Treatment and Reuse of Effluents from Palm Oil, Pulp, and Paper Mills

Article pubs.acs.org/IECR

Treatment and Reuse of Effluents from Palm Oil, Pulp, and Paper Mills as a Combined Substrate by Using Purple Nonsulfur Bacteria Pretty Mori Budiman, Ta Yeong Wu,* Ramakrishnan Nagasundara Ramanan, and Jacqueline Xiao Wen Hay Chemical Engineering Discipline, School of Engineering, Monash University, Jalan Lagoon Selatan, 46150 Bandar Sunway, Selangor Darul Ehsan, Malaysia ABSTRACT: The growth of Rhodobacter sphaeroides NCIMB8253 was investigated under photoanaerobic conditions with different concentrations of palm oil mill effluent in a dual effluent growth medium. The influence of pulp and paper mill effluent as an alternative diluting agent on the bacterial growth was also investigated. During this study, it was determined that the use of pulp and paper mill effluent as a diluting agent promoted bacterial growth compared with the use of raw palm oil mill effluent medium alone. The cell growth data for all experimental sets were well-fitted to the logistic and modified Gompertz models. The results revealed that 25% (v/v) palm oil mill effluent medium with pulp and paper mill effluent as a diluting agent, namely G5 medium, can be applied as a suitable growth medium. A maximum colony-forming-units (CFU) number of 360 × 108 CFU/mL was achieved in G5, which was higher than that for raw palm oil mill effluent and comparable with that for water-diluted palm oil mill effluent medium. Furthermore, chemical oxygen demand level (CODtotal and CODsoluble) reductions were observed in all treatments. activated sludge, and wastewater treatment plants.15 However, Rhodobacter sp. application as a biohydrogen-producing bacteria with wastewater as the fermentation substrate provides additional economic and environmental value to the treatment because of simultaneous wastewater treatment and biohydrogen production. For example, in soybean wastewater treatment, a wild strain of R. sphaeroides Z08 achieved 96% COD reduction after 10 days under sunlight irradiation, without additional nutrient addition, aeration, or adjustment of pH and temperature.11 However, R. sphaeroides O.U.001 was less efficient for COD removal, removing 48.1 and 21% during the treatment of iron-supplemented olive mill and dairy wastewater, respectively.12,13 In most cases, artificial or natural light are supplied during wastewater treatment processes employing PNS to support bacterial growth. For example, light with an intensity of 200 W/m2 was supplied using a tungsten lamp during an olive mill wastewater treatment using R. sphaeroides O.U.001 at a low concentration of olive mill effluent (2−4% (v/v)).12 Furthermore, an approximately 567% biomass increase was observed in 40% (v/v) dairy wastewater compared with a 60% (v/v) concentration of wastewater.13 These results indicate that higher light penetration into the wastewater may support the growth of PNS bacteria during wastewater treatment. Therefore, because of the high turbidity of POME, wastewater dilution is necessary before the treatment process. To reduce water consumption, the reuse of pulp and paper mill effluent (PPME) as an alternative diluting agent to replace water was investigated in this study. PPME was selected as a diluting agent because it is a lighter wastewater than POME. Thus, PPME addition was predicted to reduce POME turbidity and enhance wastewater visibility to allow POME reuse as a substrate during photofermentation.

1. INTRODUCTION During the past decade, the Malaysian palm oil industry has experienced rapid development and is known to be one of the most actively growing agro-industries in Malaysia.1 Consequently, a simultaneous upsurge of palm oil mill effluent (POME) discharge is expected to occur. For each ton of crude palm oil produced, 2.5−3 tons of POME is generated.2 Annual POME generation of 50 million tons has been reported in Malaysia, and this figure is expected to rise in the future.3,4 Therefore, direct discharge of POME into the environment has been identified as one of the most severe pollution sources in Malaysia because of its high levels of chemical oxygen demand (COD) (53 630 mg/L), oil and grease (8370 mg/L), and total suspended solids (19 020 mg/L).5 It is well-known that if the untreated waste is released into the environment, significant environmental issues will arise because of its accumulation in soil and water.6 Currently, a number of studies have been conducted by different researchers on the “zero waste” concept, considering waste avoidance and reduction technology as prime challenges rather than developing new treatment technology.7 Some studies have demonstrated that POME can be reused as an appropriate fermentation media for growing various microorganisms such as Penicillium chrysogenum (for penicillin production), Rhodobacter sphaeroides (for biohydrogen production), and Aspergillus terreus (for protease production) because it contains high levels of organic acids, carbohydrates, lipids, minerals, protein, and nitrogen.1,8 It has been hypothesized that purple nonsulfur (PNS) bacteria could be used to treat POME because of some distinctive advantages of PNS. Although PNS bacteria are frequently used in biohydrogen production,9,10 the application of PNS to treat many high-pollutant load wastewaters, namely soybean,11 olive,12 dairy,13 and brewery wastewater,14 have also been reported to be promising for reducing COD levels. Among all PNS bacteria, Rhodospirillum and Rhodoferax are known for their roles in carbon and nitrogen metabolism and are frequently found in sewage, © 2014 American Chemical Society

