2009). Li et al. (2009) reported that bioflocculant produced from Bacillus licheniformis X14 was able to remove 95% of turbidity from river water. Another study showed bioflocculant produced from mixed culture bacteria produced turbidity removal of up to 96% from river water (Buthelezi et al., 2009). However, these bioflocculants are mainly produced from an expensive carbon source such as sucrose, ethanol and yeast extract. Therefore, in this study the significant flocculating efficiency of PM-5 in river water treatment and the conversion of organic pollutant in industrial wastewater (POME) into value-added chemical (bioflocculant) by A. niger indicated the feasibility of its commercial use in real wastewater treatment industries.
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4. Conclusions
This study shows that A. niger is a bioflocculant-producing microorganism, which produced bioflocculant named PM-5. The new bioflocculant (PM-5) was optimally produced from palm oil mill effluent as carbon source and glutamic acid as nitrogen source. PM-5 showed a significant flocculating rate of kaolin suspension in the presence of Ca2+ ion. Results showed that PM-5 was successfully able to treat real river water with turbidity removal of 63%. Therefore, this study discovered significant potential in the utilization of agro-industrial waste as medium culture for bioflocculant production and improved the feasibility of commercial production.
Acknowledgments This work was supported by Mitsubishi Corporation Education Trust Fund (MCETF).
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Figure captions
Figure 1: The effect of nitrogen sources and C/N ratio on production of PM-5 bioflocculant and A. niger biomass: (a) The effect of nitrogen sources on PM-5 production with POME used in the medium as carbon source; (b) The effect of C/N ratio on production of PM-5. Figure 2: The effect of initial pH of the medium on production of PM-5. Figure 3: The effect of temperature of the medium on production of PM-5. Figure 4: The effect of metal ions on production of PM-5. Figure 5: Time course of the PM-5 production.
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PM-5 is a new bioflocculant produced from Aspergillus niger.
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POME was first time used as carbon source for bioflocculant production.
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The maximum PM-5 production was 2.6 g/L.
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PM-5 had great potential to treat river water from suspended solids.
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PM-5 combined with Ca2+ acted as stimulating agent.
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S0960-8524(14)01145-6 http://dx.doi.org/10.1016/j.biortech.2014.08.038 BITE 13801
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
10 June 2014 6 August 2014 7 August 2014
Please cite this article as: Rajab Aljuboori, A.H., Uemura, Y., Osman, N.B., Yusup, S., Production of a bioflocculant from Aspergillus niger using palm oil mill effluent as carbon source, Bioresource Technology (2014), doi: http:// dx.doi.org/10.1016/j.biortech.2014.08.038
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Production of a bioflocculant from Aspergillus niger using palm oil mill effluent as carbon source
Ahmad H. Rajab Aljubooria*, Yoshimitsu Uemuraa, Noridah Binti Osmana , Suzana Yusupb a
Centre for Biofuel and Biochemical Research (CBBR), Universiti Teknologi PETRONAS,
31750 Tronoh, Perak, Malaysia b
Biomass Processing Lab, Universiti Teknologi PETRONAS, 31750 Tronoh, Perak,
Malaysia
*
Corresponding author, Centre for Biofuel and Biochemical Research (CBBR), Universiti
Teknologi PETRONAS. Tel: +60 5 368 7645; Fax: +60 5 368 7649. E-mail address: [email protected] (Ahmad H. Rajab Aljuboori).
Abstract This study evaluated the potential of bioflocculant production from Aspergillus niger using Palm Oil Mill Effluent (POME) as carbon source. The bioflocculant named PM-5 produced by A. niger showed a good flocculating capability and flocculating rate of 76.8% to kaolin suspension could be achieved at 60 hours of culture time. Glutamic acid was the most favorable nitrogen source for A. niger in bioflocculant production at pH 6 and temperature 35 ˚C. The chemical composition of purified PM-5 was mainly carbohydrate and protein with 66.8 % and 31.4 %, respectively. Results showed the novel bioflocculant (PM-5) had high potential to treat river water from colloids and 63 % of turbidity removal with the present of Ca2+ ion. 1
Keywords: Aspergillus niger; Bioflocculant; Biopolymer flocculant; Extracellular biopolymeric substance; Microbial flocculant; Palm oil mill effluent.
