EFFECT OF FEEDING TIME OF VOLATILE FATTY ACIDS FROM ...

The 5th AUN/SEED-Net Regional Conference on Global Environment November 21st-22nd, 2012

EFFECT OF FEEDING TIME OF VOLATILE FATTY ACIDS FROM PALM OIL MILL EFFLUENT ON PRODUCTION of POLYHYDROXYALKANOATES BY Ralstonia eutropha JMP 134 IN BATCH FERMENTATION Martha Aznury1*, Azis Trianto1, Adi Pancoro2, Tjandra Setiadi1, 1

Department of Chemical Engineering Faculty of Industrial Technology, Institut Teknologi Bandung, Labtek X, Jl. Ganesha 10, Bandung 40132, Indonesia 2 School of Life Sciences and Technology, Institut Teknologi Bandung Labtek XI, Jl. Ganesha 10, Bandung 40132, Indonesia *Email : [email protected]

Abstract.Polyhydroxyalknoate (PHA) is a bioplastic from the group of polyester with physicochemical properties similar to polypropylene plastic from petroleum. This research aims to study the effect of carbon sources on PHA fermentation process performed by using Ralstonia eutropha JMP 134 in a batch bioreactor. Dynamics of on PHA production from carbon sources glucose as well as the influence of volatile fatty acids as a precursor were studied in this research. Fermentation operating condition using bioreactor 10 L maintained at a temperature of 30oC and pH of 7. The concentration of carbon source used is 40 g/L, and after 20th hours of fermentation added volatile fatty acids (VFAs) that serves as a precursor in the production of PHA. The results showed acetic acid, propionate acid, and butyric acid with concentrations are 2.79 g/L; 1.18 g/L, and 3.04 g/L, respectively. Effect of feeding time of VFAs of Ralstonia eutropha JMP 134 have differently result in batch fermentation. The result of feeding time of VFAs from POME at the 20th and 40th hour or at the 40th and 60th hour in a batch show the concentrations DCW and PHA are 2.38 g/L and 0.74 (g PHA/g DCW) or are 2.22 g/L and 0.41 (g PHA/g DCW), respectively. Effect of feeding time of VFAs at the 20th and 40th hours is higher than at the 40th and 60th hours. keywords: polyhydroxyalkanoate (PHA), palm oil mill effluent (POME), Ralstonia eutropha JMP 134, volatile fatty acids (VFAs)

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1

Introduction

One of the popular bioplastics is polyhydroxyalkanoates (PHA). The most popular type of PHA is a polyhydroxybutyrate (PHB) which can be either homopolymers or copolymers with acid valerate, P(HB-co-HV). PHB has physicochemical properties similar to polypropylene plastic (PP) which is produced from petroleum, it's just that PHB can decompose naturally produces carbon dioxide and water [1]. This makes the PHA to be a superior alternative as a substitute for petroleum-based plastics used today. PHA can be produced by several microorganisms, among others: Bacillus megaterium, Pseudomonas aeruginosa, and Ralstonia eutropha. PHA was first isolated and characterized from Bacillus megaterium in 1925 by M. Lemoigne [2] at the Institut Pasteur, Paris. Ralstonia eutropha is a Gram-negative that grows on a variety of carbon sources, including several glucose and volatile fatty acids (VFAs) [3]. The production cost of PHA is much higher compared to conventional plastics [4]. The main obstacle to mass production of PHB is located on the production cost issues are relatively expensive. One cause of the high cost of production is the cost of substrate used. Potentially to reduction cost of production can be established by using organic material from agricultural waste as much as possible [1]. Agricultural industrial wastewater is liquid waste generated from processing of agricultural industries such as palm oil and tapioca. Indonesia is the world largest producer of palm oil [5]. The palm oil extraction from the fresh fruit bunches (FFB) of palm involves a number of processing procedures: sterilization, stripping, digestion, pressing, classification, purification and vacuum drying for which large quantities of water required [6]. The process of one tonne FFB needs about 1.5 m3 of water, half of this amount ends up as palm oil mill effluent (POME) [ 6]. In the year 2011, the government is targeting production of FFB for about 35 tonnes/Ha/Y with an area of 7.8 million hectares of plantation, indicating more than 200 million tonnes of POME was generated from around 490 mills in Indonesia [7]. POME has a high organic content (more than 20,000 ppm BOD) and non-toxic that could be as a carbon source in the fermentation system [8]. There is a great potential to bioconvert POME to volatile fatty acids (VFAs), the followed by the recovery of acids for biosynthesis of PHA.On the other hand, POME substances are usually in complex forms that cannot be directly consumed by