Received: Revised: Accepted: Published: 14921

May 1, 2014 July 31, 2014 August 27, 2014 August 27, 2014 dx.doi.org/10.1021/ie501798f | Ind. Eng. Chem. Res. 2014, 53, 14921−14931

Industrial & Engineering Chemistry Research

Article

2.3. Media Preparation. Prior to the growth study, the bacteria culture was inoculated under illumination (4000 lx) and anaerobic conditions for 24 h. The culture medium used in this study consisted of (1 L) KH2PO4, 0.5 g; K2HPO4,1 g; MgSO4· 7H2O, 0.5 g; NaCl, 0.4 g; DL-malic acid, 1.0 g; sodium glutamate, 1.8 g; CaCl2·2H2O, 0.05 g; yeast extract, 10 g; ferric citrate (0.1 w/v%), 5 mL; trace element solution, 1 mL; vitamin solution, 1 mL; and HCl (37%), 0.68 mL. The trace element solution (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. The vitamin solution (1 L) contained thiamin, 500 μg; niacin, 500 μg; and biotin, 15 μg. During the fermentation experiments, three different sets of growth media were introduced, raw POME (G1), raw PPME (G8), and different dilutions of POME (G2−G7). A culture medium with POME content lower than 25% (v/v) was not attempted because the initial investigation determined that this concentration did not promote R. sphaeroides NCIMB8253 growth. POME dilution was performed using either distilled water (G2−G4) or PPME (G5−G7) as shown in Table 2. For all media, the pH was adjusted to pH 7 by the addition of 5 mol/L NaOH solution before the sterilization treatment (121 °C for 15 min) to support bacterial growth.13,14 2.4. Growth Study Procedure. The bacterial growth study experiments were performed in 100 mL Schott bottles. Illumination was provided by fluorescent lamps and was maintained at approximately 4000 lx on the outer surface of the bottle. The light illumination was measured as an average of illumination on the front and back surfaces of the Schott bottle. The temperature of the medium was maintained at 30 °C. For all experimental sets, the medium in the Schott bottle was purged with pure argon gas to create an anaerobic environment. After the sample was purged, 10% (v/v) inoculum of preactivated bacteria from the modified growth medium was transferred into a Schott bottle containing wastewater as a basal medium (Table 2). All growth media were not supplemented with any additional nutrients. 2.5. Analytical Methods. Sampling of bacterial growth was performed every 12 h during the first 3 days of the experiment, followed by 24 h sampling until the end of the experiment. For each sample, bacterial growth was monitored for pH, number of colony-forming-units (CFU), and total carbohydrate concentration. The CFU quantification was performed by spreading the broth on an agar Petri dish. Agar medium consisted of (1 L) yeast, 2.5 g; peptone, 2.5 g; and bacteriological agar, 15 g. The observation of substrate consumption was measured by the change in total carbohydrate concentration during the growth study period. These concentrations were measured by using a GENESYS 10 UV spectrophotometer (Thermo Fisher Scientific Inc., U.S.) at 490 nm via phenol-sulfuric acid method.18 Organic acids were analyzed using a high-performance liquid chromatography (Agilent HPLC model 1200 series, U.S.) equipped with a UV-DAD detector, wavelength/window 210/8 nm, reference wavelength 360/80 nm, fitted with an Agilent Zorbax SB-Aq column (4.6 mm × 150 mm × 5 μm) using 20 mM aqueous phosphate buffer pH 2.0/acetonitrile in 99:1 (v/v) as the mobile phase. Furthermore, the quality of the treated wastewater was measured by COD removal. The COD values were analyzed using the reactor digestion method (HACH Method 8000) and measured spectrophotometrically using a HACH DR2800. Total COD (CODtotal) was measured from the wastewater without undergoing filtration prior to analysis, while soluble