1. Introduction
Flocculation process is widely used in various fields of industries, including food production, downstream of fermentation process, water purification and wastewater treatment (Wang et al., 2013a; Wang et al., 2013b). Flocculating agents are generally divided into: inorganic flocculants (aluminium sulfate and polyaluminium chloride), organic synthetic flocculants (polyacrylamide), and naturally occurring flocculants (bioflocculant, chitosan and guar gum) (Aljuboori et al., 2013). Inorganic and organic synthetic flocculants are commonly used in water and wastewater industries (Zhao et al., 2013). However, the excessive use of these chemical flocculants can cause health and environmental problems (Li et al., 2013). Recently, most of bioflocculants have attracted high biotechnological attention due to their outstanding flocculation properties, biodegradability, eco-friendly and free of secondary pollution risk (Guo et al., 2013; Zhang et al., 2013). However, high cost of bioflocculant production related to expensive substrates has limited the practical application and development of bioflocculant (Zhao et al., 2012). Therefore, it is necessary to find low cost substrates such as industrial biological waste to replace conventional media substrates (sucrose, glucose, fructose) and reduce the bioflocculant production cost (Wang et al., 2013b; Zhang et al., 2013; Zhao et al., 2012). For example, Wang et al. (2013b) reported hydrolysate of corn stover was used as carbon source to produce the bioflocculant from Ochrobactrum ciceri W2. Moreover, waste 2
fermenting liquor was successfully used as media substrate to produce bioflocculant from Bacillus subtilis (You et al., 2008). Palm oil mill effluent from palm oil extraction process is one of the most abundant agroindustrial waste containing high organic matters produced in many countries including Malaysia, Indonesia and India (Saidu et al., 2013). Typically 1 tonne of Crude Palm Oil (CPO) production required 5-7.5 m3 of water and more than 50% of the water ends up as POME. Malaysia is the first exporter and second producer of CPO produced around 52.6, 51.4, 49.8, 55.7 and 55.4 million tonnes of POME in 2008, 2009, 2010, 2011 and 2012, respectively (Aljuboori, 2013). Thus, POME could be the most available and sustainable organic source for biological products industries, especially for bioflocculant production. Therefore, the aim of this study are to investigate the bioflocculant production by Aspergillus niger using POME as carbon source, to determine optimal chemical and environmental conditions for bioflocculant production and to investigate the composition and flocculating properties of bioflocculant.
2. Materials and methods
2.1 Substrate Preparation Raw POME collected from Felcra Nasaruddin, Felcra Oil Mill, Bota, Perak, Malaysia, was used as substrate (carbon source) with characteristics as follows: Total Carbon (TC) 11,891 mg L-1; Total Organic Carbon (TOC) 11,794 mg L-1 ; Total Nitrogen (TN) 290.4 mg L-1 and pH 4.6 (this sample was used for optimization of PM-5 production).
3
Additionally, two samples of POME collected from different palm oil mills were used to study the effect of POME chemical content on PM-5 production. The first sample was collected from Felcra Berhad Bidor mill (B-POME), Perak, Malaysia, with characteristics as follows: Total Carbon (TC) 12,720 mg L-1 ; Total Organic Carbon (TOC) 12,710 mg L-1 ; Total Nitrogen (TN) 570.7 mg L-1 and pH 4.5. The second sample was collected from Felcra Berhad Seberang mill (S-POME), Perak, Malaysia, with characteristics as follows: Total Carbon (TC) 9,722.5 mg L-1 ; Total Organic Carbon (TOC) 9,715 mg L-1 ; Total Nitrogen (TN) 402.1 mg L-1 and pH 4.75. Samples were analyzed in triplicate. The collected POME samples were stored at temperature of 4 ˚C.
2.2 Microorganism and growth conditions A. niger, isolated by the Department of Biotechnology and preserved at the Microbial Culture Collection Unit (UNiCC), Laboratory of Industrial Biotechnology, Institute of Bioscience, University Putra Malaysia (Selangor, Malaysia), was maintained on slant media at 4˚C and sub-cultured every 30–40 days. The medium for slant and subculture consisted of (g L-1): potato extract, 4; glucose, 20 and agar, 15. Meanwhile the initial pH was adjusted to 5.6 ± 0.2.