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The 5th AUN/SEED-Net Regional Conference on Global Environment November 21st-22nd, 2012 PHA-producing species such as Ralstonia eutropha as a representative bacterium for PHA synthesis [9]. In a previous study, reported by Aznury [11] the production of PHA with Ralstonia eutropha JMP 134 used VFAs POME as a stimulator in batch fermentations. The study is that effect of feeding time of VFAs POME affected of DCW and PHA concentrations. In this paper, we present our result on effect of feeding time of VFAs POME on PHA production by Ralstonia eutropha JMP 134.

2 2.1 2.1.1

Materil and methods Material Microorganisms

Ralstonia eutropha JMP 134 from Joint Genome Institute USA is used throughout. The Culture was maintained on Lubriath broth agar at 5 oC and sub cultured monthly.

2.1.1

Media

A mineral salts medium consisted of: 2.0 g/L (NH 4) 2SO4: 2.0 g/L KH2PO4, 0.6 g/L Na2HPO4, 0.2 g/L MgSO4.7H2O; 20 mg/L CaCl2, 10 mL/L trace metal solution, 0.1 g/L yeast extract was used. Trace metal solution consisted of: 1.3 mg/L ZnSO4.7H2O; 0.2 mg/L FeSO4.7H2O, 0.6 mg/L (NH4) 6Mo7O24.4H2O and 0.6 mg/L H3BO3. Glucose was used as a source of carbon with a concentration of 40 g/L as a medium for inoculums development and production media. Glucose, yeast extract, and salt solution were sterilized separately at 121 oC and then mixed with the inoculation aseptically. As for the pH adjusted to about 7 using 2 N HCl or 2 N NaOH [12].

2.1.2 The source of carbon, nitrogen, and VFAs The medium used in the experiment were all the same, i.e 40 g/L as carbon source, with the initial volume of 5.2 L, and urea nitrogen from with a th th th th concentration of 2 g/L, then VFAs added at 20 and 40 hours or 40 and 60 hours with a volume of 1 L, respectively.

2.2

Methods

2.2.1 Anaerobic fermentation of POME POME fermented with active anaerobic microbial seed. Active microbial seeds are added to the oil palm industry waste water with a ratio of 1 L: 4 L in a batch bioreactor with a volume of 8 liters. Anaerobic fermentation is taken and replaced with POME with a ratio of 1 L: 1 L per day. Then, POME fermented has destilation to get VFAs.

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2.2.2 Studying the growth of Ralstonia eutropha JMP 134 with concentration and the effect of feeding time of synthetic VFAs . Mineral salt medium added with glucose concentration of 40 g/L was used to develop the inoculum. Microorganisms were cultivated at a shaker speed of agitation of 150 rpm, 30oC and pH of 7 at 20th hour in the 2 L erlenmeyer contained 1.5 L medium as above. Inoculum given 10% of the total media provided. Then every hour, the sample taken to study the growth of microbes on the phases of the development of Ralstonia eutropha JMP 134. During the fermentation VFAs were added and used from synthetic acid; acetic and propionate acid. Five experiments with culture medium were conducted to investigate the influence concentration of VFAs on the growth of Ralstonia eutropha JMP 134. Their concentrations of acetid and propionate acid varied from 0 g/L, 3.3 g/L and 0.6 g/L, 6.6 g/L and 1.2 g/L, 10 g/L and 1.5 g/L, 13.2 g/L and 2.4 g/L, respectively. On the other hand, the six experiments with the same conncentration of synthetic VFAs were 6.6 g/L acetid acid and 1.2 g/L propionate acid, that its were varied of feeding time of in the 0 th, 10th, 20th, 30th, and 40th hours.