Simultaneous treatment of combined wastewater is a relatively novel treatment scheme because it has not been widely applied and studied. However, an increasing trend toward applying combined wastewater treatment has begun to occur regionally. For example, a combined treatment of industrial and domestic wastewaters has been reported in Egypt.16 The complexity of each industrial wastewater may result in the inability to use biological processes to treat the combined wastewaters. Because the efficiency of wastewater treatment is associated with microbial growth,14 growth kinetic analyses using combined wastewater as a substrate must be performed to predict the trend and growth of bacteria to improve the treatment process. Furthermore, it is important to examine essential parameters during the anaerobic bioconversion process, such as biomass yield and substrate utilization rate, which lead to higher wastewater treatment efficiency.17 In this study, the potential of combining POME and PPME as a growth medium for PNS bacteria was investigated and compared with raw POME, raw PPME, and a distilled waterdiluted system. Additionally, COD removal from the wastewater was also evaluated. A kinetic analysis examining bacterial growth for all investigated media was conducted to describe the growth of PNS bacteria during wastewater treatment by fitting the growth data into various growth models. Additionally, the relationships between bacterial growth and COD removal were predicted using a regression analysis.

2. MATERIAL AND METHODS 2.1. Collection of Wastewaters. POME and PPME were collected from the Seri Ulu Langat Palm Oil Mill Sdn. Bhd. Dengkil and Muda Paper Mills Sdn. Bhd. Kajang, respectively. The characteristics of these two effluents are shown in Table 1. Table 1. Characteristics of POME and PPMEa

a b

parameter

unit

POME

PPME

pH COD turbidity total suspended solids total carbohydrate total organic carbon total nitrogen C/N ratio total phenol color Cu Fe Zn Mn Mg Al

− mg/L NTU mg/L mg/L mg/L mg/L − mg/L − mg/L mg/L mg/L mg/L mg/L mg/L

4.3 ± 0.3 84,450 ± 19,500 67,500 ± 1,910 19,610 ± 7,900 27,500 ± 2,250 4,251 ± 112.70 650 ± 300.00 6.54 ± 3.43 n.d.b dark brown n.d.b 70.7 ± 1.65 7.53 ± 1.07 6.47 ± 1.43 1,144 ± 7.00 334 ± 22.65

6.15 ± 1.3 2,716 ± 125 4,700 ± 141 841 ± 878 353.99 ± 18.94 473.3 ± 18.56 3.70 ± 3.65 128 ± 22 5.25 ± 0.35 pale yellow n.d.b 0.50 ± 0.01 0.12 ± 0.01 0.09 ± 0.01 3.28 ± 1.08 33.43 ± 1.10

POME, palm oil mill effluent; PPME, pulp and paper mill effluent. n.d., not detectable.

2.2. PNS Bacteria. The PNS bacteria used in this study were R. sphaeroides NCIMB8253. These bacteria were obtained from the Faculty of Engineering and Built Environment, National University of Malaysia. The bacteria were cultivated on agar slants and incubated for 24 h at 30 °C with 4000 lx illumination (WalkLAB Digital Lux meter, Trans Instruments (S) Pte. Ltd., Singapore). The bacteria were then maintained and preserved at 4 °C. 14922

dx.doi.org/10.1021/ie501798f | Ind. Eng. Chem. Res. 2014, 53, 14921−14931


Article

Table 2. Growth Media Formulationa mediumc

POME concentration (v/v)

diluting agent

turbidity (NTU)

CODtotal (mg/L)

CODsoluble (mg/L)

G1 G2 G3 G4 G5 G6 G7 G8b

100% 25% 62.5% 75% 25% 62.5% 75% 0%

− distilled water distilled water distilled water PPME PPME PPME −

67 500 ± 1 910 6 800 ± 396 11 800 ± 849 16 500 ± 142 8 000 ± 248 12 900 ± 707 16 200 ± 778 4 700 ± 141

84 450 ± 19 500 44 100 ± 849 54 050 ± 212 58 750 ± 750 58 750 ± 1 768 62 250 ± 778 71 100 ± 1 414 2 716 ± 125

72 785 ± 2355 33 940 ± 1 640 43 060 ± 1 499 55 775 ± 2 015 45 250 ± 2 051 50 825 ± 601 62 800 ± 2 546 1903 ± 60

a

POME, palm oil mill effluent; PPME, pulp and paper mill effluent. bG8 medium contained 100% raw PPME. cNo additional nutrients were added into the medium.

Figure 1. Variation of CFU number in (a) POME and distilled water-diluted media and (b) PPME and PPME-diluted media during R. sphaeroides NCIMB 8253 growth (n = 3).

2.6. Growth Models. Bacterial growth curves were obtained by plotting the CFU number versus time for all types of growth media. In this study, R. sphaeroides NCIMB 8253 growth curves were fitted into three different growth model equationsthe

COD (CODsoluble) was contained in the supernatant (or the soluble portion) of the wastewater after centrifugation. Experimental data are expressed as the average of the values obtained from three replications of each experimental set. 14923



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Figure 2. pH changes in (a) POME and distilled water-diluted media and (b) PPME and PPME-diluted media during R. sphaeroides NCIMB 8253 growth (n = 3).