2.3 Production of bioflocculant The production medium consisted of (g L-1): POME, 10 (TOC); glutamic acid, 7.92; MgSO4·7H2O, 0.5; KCl, 0.5; FeSO4, 0.01; K2HPO4, 1.0; and its initial pH was adjusted to 6.0. The fungus was cultured in 100-mL Erlenmeyer flasks containing 50 ml of medium and incubated in a shaker at 150 rpm for 3 days at 32 ˚C. Samples were taken at different 4
time intervals to determine flocculation rate and fungal biomass weight. The biomass samples were filtered and dried at 105 ˚C in an oven for 2 hours. Distilled water was used to prepare all medium solutions and the media were sterilized at 121 ˚C for 20 minutes.
2.4 Optimization of culture conditions of A. niger for bioflocculant production Six factors inclusive nitrogen source, C/N ratio, initial pH, culture temperature, metal ions and culture time were investigated. To determine the effect of nitrogen sources on bioflocculant production, glutamic acid was replaced with (NH4)2SO4, NH4NO3, NaNO3, urea and yeast extract (1 g L-1 nitrogen source). For the C/N ratio, different concentrations of POME (TOC) were used in order to get the different C/N ratio of 0/1 to 40/1. The initial pH of production media were adjusted at 3 to10. Temperature of production media were adjusted at 25 to 40 ˚C. To study the effect of metal ions on bioflocculant production, KCl was replaced with NaCl, CaCl2, MgCl2, MnCl2 and FeCl3 at the same concentration. The effect of time course of bioflocculant production was investigated between 0 and 96 hours. All experiments were conducted in duplicate.
2.5 Purification of the bioflocculant Two volumes of cold ethanol (at 4˚C) were added to 1 L culture broth (supernatant). The precipitate was dissolved in 100 ml deionized water and 50 mL of 2% cetylpyridinium chloride solution (CPC) was added to the solution and thoroughly mixed. After three hours, the precipitate was collected and dissolved in 100 mL of 0.5 M NaCl. Two volumes of cold ethanol were added and the precipitate was washed with ethanol, dissolved in 5 mL of deionized water and vacuum-dried (Aljuboori et al., 2013; Deng et al., 2005). 5
2.6 Analysis of bioflocculant The total sugar content of bioflocculant was determined according to the phenol sulfuric acid method using glucose as standard (Dubois et al., 1956; Krishnaveni et al., 1984). The total protein content was determined by the Bradford method with bovine serum albumin as standard (Bradford, 1976). The functional groups of bioflocculant were determined with a Spectrum One FT-IR spectrometer (PerkinElmer, USA). Elemental analysis of PM-5 was carried out with a PerkinElmer Series II CHNS/O 2400 elemental analyzer (USA).
2.7 Determination of flocculating rate of bioflocculant A kaolin suspension was used to determine the flocculating rate of the bioflocculant in culture broth. 2 grams of Kaolin clay (Merck, Germany) was suspended in 1 L of deionized water. One ml of culture broth and 0.5 ml of CaCl2 (10 mmol/L) were added to 198.5 ml of kaolin suspension in a 500-ml beaker and the pH value was adjusted to 7.0 using 1 M NaOH or HCl. The mixture was stirred at 200 rpm for 1 min, slowly stirred at 60 rpm for 5 min, and allowed to stand for 10 min using jar tester (FC6S, VELP SCIENTIFICA, Italy). The optical density (OD) of the supernatant was measured with a spectrophotometer (DR5000 UN-VIS, HACH, USA) at 550 nm. In the control experiment, 1 ml of culture broth was replaced with 1 ml of fresh culture medium. The flocculating rate was calculated according to the following equation: Flocculating rate (%) = (A550 - B550)/A550 X100
(1)
where A550 and B550 were the OD550 (optical density at 550 nm) of control and sample supernatant, respectively.