2.2.3 Effect of feeding time of stimulator synthetic VFAs or VFAs from POME Cultivation was carried out in a 10 L fed-batch bioreactor with the aerobic condition, temperature of 30oC and pH of 7 at t = 0 until t = 20th hours . The concentration of glucose as a carbon source used was 40 g/L with the initial volume of 5.2 L medium, and 0.8 L of inoculums. Effect of feeding stimulator of VFAs from POME on PHA production was examined by carrying out batch fermentation. In run of cultivation, the feeding time strategies of VFAs as a stimulator was 20th and 40th hours in batch fermentation. The cultivations were carried out without pH control, aerobic condition, and temperature of 30oC. The research was conducted by using a substrate concentration of glucose 40 g/L. VFAs concentration consisting of acetic acid, propionate acid, butyric acid have 2.79 g/L, 1.18 g/L, and 3.04 g/L, respectively. Media production and inoculum cultivated at 150 rpm agitation speed, temperature 30oC (Water bath circular, JEIO MC-11, TECH JEIO Korea) and pH meter (Electronic JENCO LTD, USA) at pH 7 for 200 th hour in a bioreactor Biostat V type E10 with volume capacity of 10 L. Medium volume production has 5.2 L and 0.8 L of inoculum. VFAs of POME are feeding time in the logarithm phase of Ralstonia

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The 5th AUN/SEED-Net Regional Conference on Global Environment November 21st-22nd, 2012 eutropha JMP 134 on PHA production. VFAs are given by the volume of each 1 L VFAs at 20th and 40th hours or 40th and 60th hours to the batch fermentation. Synthetic VFAs were acetic acid (JT Baker), propionate acid (Merck), and butyric acid (Sigma-Aldrich) added in the fermentation include with concentrations 2.79 g/L; 1.18 g/L; and 3.04 g/L, respectively. VFAs were added at the 20th and 40th hours. After fermentation is complete, the cells containing PHA is separated from the fermentation medium by centrifugation. Supernatant was collected for analysis of glucose, acetic acid, propionate acid and butyric acid consumption by using HPLC (Waters, USA). Debris of cells used for analysis of DCW and PHA content. Cell debris in the wash with akuadest for 2 times and then dried at 50 oC for 2-3 days. After drying weighed DCW. PHA was using crotonic acid analysis. PHA dissolved in chloroform and concentrated H2SO4 was added to form acids that can be measured crotonic acid at spectrophotometer with a wavelength of 235 nm.

2.2.4 Nitrogen Analysis POME was also analyzed for total nitrogen content. Analysis of total nitrogen content using a Buchi (Büchi 412 scrubbers, Büchi 435 digestion unit, Büchi 339 distilation unit, Germany).

2.2.5 Biomass Analysis Cell growth was analyzed using optical density spectrophotometer (Genesys 10 UV-Visible, Rochester USA) in the culture medium at a wavelength of 600 nm. To determine cell dry weight, samples were centrifuged and the precipitate was dried in a hot air oven at 50oC for 3 days.

2.2.6 Analysis and VFAs The content of residual VFAs was determined and described in standard methods [13].

2.2.7 Analysis of PHAs Samples were collected at every 20 hour intervals during incubation and pH was measured using an Ecoscan Hand-held series pH meter (Eutech Instruments, Singapore). At 20th until 60th hours, 50 ml samples were collected to determine cell dry weights. In determining the concentration of PHA, biopolymer contained in the cells extracted with the addition of sodium hypochlorite and chloroform on the cell as has been described by Jacquel N.,et al [14]. PHA dissolved in chloroform was analyzed by concentration crotonic acid conducted by Slepecky [15].

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2.2.8 Experimental Set-Up The set-up consisted of two bench-scale reactors and a distillation equipment (Fig. 1). The acidogenic fermentation of POME was carried out in a batch reactor under anaerobic conditions. PHA-accumulating by Ralstonia eutropha JMP 134 was carried out in an aerobic batch fermentation. (1) Acidogenic fermentation

Distillation POME

Anaerobic fermentation

Fermented POME

Mineral medium Ralstonia eutropha Glucose

VFAs

Aerobic batch fermentation

(2) PHA production

PHA

Figure 1 Two-step PHA production process from POME by Ralstonia eutropha JMP 134.

3

Results and Discussions

The experiment was designed to investigate: the growth of Ralstonia eutropha JMP 134 with effect of feeding time of concentration synthetic VFAs. Effects of feeding time of VFAs from POME on PHA production by Ralstonia eutropha JMP 134.