The Weibull model21 is described as

logistic, Gompertz, and Weibull modelsusing a software program (OriginPro 8.5, USA). The logistic model equation has been widely suggested to be the best descriptive model for bacterial growth characteristics. The equation for the logistic model19 is X max X= ⎡ ⎤ X max ⎢⎣1 + exp( −kct ) Xo − 1 ⎥⎦ (1)

(

X = X max − (X max − Xo) exp[−(kt )δ ]

where X is the cell concentration in CFU (CFU/mL) at time t (h), Xmax the maximum cell concentration (CFU/ml), Xo the minimum cell concentration (CFU/ml), k the specific growth rate (h−1), and δ the inflection point parameter.

)

3. RESULTS AND DISCUSSION Observations on changes in pH, CFU number, and total carbohydrate concentration were conducted to investigate the growth behavior of R. sphaeroides NCIMB 8253 in various wastewater media up to 144 h. Furthermore, a kinetics analysis was performed to predict bacterial growth. COD removal was determined to justify wastewater treatment efficiency using PNS bacteria. 3.1. Growth Variation in Terms of CFU Number. In this study, cell growth was expressed as the CFU number rather than the commonly used dry cell weight. Because of the high amount of total suspended solids present in the POME (Table 1), the measurement of dry cell weight was inaccurate for representing

where X is the cell concentration in CFU (CFU/mL), t time (h), kc the apparent specific growth rate (h−1), Xo the initial cell concentration (CFU/mL), and X max the maximum cell concentration (CFU/mL). The equation for the modified Gompertz model20 is ⎧ ⎡R e ⎤⎫ X = X max exp⎨−exp⎢ max (λ − t ) + 1⎥⎬ ⎣ X max ⎦⎭ ⎩ ⎪

⎪

⎪

⎪

(3)

(2)

where X is the cell concentration in CFU (CFU/mL) at time t (h), Rmax the maximum cell concentration growth rate (CFU/ (ml.h)), λ the lag time of cell growth (h), and Xmax the maximum cell concentration (CFU/ml). 14924



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Table 3. Organic Acid Measurements acetic acid (g/L)

butyric acid (g/L)

formic acid (g/L)

malic acid (g/L)

medium

initial

final

initial

final

initial

final

initial

final

G1 G2 G3 G4 G5 G6 G7 G8

110.91 ± 0.14 41.20 ± 2.24 66.06 ± 0.63 83.18 ± 1.82 35.20 ± 0.72 55.47 ± 1.34 76.47 ± 0.44 13.24 ± 0.22

116.98 ± 7.07 4.71 ± 2.49 60.48 ± 0.74 83.54 ± 1.54 12.22 ± 0.59 56.45 ± 7.24 79.82 ± 3.9 5.99 ± 0.13

1.29 ± 0.1 2.87 ± 0.98 2.92 ± 0.8 3.75 ± 0.44 2.40 ± 0.75 2.79 ± 0.1 1.96 ± 0.09 3.70 ± 0.52

1.34 ± 0.25 2.00 ± 0.83 3.68 ± 0.52 11.00 ± 3.36 2.48 ± 0.62 9.71 ± 0.49 6.01 ± 0.27 2.26 ± 0.43

10.75 ± 0.41 11.10 ± 0.65 10.62 ± 0.02 12.89 ± 1.83 42.79 ± 1.89 12.59 ± 0.14 13.37 ± 0.49 6.67 ± 0.23

12.42 ± 3.27 7.81 ± 0.67 8.48 ± 0.74 7.95 ± 0.92 3.83 ± 0.01 10.69 ± 1 16.55 ± 2.34 4.61 ± 0.16

17.76 ± 0.28 7.36 ± 0.09 9.32 ± 0.01 13.03 ± 0.84 6.29 ± 0.08 6.70 ± 0.01 10.24 ± 0.31 2.42 ± 0.1

17.03 ± 8.67 12.48 ± 4.14 12.78 ± 1.66 27.00 ± 0.66 10.20 ± 5.39 12.79 ± 1.75 11.63 ± 2.71 2.52 ± 0.15

Figure 3. Substrate consumption yield variations in (a) POME and distilled water-diluted media and (b) PPME and PPME-diluted media during R. sphaeroides NCIMB 8253 growth (n = 3).

