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Chemical Industry & Chemical Engineering Quarterly www.ache.org.rs/CICEQ Chem. Ind. Chem. Eng. Q. 23 (4) 507−514 (2017) CI&CEQ

DUNG VAN NGUYEN FED-BATCH FERMENTATION OF NIPA SAP HARIFARA TO ACETIC ACID BY thermoacetica RABEMANOLONTSOA (f. thermoaceticum) SHIRO SAKA

Department of Socio-Environ- Article Highlights mental Energy Science, Graduate • Acetic acid could be produced from the underutilized nipa sap using Moorella thermo- School of Energy Science, Kyoto acetica University, Yoshida-honmachi, • High substrate concentration in batch fermentation caused low conversion efficiency • Sakyo-ku, Kyoto, Japan Fed-batch fermentation provided over 42.6 g/L of acetic acid with high conversion efficiency of 87% • High feeding rate improved acetic acid productivity as compared with the low feeding SCIENTIFIC PAPER rate • The current method shows much higher efficiency than the traditional vinegar process UDC 633.8:58:663:547.292

Abstract An efficient process for conversion of nipa sap to acetic acid was developed. Nipa sap was hydrolyzed with invertase and provided glucose as well as fructose as main sugars. Batch fermentation of glucose and fructose was inadequate with increased substrate concentration. By contrast, fed-batch technique on hydro- lyzed nipa sap with high feeding rate drastically increased acetic acid concen- tration and productivity to be 42.6 g/L and 0.18 g/(L/h), respectively. All the sugars in hydrolyzed nipa sap were consumed, with acetic acid yield of 0.87 g/g sugar. Overall, nipa sap as hydrolyzed with invertase was efficiently fermented to acetic acid, which is a valuable chemical and a potential biorefinery intermediate. Keywords: nipa sap, fed-batch fermentation, , acetic acid, biorefinery.

Acetic acid is an important chemical widely used ments such as coastal areas, river estuaries and as a solvent [1], food [2], or as a precursor for the mangrove forests with little care and investment synthesis of paints, plastics, fibers [3], deicers [4] and [11,12]. By tapping the stalk of nipa palm, sugar-rich pharmaceutical products [5]. Recently, acetic acid has sap can be obtained daily with high yield of around been explored for biodiesel [6] and for bioethanol 1.3 L/(palm/day) and average sugar content of 16.4% production after its hydrogenation over a metal [13]. Nipa palm can provide sap all year long and its catalyst [7-10]. Due to these increasing interests in annual productivity is estimated to be 169,000 biorefineries, acetic acid production through biological L/(ha/year) [13], equivalent to 181 t/(ha/year) [14], which processes has received considerable attention. Cost- is higher than sugarcane juice, 70 t/(ha/year) [15]. effective acetic acid production necessitates As reported by Tamunaidu and Saka [14], competitive and available feedstocks. chemical components of nipa sap include sucrose, Nipa (Nypa fruticans) is an abundant palm spe- glucose, fructose, and minor amounts of organic and cies that can naturally grow in brackish water environ- inorganic compounds. Although these constituents can be used for various purposes, the sap is collected

limitedly by local communities for their traditional use Correspondence: S. Saka, Department of Socio-Environmental Energy Science, Graduate School of Energy Science, Kyoto without any industrial applications [12,16]. The under- University, Yoshida-honmachi, Sakyo-ku, Kyoto 606-8501, Japan. utilized nipa sap could, thus, be a good raw material E-mail: [email protected] for acetic acid production. Paper received: 3 January, 2017 Paper revised: 26 January, 2017 Bio-derived acetic acid can be obtained from dif- Paper accepted: 29 January, 2017 ferent sugars through vinegar production using yeasts https://doi.org/10.2298/CICEQ170103003N and Acetobacters [17]. However, the conversion effi-