3.1

Anaerobic fermentation of POME

Palm oil mill effluent after fermented to distilled to obtain pure compounds VFAs. The results of this distillation analyzed using HPLC (Waters, USA) to determine the type and consentration acidic compounds by comparing the standard of acetic acid (JT Baker), propionate acid (Merck), and butyric acid (Sigma-Aldrich). Figure 2 shows concentration of acetic acid, propionate acid and butyric acid from POME, fermented POME, and distillated POME.

706


3,50 3,00

asetic acid propionate acid butyric acid

g/L

2,50 2,00 1,50 1,00 0,50

0,00 POME

Fermented POME

Distillated POME

Figure 2 Comparison acetic acid, propionate acid, and butyric acid from POME, fermented POME, destilated POME

POME fermented anaerobic on VFAs production for 1 day and at pH 4.43. POME and fermented POME also contain other fatty acids such as citric acid, formic acid, and malonic acid, while for distillation POME had acetic acid, propionate acid and butyric acid. The results VFAs from POME had acetic acid, propionate acid and butyric acid that the concentration were 1.41 g/L; 0.72 g/L, and 0.29 g/L, respectively. While fermented POME produced acetic acid, propionate acid and butyric acid were 2.97 g/L; 1.34 g/L, and 3.17 g/L, respectively. Destilated POME produced acetic acid, propionate acid and butyric acid, were 2.79 g/L; 1.18 g/L, and 3.04 g/L, respectively. VFAs from destilated POME would used as percursor for effect of fedding time of Synthetic VFAs in batch fermentation at 20th and 40th (3.3.1).

3.2

The growth of Ralstonia eutropha JMP 134 in synthetic VFAs

Effect of feeding time and the differently of concentration synthetic VFAs on Ralstonia eutropha JMP 134 growth were respectively in the Figures 3 and 4.

3.2.1

Effect of feeding time of synthetic VFAs on Ralstonia eutropha JMP134 growth

In the Figure 3 shows the six cultivations had divided almost the same of 2 kinds of increase or decrease lines. The first decrease line was between in the 0th hour as same as in the 10th hour adding of synthetic VFAs. It can be argued that during in the initial of feeding time, the Ralstonia eutropha JMP 134 was stops or slow-down of metabolic rates than it show a death phase. The

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The 5th AUN/SEED-Net Regional Conference on Global Environment November 21st-22nd, 2012 metabolic activity was decrease because of synthetic VFAs as an inhibitor in cultivation on the feeding time of the 0 th hour or 10th hour adding. The second increase line had 4 lines that its were between of feeding time of synthetic VFAs in the 20th hour, 30th hour, 40th hour, and without VFAs. The main constraint on Ralstonia eutropha JMP 134 growth by the feeding time its was increase, that the culture had adapted to synthetic VFAs performance as a stimulator and show in the increase of DCW. Effect of feeding time of synthetic VFAs on Ralstonia eutropha JMP 134 growth had in the exponential phase. The results seems to indicate of feeding time of synthetic VFAs that did not used in the initial cultivation, because the Ralstonia eutropha JMP 134 need the carbon source from glucose for growth. The strategies of feeding time of VFAs have the advantage of keeping in the right time of the culture in the exponential phase, causing the performance of Ralstonia eutropha JMP 134 growth is increase. without VFAs

7

add VFAs in the 0th hour add VFAs in the 10th hour

6

add VFAs in the 20th hour add VFAs in the 30th hour

DCW (g/L)

5

add VFAs in the 40th hour

4 3 2 1 0 0

10

20

30

40

50

60

70

Fermentation time (hour)

Figure 3 Effect of feeding time of synthetic VFAs on Ralstonia eutropha JMP 134 growth as a function of fermentation time (hour) and DCW (g/L). For the cultivation: the concentration of glucose = 40 g/L, concentration of acetate acid and propionate acid were 6.6 g/L and 1.2 g/L, respectively, and batch reactor.