Figure 1 shows that after the addition of inoculum, there were no significant changes in bacterial cell growth during the lag phase because of the adaptation of R. sphaeroides to the new environment. The lag phase was observed over 0−24 h for all distilled water-diluted treatments (Figure 1a). The exponential phase began at 24 h, and CFU continued to increase throughout the rest of the growth period. However, as the turbidity of

bacterial growth. Therefore, CFU measurements were performed to obtain an accurate representation of bacterial growth in the effluent media. Figure 1a displays the variation of CFU for all treatments including raw POME (G1) and distilled waterdiluted treatments (G2−G4). Figure 1b shows the variation of CFU for raw PPME (G8) and PPME-diluted treatments (G5−G7). 14925



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Figure 4. Growth kinetics of R. sphaeroides NCIMB 8253 in (a) G1, (b) G2, (c) G3, (d) G4, (e) G5, (f) G6, and (g) G7 fitted with the logistic model (dotted curve), modified Gompertz model (dashed curve), and Weibull model (solid curve).

488 × 108 CFU/mL at 144 h, proving that dilution decreased the turbidity of POME, thus increasing its suitability for reuse as a medium during photofermentation. Better bacterial growth occurred because of the enhanced light penetration and a faster adaptation period for PNS bacteria in the diluted media.12,13

the medium increased with increased POME concentrations (Table 2), PNS bacterial growth was inhibited by decreased light penetration. Furthermore, the highest bacterial growth was observed in the most diluted medium, G2, containing 25% (v/v) POME. G2 generated the highest CFU number, 14926



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Table 4. Comparison of Experimental and Growth Model Parameters for Different POME Concentration Mediaa treatment parameters experiment Xo Xmax logistic model R2 Xo Xmax kc modified Gompertz model R2 Xmax Rmax λ Weibull model R2 Xo Xmax k δ a

G1

G2

G3

G4

G5

G6

G7

4.0 70.7

8.0 488.0

4.0 174.7

4.0 90.7

6.7 359.2

8.0 137.2

13.0 88.0

0.982 2.2 69.6 0.074

0.986 27.9 487.1 0.043

0.985 6.5 191.8 0.042

0.973 5.7 93.3 0.041

0.990 22.2 343.7 0.104

0.896 22.7 124.8 0.038

0.928 1.794 80.2 0.127

0.931 68.13 1.178 15.6

0.986 545.6 4.818 12.7

0.986 235.7 1.647 25.9

0.983 101.8 0.899 12.5

0.992 349.7 8.748 4.9

0.922 130.0 2.679 8.5

0.909 79.7 3.841 22.3

0.984 8.2 67.8 0.018 3.488

0.980 38.6 496.2 0.012 2.088

0.982 4.9 207.1 0.009 2.157

0.978 0 106.9 0.011 1.491

0.979 0 348.6 0.033 1.553

0.951 0 130.9 0.003 0.640

0.934 14.9 78.7 0.029 7.954

POME, palm oil mill effluent.

As depicted in Figure 1a, a significantly higher CFU number was also observed for the G2 medium during R. sphaeroides NCIMB 8253 growth. In comparison with G1, G3, and G4, the pH values remained at approximately pH 6.0 with a slight decrease after 84 h to pH 5.8. This pH decrease may be due to the lower light penetration in the treatments with higher POME concentrations, as indicated by higher turbidity values (Table 2). Lower light penetration results in Rhodobacter growth inhibition and acidic byproduct generation from the dark fermentative pathway, leading to the production of organic acids and decreased pH values.26 Organic acid production during fermentation was measured and is reported in Table 3. A substantial organic acid production was observed for the treatment with the highest turbidity (G1), which led to decreased pH during the treatment process (Figure 2a). In contrast, decreased pH was not observed for the treatment with the lowest turbidity (G8), in which the pH values for the raw PPME treatment (G8) fluctuated near the initial pH value of 7.0 throughout the growth period. For the G8 treatment, as the organic acids in the medium were consumed (except minor production of malic acid, Table 3), the pH level remained in the alkaline zone (Figure 2b). In general, as the dilution factor decreased, a more prominent pH reduction was observed because of organic acid production. For PPME-diluted treatments (G5−G7), V-shaped curves were obtained for all treatment sets. The pH continued to experience a decrease after inoculum addition up to 36 h; however, the pH increased to 5.7 and remained almost constant at 48 h and beyond. 3.3. Variation of Substrate Consumption Yield. Substrate consumption yield is defined as the difference in CFU number over the difference of total carbohydrate concentrations over a period of time. Total carbohydrates decreased in all treatments because of substrate consumption during R. sphaeroides NCIM8253 growth. Figure 3 displays the variation of substrate consumption yield versus time for the (a) distilled-water diluted and (b) PPME-diluted treatments up to 144 h fermentation. Figure 3a shows that the highest substrate consumption yield (15.8 × 1012 CFU/g carbohydrate) was obtained in the G2