6
2.8 Purification of river water Different concentration (1 to 50 mg L-1) of PM-5 bioflocculant and CaCl2 (5 mmol/L) were added into 200 ml real river water obtained from Perak river, Perak, Malaysia, mixed at 200 rpm for 1 min, and then at 60 rpm for another 5 min, then allowed to stand for 10 min using 6-breaker jar tester (FC6S, VELP SCIENTIFICA, Italy), and the supernatant was taken for analysis. Turbidity was measured according to the procedure of turbidity meter (2100Q, HACH, USA). The pH value of water was measured by a pH meter (OAKTON, EUTECH INSTRUMENTS, SINGAPORE). The Total suspended solids (TSS) of river water was determined by 2540 D (APHA, 2005). The turbidity removal was calculated as follows: Turbidity removal efficiency (%) = (Ca- Cb)/Ca X100
(2)
where Ca is the initial turbidity value and Cb is the turbidity value after treatment.
3. Results and discussion
3.1 Bioflocculant production
3.1.1 Effect of POME, nitrogen sources and C/N ratio on bioflocculant production As shown in Fig. 1(a and b), POME was a good carbon source for bioflocculant production by A. niger with the flocculating rates up to 80%. In order to maximize the bioflocculant production, organic and inorganic nitrogen sources were used to find the most favorable nitrogen source for A. niger. Glutamic acid (organic nitrogen) was efficiently used in the production of PM-5 with the flocculating rate up to 81%. Although yeast extract and urea 7
were favorable nitrogen sources for biomass growth, the flocculating rate showed not higher than 65%. Similarly, Ugbenyen et al. (2012), reported yeast extract and urea used for bioflocculant production were poorly utilized by Cobetia sp. Moreover, the low productions of PM-5 and biomass growth were observed when (NH4)2SO4, NH4NO3 and NaNO3 were used as inorganic nitrogen sources. This result is in agreement with Liu et al. (2010) finding, where inorganic nitrogen sources ((NH4)2SO4, NH4NO3 and NaNO3) led to poor production of bioflocculant and cell growth of Chryseobacterium daeguense W6. Glutamic acid was the most favorable nitrogen source for PM-5 production and used in the following experiments. The effect of C/N ratio on bioflocculant production was investigated in this study. Results showed the production of bioflocculant gradually increased as the C/N ratio increased up to 20/1 with flocculating rate of 79% (Fig.1b), but further increased in C/N ratio slightly decreased the PM-5 production. Thus, 20/1 ratio was chosen for the next experiments.
3.1.2 Effect of the initial pH, temperature and metal ions on PM-5 production For most microorganisms the microbial product such as bioflocculant is regularly increase between minimum and the optimum pH, and a corresponding regularly decrease in microbial product between the optimum and the maximum pH. This reflects the effect of [H+] change on enzymatic reaction rates and nutrient absorption. Fig. 2 shows the effect of initial pH of culture medium on PM-5 production. The production of PM-5 dramatically increased as the pH increased from 3 to 6, and about 77% of highest flocculating rate was recorded at pH 6. While, culture medium with initial pH between 7 to 10 showed low bioflocculant production. Similarly, Mabinya et al. (2012), reported that bioflocculant 8
produced by Arthrobacter sp. at acidic pH range was higher than basic pH range. Thus, pH 6 was selected as the initial pH in the following experiments. Physical conditions affect the microbial growth and productivities, as enzymatic activity of microorganism depends on temperature of culture medium. The production of bioflocculant (PM-5) rapidly increased as the temperature of culture medium increased from 25 to 30 ˚C, and then slightly increased as the temperature reached 35˚C (Fig. 3). The highest flocculating rate was 76.3% at 35˚C, further increased in culture medium temperature (40˚C) significantly affected the PM-5 production. This may be due to the enzymes of bioflocculant production are deactivated at 40˚C (Xia et al., 2008). The optimal temperature range for bioflocculant-producing microorganisms was reported to be between 25 to 37˚C (Nam et al., 1996; Salehizadeh and Shojaosadati, 2001; Wu and Ye, 2007). However, the A. niger growth gradually decreased as the temperature increased from 25 to 40˚C. As shown, the optimum temperature for PM-5 production was 35 °C, this temperature was chosen for the following experiments. In addition, PM-5 production by A. niger was stimulated in the presence of different metal ions Mn2+, Ca2+, K+, Mg2+ and Na+ in culture medium with the flocculating rate of 74.7, 70.6, 69, 66.4 and 56.2 %, respectively. In contrast, Fe3+ poorly stimulates the PM-5 production with the flocculating rate of 26.1% (Fig. 4). Ugbenyen and Okoh (2013) also reported, metal ions such as Ca2+, Mn2+, K+, Mg2+ and Na+ were significantly stimulated the bioflocculant production by Bacillus sp. Although Mn2+ was the most favorable metal ion for PM-5 production, A. niger growth was the lowest in comparison with other metal ions
9
and control (no metal ion added) culture medium. Hence, as Mn2+ was the most favorable metal ion and used in culture medium for the following experiments.