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3.2.2

Effect of concentration of synthetic VFAs on Ralstonia eutropha JMP 134 growth

In the beginning result of feeding time of synthetic VFAs were selected in the 20th hour. The continuous effect of feeding strategy of synthetic VFAs concentration on the growth of Ralstonia eutropha JMP 134 are investigated. The composition of the VFAs from acetate and propionate acids were based on the results of the anaerobic fermentation of palm oil mill effluent (POME) that it had of 6.6 g/L acetate acid and 1.2 g/L propionate acid [16]. Figure 4 shows that the dynamic on Ralstonia eutropha JMP 134 growth by carbon source used 40 g/L glucose and the effect of feeding concentration of VFAs from POME. For the control (without adding VFAs) and the low concentration of synthetic VFAs (3.3 g/L acetat acid and 0.6 g/L propionate acid) was increase without slowing down metabolit rate after adding VFAs. Then, in the medium and higher of the concentration of 6.6 g/L and 1.2 g/L, 10 g/L and 1.5 g/L, 13.2 g/L and 2.4 g/L acetate acid and propionate acid, respectively had in the adaptation phase after adding VFAs in the 25 th hour. After adaptation phase, the Ralstonia eutropha JMP 134 was going to growth. In Figure 4 shows the growth of Ralstonia eutropha JMP 134 on condition without VFAs and concentration VFAs were 3.3 g/L acetate acid and 0.6 g/L propionate acid that in the 60th hour had indication of the static phase due to a reduced carbon source. On the other hand, the concentration VFAs with 6.6 g/L acetate acid and 1.2 g/L propionate acid to shows increase in dry cell weight until 70th hours. As for the concentration of 12.3 g/L acetate acid and 2.4 g/L propionate acid shows the number of cells that it was slow-down of metabolic rates, seen by the number of dry cell weight decreased (Figure 4). Effect of high concentrations VFAs will be inhibitory or toxic thus may result in a low growth rate and PHA content [17]. These results indicate that the concentration of VFAs from POME were 6.6 g/L and 1.2 g/L, 10 g/L and 1.5 g/L acetate acid and propionate acid, respectively, that the concentrations were used to the next step for PHA productions. An interesting result was that the metabolic rates was going slow-down after adding of VFAs with the high concentration in a batch reactor. It was shown that the feeding strategy used a fed-batch reactor had the advantage of keeping at constant maximum rates rather than in a batch reactor.

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Without VFAs

7,00

acet.acid=3.3 g/L; pro.acid = 0.6g/L acet.acid=6.6 g/L; pro.acid = 1.2 g/L acet.acid=10 g/L; pro.acid = 1.5 g/L

6,00

acet.acid=13.2 g/L; pro.acid = 2.4 g/L

5,00

DCW(g/L)

4,00 3,00 2,00 1,00 0,00

0

10

20

30

40

50

60

70

fermentation time (hour)

Figure 4 The dynamic of Ralstonia eutropha JMP 134 growth as a function of fermentation time and volatile fatty acid synthetic variations (acetate and propionate acids

DCW in Glucose with VFAs (13,2 g/L acetid acid and 2,4 g/L propionate acid) had 2.67 g/L, 3.20 g/L, and 2.15 g/L, respectively at the 20th, 60th, and 70th hours. Effect of feeding high concentration VFAs of shows the number of cells undergoing death, seen by the number of DCW decreases. These data show effect of VFAs high concentrations will inhibit microbial growth or be toxic to microbial death. It is already described in the discussion as above. The number of protons that are added can be calculated between a mixture of weak acids based on the Henderson-Hasselbach equation.

pKa acetic acid = 4,76; BM = 60,05 pKa propionate acid = 4,87; BM = 74,06 [

]

√

[

]

[

]

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The 5th AUN/SEED-Net Regional Conference on Global Environment November 21st-22nd, 2012 To the concentration of weak acids undissociated be treated the same as the initial concentration. So that the calculation results in Table 1: Table 1

Substrat Run I Run II Run III Run IV

Calculation of number of protons in the cytoplasm Precursor Cells from Various concentrations of synthetic VFAs