A similar trend was also observed in the PPME-diluted medium (Figure 1b). The PPME-diluted medium containing 25% (v/v) POME, G5, had a significantly better bacterial growth profile compared with that of other media (G6−G8). The CFU values for the G5 medium demonstrated that the bacteria began their exponential phase at 12 h and reached stationary phase at 36 h, with a maximum CFU value of 360 × 108 CFU/mL. However, another study demonstrated that under aerobic conditions the growth of R. sphaeroides O.U.001 was not significantly influenced by the presence of light.22 Rather, agitation, pH and temperature were more important for enhancing R. sphaeroides O.U.001 growth in a modified growth medium. Conversely, CFU values in G8 decreased after 60 h of fermentation. Decreased CFU values indicated that the bacterial cells had deteriorated and were in the death phase.23 Furthermore, it was suspected that bacterial cell deterioration occurred in raw PPME or G8 because of the lack of nutrient content with a higher C/N ratio (Table 1), which was not favorable for bacterial growth. 3.2. Variation of pH in Different Growth Media. pH is one of the most important physical parameters that can be observed during bacterial growth. pH changes aid in the determination of the bacterial community’s metabolic activity in the medium and its effectiveness for electron transport, as well as influence the ionic forms of the active site of enzymes.24 Furthermore, the observation of pH values during bacterial growth can be used as an indication of cell growth because an increase in pH is generally associated with the evolution of cell growth.25 Figure 2 shows the pH variation during 144 h for all of the treatments investigated in this study, including raw POME (G1), raw PPME (G8), and diluted POME (G2−G7). For treatments that were diluted with distilled water (Figure 2a), pH values remained at 6.0 from 0 to 60 h of the growth period. The pH value began to increase afterward for the G2 treatment medium, whereas the other treatments experienced decreases in pH values. An increase in pH for G2 may indicate better bacterial growth compared with the other treatments, which had higher POME concentrations (G1, G3 and G4). 14927



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Figure 5. Changes in the total (■, ●) and soluble (□,○) COD levels in the G2 (squares) and G5 (circles) media during R. sphaeroides NCIMB 8253 growth (n = 3).

high kc values, 0.043 and 0.104 h−1, respectively. In comparison with another wastewater study, the kc value of G5 was comparable with R. sphaeroides O.U.001 in 2% olive mill wastewater medium (0.109 h−1).27 The R2 values for the logistic model fits were approximately 0.9, indicating excellent agreement between the experimental results and the model. Furthermore, in comparison with the experimental Xmax values, the logistic model predictions agreed well with the experimental results for all treatments. However, discrepancies between the experimental and predicted model Xo values were observed, which should be taken into consideration. When the modified Gompertz model was applied, a similar Xmax value trend was also observed; G2 and G5 yielded the highest Xmax values, 545.6 and 349.7 CFU/ml, respectively. Additionally, the G2 and G5 sets had the highest maximum cell concentration growth rates (Rmax), 4.818 and 8.748 CFU/(ml.h), respectively. This model enables the prediction of lag time, and the smallest lag time value (4.9 h) was in the G5 treatment because bacterial growth was observed during the early growth period (Figure 1b). The R2 values for the modified Gompertz model were greater than 0.9, indicating excellent agreement between the experimental results and the model. In comparison with the experimental Xmax values, the modified Gompertz model predictions deviated more from the experimental results for all treatments when compared with those of the logistic model. Using the Weibull model, similar Xmax value trends were also observed; G2 and G5 yielded the highest Xmax values, 496.2 and 348.6 CFU/ml, respectively. However, when specific growth rate (k) values are compared, G5 achieved the highest value at 0.033 h−1, whereas the k value for G2 was significantly lower at 0.012 h−1. Furthermore, the k values predicted by the Weibull model were significantly lower compared with those of the logistic model. Slightly lower R2 values were calculated for all treatment sets compared with those of the other two models, indicating marginally poor agreement between the experimental results and the Weibull model. Among these three models, it was concluded that the kinetics of actual PNS bacterial growth agreed best with the logistic model compared with the other two models based on their R2 and Xmax values.