3.1.3 Time course of PM-5 production Fig. 5 shows the time courses of flocculating rate, growth and pH of A. niger culture broth. In general, the flocculating rate of the culture broth increased in parallel with cell growth. The flocculating rate increased dramatically after 24 hours and reached maximum value of 77% at early stationary phase (60 h) and decreased gradually thereafter. This may be due to enzymatic activity and cell lysis (Aljuboori et al., 2013; Xiong et al., 2010). This phenomenon showing the PM-5 produced by biosynthesis during A. niger growth (Okaiyeto et al., 2013). Studies similarly reported that bioflocculants from Rhodococcus R3 (Guo et al., 2013), Serratia ficaria (Gong et al., 2008) and Ochrobactium ciceri W2 (Wang et al., 2013b) were produced by biosynthesis during cell growth. The biomass growth was rapidly increased from 12 to 24 h of culture time then slowly increased and reached stationary phase with maximum biomass growth of 3.82 g L-1. Referring to pH, the pH profile was relatively changed during PM-5 production. Results showed as the flocculating rate increased the pH level decreased from 5.89 to 4.4 within 72 h, which might be due to the presence of organic acid components of the bioflocculant (Lu et al., 2005; Xiong et al., 2010). About 2.6 g L-1 of purified PM-5 was obtained from culture broth when the flocculating rate reached the maximum level at 60 h. Moreover, this study investigated the effect of POME chemical content on PM-5 production. B-POME and S-POME are two different samples with different chemical content were used to produce PM-5 by A. niger at optimum conditions. This study found 10
that B-POME produced 2.73 g L-1 of purified PM-5 with average flocculating rate of 77.2%. While S-POME produced 2.21 g L-1 of purified PM-5 with average flocculating rate of 62.6%. These results shows that POME with high TOC concentration produced high concentration of PM-5 by A. niger.
3.2 Chemical analysis of PM-5 The phenol-sulfuric acid method showed that purified PM-5 bioflocculant contain 66.8 % of carbohydrate and Bradford method determined about 31.4 % of total protein in PM-5. The elemental analysis of purified PM-5 showed that the mass proportion of C, H, O and N, were 25.2%, 4.6%, 69.1% and 1.1% (w/w), respectively. The IR spectrum of purified PM-5 showed a broad stretching peak at 3430 cm-1 which is an indication of −OH stretching from hydroxyl group. A weak peak at 2927 cm-1 indicated C-H asymmetrical stretching vibration and known to be typical of carbohydrate derivatives and the peak at 1631 cm-1 displayed a carboxylate anion group. Another peak at 1413 cm-1 could be attributed to the symmetric stretching of the ̶COO−group. A weak peak at 1324 cm-1 showed carboxyl group. The behavior of the wide peaks in 1078 - 1047 cm-1 range, represents the stretch vibration of CO in the carbohydrate region. The presence of hydroxyl and carboxyl groups in purified PM-5 bioflocculant are preferable functional groups for the bioflocculation process and induce high binding capacity (Guo et al., 2014). Metal ions are stimulating agents for the flocculating activity of cation-dependent bioflocculant. Therefore, Ca2+ ion induced the flocculating activity of PM-5 on kaolin particles by neutralizing and stabilizing the negative charge of those functional groups and thereby bridging mechanism occurs when the kaolin particles adsorbed onto the bioflocculant chain (Gao et al., 2006). 11
3.3 Purification of river water Real river water with initial turbidity of 73.2 NTU, Total Suspended Solids of 117 mg L-1 and pH 6.9 was used to investigate the effect of PM-5 on turbidity removal. Results showed PM-5 bioflocculant produced by A. niger could effectively use to reduce the turbidity of river water. The turbidity removal was increased dramatically as the PM-5 concentration increased from 20 to 35 mg L-1 and recorded the highest removal of turbidity of 63%. This is due to high concentration of PM-5 in the presence of Ca2+ ion that provided more bridging sites to colloids and particles (Doherty et al., 2003). Thereafter the turbidity removal was sharply decreased as the PM-5 concentration increased beyond 35 mg L-1 (overdose), due to restabilization of colloid complex (Renault et al., 2009). Li et al. (2009) reported that bioflocculant produced from Bacillus licheniformis X14 was able to remove 95% of turbidity from river water. Another study showed bioflocculant produced from mixed culture bacteria produced turbidity removal of up to 96% from river water (Buthelezi et al., 2009). However, these bioflocculants are mainly produced from an expensive carbon source such as sucrose, ethanol and yeast extract. Therefore, in this study the significant flocculating efficiency of PM-5 in river water treatment and the conversion of organic pollutant in industrial wastewater (POME) into value-added chemical (bioflocculant) by A. niger indicated the feasibility of its commercial use in real wastewater treatment industries.