Acetic acid (g/L) 3,3 6,6 10 13,2

Propionate acid (g/L) 0,6 1,2 1,5 2,4

Ka acetic acid

Ka propionate acid

1,74 x 10-5

1,35 x 10-5

[H+] Molar 1,03 x 10-3 1,46 x 10-3 1,78 x 10-3 2,06 x 10-3

Proton concentration in run I, II, and IV were 1.03 x 10-3 M, 1.46 x 10-3 M, and 2.06 x 10-3 M, respectively. These data show the number of protons is added to the IV run worth two times more than run I. High proton concentration means greater energy required to remove the proton from the cytoplasm to the cell medium. High acid concentration will inhibit cell growth. This was explained the decrease in the amount of DCW run IV. Mechanisms of high concentrations of VFAs would affect cell growth can be seen in Figure 3. Concentrations of DCW on run III had 2.67 g/L, 2.20 g/L, and 3.59 g/L, respectively, at the 20th, 25th, and 40th hour. Run III was added to the VFAs in 20th to 40th hour to show the growth of the static, undergoing a process of adaptation to a carbon source of precursor VFAs. DCW concentrations were 4.43 g/L and 5.71 g/L on the run II at the 50th to 60th hours. It showed a rapid growth because of Ralstonia eutropha JMP 134 are adapted to the carbon source as VFAs. The results were the addition of feeding time VFAs at the 40th hour. Studies continue to observe the PHA content on compositions and concentrations of the synthetic VFAs; acetic acid, propionate acid, and butyric acid were 2.79 g/L; 1.18 g/L, and 3.04 g/L, respectively. Synthetic VFAs added based on the results of fermentation anaerobic POME (3.1) and added at the 20th and 40th hours based on the discussion 3.2.1. Comparison of synthetic VFAs with medium volume production were 1L: 6L, respectively.

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3.3

Comparison of Synthetic VFAs or VFAs from POME on DCW and PHA Productions.

Batch Fermentation of Ralstonia eutropha JMP 134 was added synthetic VFAs and POME at the 20th and 40th hours. Synthetic VFAs are acetate, propionate, and butirate acids. The concentration of acetate, propionate, and butirate acids are 2.79 g/L, 1.18 g/L, and 3.04 g/L, respectively. The addition of precursor VFAs were based on the conclusions at 20th and 40th hours from the analysis results of the discussion 3.2.1 and also as compared at 40th and 60th hours. 3.3.1

Effect of fedding time of Synthetic VFAs in batch fermentation at 20th and 40th hours on DCW and PHA productions.

Fermentation Ralstonia eutropha JMP 134 shows the consumption of glucose, acetic acid, propionate acid and butyric acid in Figure 5. Fermentation of glucose consumption showed a initial concentration of was 40 g/L and decreased until consentration glucose was 18.38 g/L at 40th hour. Glucose and ammonium (limited quantities) consumed in large quantities during the growth phase cells [16]. VFAs were added to directly consum by bacteria at the 20th and 40th hours. Ralstonia eutropha JMP 134 ceased to consumption propionate acid at the 100th hour, while acetic acid consumption at the 140th, and butyric acid at 160th hour. While the concentration of glucose has been consumed by bacteria amounted to 0.24 g/L at the 160th hour. Initial concentration butyric acid had 1.69 g/L to 0.5 g/L during the fermentation at 40th to 80th hours. Butyric acid consumption was faster consumed than acetic acid and propionate acid by the cell. Effect of addition of VFAs as a precursor suggests that the ability of cells to consume each of the different acids.

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asetic acid propionic acid butyric acid glucose

2 1,8 1,6 1,4 1,2 1 0,8 0,6 0,4 0,2 0 0

45 40 35 30 25 20 15 10 5 0

glucose (g/L)

acetic , propionic, butyric acid (g/L)


20 40 60 80 100 120 140 160 180 200 time (h)

Figure 5 Effect of feeding time of precursor synthetic VFAs in batch fermentation at the 20th and 40th of the consumption of glucose, acetic acid, propionate acid, and butyric acid.

Acetic acid and propionate acid on the production of PHA activated by the same enzyme, acetate-CoA ligase (EC 6.2.1.1), in contrast with butyric acid that was activated by butyrate CoA ligase [18]. Butyric acid was the first consumed as compared to acetic acid and propionate acid. 3- hydroxybutyrate (3HB) units can be synthesized from butyric acid directly via acetoacetyl-CoA without its prior degradation to acetyl-CoA. Here both NADHdependent and NADPHdependent acetoacetyl-CoA reductases are involved , and β-ketothiolase is not involved [19]. VFAs difference consumed by the cells is also discussed [16], in which formic acid, acetic acid and propionate acid in the fermentation to produce PHA. Acetic acid and propionate acid was more quickly consumed to produce PHA, where as formic acid consumed less and after the consumption of acetic acid and propionate acid. Effect of synthetic VFAs concentrations were added to the cell cytoplasm was calculated with the Henderson-Hasselbach equation. Proton concentration in the cytoplasm of cells of the concentration of the added synthetic VFAs based on Henderson-Hasselbach equation is 1.24 x 10-3 Molar. Proton concentration was not going to cause bacterial cell death as described in the discussion 3.2.2.