treatment at the 84 h growth period because of the significant increase in CFU number during this period, as seen in Figure 1a. However, treatments with higher POME concentrations had relatively lower substrate consumption yields, indicating bacterial growth inhibition. This suppressed bacterial growth may occur because of the increased turbidity in these treatments, resulting in the inhibition of light penetration (Table 2). Furthermore, bacterial growth suppression may be associated with the presence of water-soluble antioxidants, phenolic acids, and flavonoids in the POME, which may inhibit the growth of microorganisms.1 Therefore, it is suggested that for POME to be reused effectively as a fermentation medium, POME dilution is necessary. A similar trend was also observed in PPME-diluted treatments (Figure 3b). The G5 treatment set achieved the high substrate consumption yield, 5.7 × 1012 CFU/g carbohydrate, at 60 h. Meanwhile, the higher POME concentration treatments (G6 and G7) produced lower substrate consumption yields. The highest substrate consumption yield (27.2 × 1012 CFU/g carbohydrate) was obtained in raw PPME (G8) at 24 h because only a very small amount of total carbohydrate was consumed by the bacteria. However, a drastic decrease of substrate consumption yield was observed in G8 after 60 h of treatment, followed by negative yields during 96−144 h, which occurred because of bacterial deterioration during this period. A lack of nutrients in the G8 medium was suspected of causing bacterial growth inhibition and bacterial structure deterioration at the end of the growth period, as shown in Figure 1b. 3.4. Kinetics Models. Kinetic growth curves were obtained by plotting the CFU number versus time for all types of growth media. R. sphaeroides NCIMB 8253 growth values were fitted to the logistic model (eq 1), modified Gompertz model (eq 2), and Weibull model (eq 3). Figure 4 shows the results of the kinetic model fitting into the CFU number data for the G1−G7 treatment sets. Data fittings were applied to verify the accuracy and the agreement of the model with the experimental results. The model parameters for each set of growth media are listed in Table 4. Using the logistic model, the highest Xmax value for the distilled water-diluted and PPME-diluted treatment sets were 487.1 and 343.7 CFU/ml, respectively. A similar trend was also observed for the specific growth rate results (kc). The treatments with the highest dilution factors (G2 and G5) generated considerably 14928



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3.5. Treatment of Combined Effluents Using R. sphaeroides NCIMB 8253. A reduction of total (CODtotal) and soluble (CODsoluble) COD in the effluent after the photofermentation process was expected because of organic matter consumption by the PNS bacteria. Further observation of COD removal was conducted in the most suitable growth medium that was diluted by different diluting agents (i.e., G2 and G5 medium). COD reductions throughout the growth period in the G2 and G5 media is depicted in Figure 5. Figure 5 shows a decreasing trend of CODtotal and CODsoluble in both media, indicating organic matter consumption by R. sphaeroides (Figure 3). A significant decrease in CODtotal in the G5 medium was observed at 12 h, corresponding well with the beginning of the exponential phase during bacterial growth, as shown in Figure 1b. COD reduction in the G5 medium began to slow after 36 h, indicating the beginning of the stationary phase (Figure 1b). A similar trend was also observed for CODsoluble throughout the growth period. When the fermentation time was accounted for, it was determined that CODtotal removal for the G2 and G5 media was comparable to that reported previous studies (Table 5). In this

Figure 6. Schematic diagram of the COD balance calculation.

Table 6. COD Balance for the Optimal Treatment Processes input COD (g COD/L) medium

COD influenta

G2

44.10

G5

58.75

Table 5. Comparison of COD removal wastewater

time of CODtotal PNS bacteriaa fermentation (h) removal (%) reference

olive mill R. sphaeroides wastewater O.U.001 dairy wastewater photosynthetic mixed culture chemical photosynthetic wastewater mixed culture dark fermenter R. capsulatus effluent DSM 170 G2 medium R. sphaeroides NCIMB 8253 G5 medium R. sphaeroides NCIMB 8253 a

54

30.2

12

24

16.27

28

24

12.76

28

240

68

29

144

50.1 ± 2.6 this study

144

52.9 ± 1.9 this study

output COD (g COD/L) COD effluentb acetic acid butyric acid formic acid malic acid other productse acetic acid butyric acid formic acid malic acid other productse

5.04 3.64 2.73 8.99 1.60 13.08 4.51 1.34 7.34 1.38

COD gasc

COD sludged

0.06

22.04

0.14

30.96

a

COD influent includes total COD of solids and colloidal particles. COD effluent includes total COD of organic materials and solids, calculated by eq 5. cCOD gas includes total COD of hydrogen gas produced, calculated by eq 5. Volumes of H2 obtained were 78.3 and 195.6 mL for G2 and G5 medium, respectively. CH4 and H2S were not detected. dCOD sludge includes total COD of solids and newly grown biomass. eOther products may include total COD of metabolites. b

From eq 4, the theoretical COD calculation can be summarized in eq 5 below.

PNS bacteria, purple nonsulfur bacteria.