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4. Conclusions
This study shows that A. niger is a bioflocculant-producing microorganism, which produced bioflocculant named PM-5. The new bioflocculant (PM-5) was optimally produced from palm oil mill effluent as carbon source and glutamic acid as nitrogen source. PM-5 showed a significant flocculating rate of kaolin suspension in the presence of Ca2+ ion. Results showed that PM-5 was successfully able to treat real river water with turbidity removal of 63%. Therefore, this study discovered significant potential in the utilization of agro-industrial waste as medium culture for bioflocculant production and improved the feasibility of commercial production.
Acknowledgments This work was supported by Mitsubishi Corporation Education Trust Fund (MCETF).
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Figure captions
Figure 1: The effect of nitrogen sources and C/N ratio on production of PM-5 bioflocculant and A. niger biomass: (a) The effect of nitrogen sources on PM-5 production with POME used in the medium as carbon source; (b) The effect of C/N ratio on production of PM-5. Figure 2: The effect of initial pH of the medium on production of PM-5. Figure 3: The effect of temperature of the medium on production of PM-5. Figure 4: The effect of metal ions on production of PM-5. Figure 5: Time course of the PM-5 production.
17
Biomass
100 90 80 70 60 50 40 30 20 10 0
a 14
12 10 8 6 4 2 0 Yeast Extract
Glutamic acid
Urea
(NH₄)₂ SO₄
NH₄NO₃
Nitrogen Source
18
NaNO₃
Control (no nitrogen added)
Biomass (g/L)
Flocculating rate (%)
Flocculating Rate
Flocculating rate
Biomass
100
b
90
12
70
10
60 8
50 40
6
30
4
20 2
10 0
0 0/1
1/1
2/1
4/1
6/1
8/1 10/1 20/1 30/1 40/1
C/N ratio
19
Biomass (g/L)
Flocculating rate (%)
80
14
Biomass
100
20
90
18
80
16
70
14
60
12
50
10
40
8
30
6
20
4
10
2
0
0 3
4
5
6
7 pH
20
8
9
10
Biomass (g/L)
Flocculating rate (%)
Flocculating rate
Biomass
100
15
90
13
80
11
70 60
9
50
7
40
5
30
3
20 10
1
0
-1 25
30
35
Temperature (˚C)
21
40
Biomass (g/L)
Flocculating rate (%)
Flocculating rate
Flocculating rate
Biomass
100
Flocculating rate (%)
80
12
70
10
60 8
50 40
6
30 20
4
10
2
0
0 FeCl₃
MnCl₂
CaCl₂
NaCl
Metal ions
22
MgCl₂
KCl
Control (no metal ion)
Biomass (g/L)
14
90
Biomass
pH
90
14 13 12 11 10 9 8 7 6 5 4 3 2 1 0
Flocculating rate (%)
80 70 60 50 40 30 20 10 0 0
12
24
36
48 Time (h)
23
60
72
84
96
Biomass (g/L) pH
Flocculating rate
Highlight
•
PM-5 is a new bioflocculant produced from Aspergillus niger.
•
POME was first time used as carbon source for bioflocculant production.
•
The maximum PM-5 production was 2.6 g/L.
•
PM-5 had great potential to treat river water from suspended solids.
•
PM-5 combined with Ca2+ acted as stimulating agent.
24