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DCW (g/L), PHA(g/L)


4 3,5 3 2,5 2 1,5 1 0,5 0

DCW(g/L) PHA (g/L)

0

50

100

150

200

time (h) Figure 6 Effect of feeding time of synthetic VFAs at the 20th and 40th hour on DCW and PHA productions

Figure 6 shows the addition of precursor VFAs in batch fermentation on DCW and PHA production at the 20th and 40th hour. The concentration of DCW 0.29 g/L, 0.93 g/L, 0.81 g/L, and 0.88 g/L, respectively at the 20th, 40th, 60th, and 80th hours. Fermentation to continue to increase until had the higher value its was 3.52 g/L at 180th hour. PHA concentrations are determined by analysis crotonic acid in the adaptation phase to 60th hour PHA namely the amount of 0.1 g/L. Subsequently the cells increased as the length of time of fermentation. The highest concentration of PHAs was 1.04 g/L but the number of cells that have decreased 1.39 g/L at the time 200th hour. The results of PHA content of DCW was 0.75 (g PHA/g DCW) at the time 200th hour. 3.3.2

Effect of feeding time of precursor VFAs from POME at the 20th and 40th hours on DCW and PHA productions

VFAs from POME were added 1 liter in the batch fermentation at the 20th and 40th hours. Consumption of glucose, acetic acid, propionate acid, and butyric acid analyzed at the 0th to 200th hours. Figure 7 shows the cells to consume glucose and VFAs from POME. The concentration of glucose consumed by bacteria as source of growth at the 0th to 160th hours. Glucose concentration shows a very drastic decrease in the concentration from 20.77 g/L to 3 g/L at the 60th to 80th hours. The concentration of glucose decreased indicates that the cells require a source of the other substrates on PHA production. VFAs consumption were an after adaptation phase at the 60th and 80th hour. The increase of consumption VFAs were need cell on PHA production. VFAs

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acetic acid propionic acid butyric acid glucose

acetic, propionic, butyric acid (g/L)

3,5 3

2,5 2

1,5

1

0,5 0 0

45 40 35 30 25 20 15 10 5 0

glucose (g/L)

consumption ceased when glucose had also begun to run out can be seen at the 120th hour.

20 40 60 80 100 120 140 160 180 200 time (h)

Figure 7 Effect of feeding time of precursor VFAs from POME in batch fermentation at the 20th and 40th of the consumption of glucose, acetic acid, propionate acid, and butyric acid.

Figure 8 shows concentration of DCW and PHA increased during the first reflected the cell growth at the 20 th hour. VFAs from POME did not significantly affect the growth of bacteria due to the concentration of DCW at the 20th and 40th hours were 0.29 g/L and 0.4 g/L, respectively. VFAs had not innhibit cell on growth phase. While the highest number of DCW fermentation occurs in the 200th hour was 3.66 g/L. Graph the rate of cell growth has been through a phase when compared to static fermentation at the 160th, and 180th were 3.57 g/L, and 3.66 g/L, respectively. So the increase ranged only from 0.09 g. A small increase was indicates that the cell has experienced a static phase. Butyric acid was consumed at higher rates than the remaining organic acids, thereby being first exhausted.

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DCW (g/L), PHA (g/L)

4 3 2

DCW(g/L) PHA (g/L)

1 0

0

50

100

150

200

time (h) Figure 8 Effect of feeding time of precursor VFAs from POME at the 20th and 40th hours on production of DCW and PHA

The higest consentration and content of PHAs were 2.76 g/L and 0.75 (g PHA/g DCW) at the 200th hour. PHAs were also the rate of increase began to decline from from 160th to 200th hours and ranged from 0.28 to 0.35. PHAs content in the cell begins to decline or already depolymerization by cells for energy. The cells harvest to produce PHA at the 200th hour. 3.3.3

Effect of feeding time of precursor VFAs from POME at the 40th and 60th hours on DCW and PHAs productions

asetic acid propionic acid butyric acid glucose

acetic, propionic, butyric acid (g/L)

3,5 3

2,5 2

1,5 1

0,5 0 0

40 35 30 25 20 15 10 5 0

glucose (g/L)

Fermentation with feeding time of VFAs from POME were at the 40th and the 60th hours. Effect of feeding time of VFAs from POME were the rate of glucose and VFAs consumption. The study also looked at effects on the amount of DCW and PHA levels in the cell.