⎛ g COD ⎞ 8(4n + a − 2b − 3d) Theoretical COD, ⎜ ⎟= (12n + a + 16b + 14d) ⎝ g CnHaOb Nd ⎠ (5)

study, 50−53% CODtotal removal was achieved during the 144 h photofermentation process. In a previous study, a photosynthetic mixed culture required 24 h to reduce CODtotal 16.27% and 12.76% in dairy and chemical wastewater, respectively.28 Additionally, another PNS bacteria, R. sphaeroides O.U.001, removed 30.2% CODtotal in olive mill wastewater during a 54 h process without the addition of iron or molybdenum.12 A higher CODtotal removal (68%) was reported during the fed-batch photofermentation of dark fermenter effluent using R. capsulatus DSM170.29 However, 10 days of operation and additions of both iron (0.102 mM) and molybdenum (0.165 μM) were required to achieve this removal amount.29 In this study, the COD balance was constructed based on the input and output of the COD value (Figure 6). Table 6 summarizes the COD balance for the G2 and G5 treatments. The biomass concentration and fermentative products were converted into COD concentration based on the theoretical oxygen demand for each compound.30 The theoretical amount of COD was calculated based on the following complete oxidation reaction of organic compounds.30 1 CnHaOb Nd + (4n + a − 2b − 3d)O2 4 1 → nCO2 + (a − 3d)H 2O + d NH3 (4) 2

Higher COD values in the COD effluent and COD sludge were obtained for the G5 medium compared with the G2 medium (Table 6). This primarily occurred because of the higher amount of residual acetic and butyric acid in the G5 effluent (Table 3), which were not readily consumed by R. sphaeroides NCIMB 8253 in this medium. Additionally, increased sludge generation is expected in the G5 medium because of the higher amount of total suspended solids in the combined effluents. Measured COD removal (total and soluble) is depicted versus the corresponding CFU number for the G2 and G5 media in Figure 7. A simple linear equation (y = ax + b) was fitted to these data to predict the relationship between these two parameters. Lu et al.14 reported that microbial growth could be associated with wastewater treatment efficiency. The present study determined that the CODtotal and CODsoluble removal in the G5 medium (Figure 7b) had stronger linear correlations with their CFU number, as evidenced by higher R2 values, 0.9088 and 0.9796, respectively. The present study also found lower R2 values, 0.7708 and 0.8002, for CODtotal and CODsoluble removals, respectively, for the G2 medium (Figure 7a). The reason for this result may be because the organic matter removal was 14929



Article

Figure 7. Linear correlation between CFU number and COD removal for (a) G2 and (b) G5.

could also be introduced as a final treatment step to further reclaim the treated effluent.

not solely dependent on Rhodobacter growth but was also possibly dependent on other enzymatic activities. For example, hydrogen or poly β-hydroxybutyrate production during the fermentation process could also cause organic matter consumption.31 From this study, the final COD values for the optimal treatment were 220 and 276.5 mg/L for the G2 and G5 media, respectively. In comparison with the Malaysian Environmental Quality Act 1974 Standard A discharge limit (50 mg/L),32 these final COD values are relatively higher than the allowable amount. Therefore, a post-treatment option such as a combination of coagulation−flocculation treatment using a natural coagulant and membrane separation process is recommended to further reduce COD levels. POME coagulation−flocculation treatment has been widely studied by several researchers. Recently, the performance of natural coagulants such as unmodified rice starch4,33 and Cassia obtusifolia seed gum34 have shown promising results for reducing COD levels and recovering sludge from the effluent. The recovered sludge could be further transformed into stabilized and matured organic fertilizer via vermitechnology.5 Then, an ultrafiltration membrane process35

4. CONCLUSIONS This study demonstrated that R. sphaeroides NCIMB 8253 were able to grow adequately in POME diluted with either water or PPME. A concentration of 25% (v/v) POME was recommended as the optimal concentration for encouraging PNS bacterial growth. A maximal CFU number of 488 × 108 and 360 × 108 CFU/mL was obtained for water-diluted and PPME-diluted media, respectively. In the raw POME medium (G1) with 67500 NTU, the growth of R. sphaeroides NCIMB8253 bacteria was strongly inhibited by inefficient light penetration, and the maximum CFU number was only 68 × 108 CFU/mL. Raw PPME medium (G8) did not support bacterial growth because of a lack of nutrients. The kinetic CFU number fittings demonstrated that the logistic model best fit the experimental data and was able to represent bacterial growth behavior in various media. Additionally, a moderate reduction in CODtotal and CODsoluble values observed in the G2 and G5 media indicated an additional advantage for using diluted POME as an alternative bacterial growth medium. 14930



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AUTHOR INFORMATION

Corresponding Author

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

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Monash University Malaysia for providing Pretty Mori Budiman and Jacqueline Xiao Wen Hay with PhD scholarships.

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REFERENCES

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Treatment and Reuse of Effluents from Palm Oil, Pulp, and Paper Mills

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