20 40 60 80 100 120 140 160 180 200 time (h)

Figure 9 Effect of feeding time of precursor VFAs from POME in batch at the 40th and 60th hours of the consumption of glucose, acetic acid, propionate acid, and butyric acid.

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DCW (g/L), PHA (g/L)

Figure 9 shows glucose consumption at the 20th and the 40th hours were 27.14 g/L to 2.98 g/L, respectively. The consumption glucose was indicating that cells was growing very fast. Additions to the VFAs at 40th and 60th hours the cell shown that consumed directly VFAs have decreased each time the fermentation. Glucose consumption began to cease when the fermentation to 140th hours. While the consumption of acetic acid, propionate acid and butyric acid ceased at the 180th, 100th, and 180th hours, respectively.

2,5 2 DCW (g/L) PHA (g/L)

1,5 1 0,5 0

0

50

100 time (h)

150

200

Figure 10 Effect of addition of precursor VFAs from POME in batch at the 40th and 60th hours on DCW and PHA productions

Figure 10 shows concentration of DCW and PHA when adding to the VFAS at 40th and 60th hours. Effect of time addition of VFAs was seen in the number of DCW just to increase ranged from 0.24 at the 80th to 100th hours. The increase DCW was 1.14 g/L at the 120th hour. While the highest number of DCW were 2.22 g/L at the 200th hour. The higest PHA was 0.92 g/L at the 200th hour. The level PHA increased of was 0.02 range at the 180th to 200th hours. The harvest in the fermentation was the 200th hour.

3.4

Comparison of yield cells and PHA production from synthetic precursors of VFAS or VFAS from POME

Comparison of the three variables of time and the addition of synthetic VFAs or VFAs from POME were yield cell, yield PHA, and PHA content (Table 2). Table 2 summarizes the VFAs were used of feeding time in the 20 th and 40th hours in PHA production was higher than from feeding time in the 40th and 60th hours.

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The 5th AUN/SEED-Net Regional Conference on Global Environment November 21st-22nd, 2012 Table 2

Comparison of yield cell and PHA production of synthetic VFAs or VFAs from POME added in Batch fermentation

Yield cell (Cmol biomassa/mol substract) Yield PHA(Cmol product/mol substract) PHA content (g PHA/g DCW)

Synthetic VFAs

VFAs20-40 POME

VFAs40-60 POME

0.09 0.07 0.74

0.10 0.08 0.75

0.05 0.03 0.41

VFAs20-40 , added VFAs at the 20th and 40th hour VFAs40-60 , added VFAs at 40th and 60th hour

The higher yield cell, yield PHA , and PHA content were precursor VFAs from POME at feeding time the 20th and 40th hours than the other two variables. Yield cell, yield PHA, and PHA content were 0.10 (Cmol biomassa/mol substract), 0.08 (Cmol product/mol substract), and 0.75 (g PHA/g DCW), respectively. While the lowest yield cell, yield PHA, and PHA content were fermentation with VFAs from POME at the 40th and 60th hours. Yield cell, yield PHA, and PHA content were 0.05 (Cmol biomassa/mol substract), 0.03 (Cmol product/mol substract), and 0.41 (g PHA/g DCW), respectively. Hassan, et al [20] conducted fermentation POME with Rhodobacter sphaeroides in batch anaerobic during 3000 hours to get the content of PHA at 0.6 (g PHA/g DCW). Effect of feeding time of synthetic VFAs or VFAs from POME was significantly in yield and PHA content when precusor was fed at the 20 th and 40th hours.

4

Conclusions

PHA content obtained depending on the substrate as a precursor VFAs, VFAs feeding and duration time of fermentation would affect the amount of PHA content. Mode of cultivation will also determine the number of cells that grow and will affect the content of PHA. The effect of fed VFAs into a batch fermentation was investigated in detail with respect to its addition time and initial concentration as its was growth or inhibit cell to growth. Other hand, PHA formed from secondary metabolism so it would be better used by the fed batch methodology. Fed batch will increase the PHA content also to prevent the death of Ralstonia eutropha JMP 134. Future investigations need to focus in fed batch fermentation and feeding strategies at appropriate time interval.

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[2]

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[5]

[6]

[7]

[8] [9]

[10]

[11] [12]

[13]

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