507 D. VAN NGUYEN et al.: FED-BATCH FERMENTATION OF NIPA SAP… Chem. Ind. Chem. Eng. Q. 23 (4) 507−514 (2017) ciency of sugars to acetic acid is as low as 27% [18] Hydrolysis of nipa sap due partly to CO2 emission [3] as can be seen in the Sucrose in nipa sap was hydrolyzed to glucose following equation: and fructose by 3.92 vol.% invertase solution at room

2CO2 2O2 temperature for 30 min. C H O 2C H OH 2CH COOH 6 12 6 2 5 3 Batch fermentation of standard sugars 2H2O Batch fermentations using glucose and fructose To avoid carbon loss during fermentation of nipa as carbon sources were first studied to investigate the sap, a novel process involving hydrolysis of the effects of high substrate concentration on free cells of sugars in nipa sap and subsequent fermentation with M. thermoacetica. For this purpose, a standard sugar Moorella thermoacetica has been developed [19]. The solution containing 100 g/L each of glucose and fruc- process does not release CO2 during fermentation tose was used. Fermentation was accomplished in and most carbon atoms in the sugar can be converted 500 mL DPC-2A fermenters. The composition and to acetic acid according to the following reaction preparation of nutrients in the fermentation medium [3,20]: are detailed by Rabemanolontsoa et al. [24]. The nutrients include 5 g/L yeast extract, 0.1 g/L cys- C6H12O6 3CH3COOH teine·HCl·H2O, 1 g/L (NH4)2SO4, 0.25 g/L MgSO4·7H2O, Initial application of this novel process on nipa 0.04 g/L Fe(NH4)2(SO4)2·6H2O, 0.24 mg/L NiCl2·6H2O, sap provided 9.9 g/L of acetic acid from 10.2 g/L 0.29 mg/L ZnSO4·7H2O, 0.017 mg/L Na2SeO3 and substrates, corresponding to 98% conversion effici- 1 mg/L resazurin. ency of the sugars to acetic acid [19]. To be more The fermenters as well as the substrate and effective for industrial-scale production, it is important nutrient solutions were sterilized in an autoclave at to improve the product concentration. For that matter, 121 °C for 20 min. Then, they were moved to a glove substrate concentration should be increased. How- box which provided an anaerobic environment by ever, many microorganisms can be inhibited by high filling with N2 gas. A working volume of 200 mL was substrate concentration [21,22], but evidence of such obtained by pouring 20 mL of M. thermoacetica inoc- effects on M. thermoacetica is scarce. Thus, the first ulum, 20-50 mL of standard sugar solution, adequate objective of this study is to investigate the effects of volumes of nutrient solution and water into each of high substrate concentration on batch fermentation by the fermenters. M. thermoacetica. Then, fed-batch technique was Fermentations were performed anaerobically at explored to improve acetic acid concentration from 60 °C with a stirring rate of 300 rpm. The pH was hydrolyzed nipa sap. controlled at 6.5±0.1 by automatic titration with oxy- gen-free 2 M NaOH. To correct for the acetic acid EXPERIMENTAL produced from the nutrients, inoculum and invertase, fermentations without substrates were accomplished Materials under the same conditions. Samples were collected Nipa sap was collected from Sarawak, Malaysia at specific time intervals and were immediately frozen and stored in concentrated and frozen state. To pre- at –31 °C until analysis with high-performance liquid pare the substrate for fermentation, 200 g of the con- chromatography (HPLC). centrated nipa sap was diluted with Milli-Q water to a total volume of 1 L in order to reach a sugar concen- Fed-batch fermentation of hydrolyzed nipa sap tration similar to that of the natural sap. Fed-batch fermentation was started with similar Invertase solution (EC No.3.2.1.26) with a mini- nutrient concentrations as in batch fermentation and mal activity of 4 units/mL was obtained from Wako approximately 20 g/L sugars from 28 mL hydrolyzed Pure Chemical Industries Ltd., Osaka, Japan. M. ther- nipa sap. In addition, oxygen-free feeding medium moacetica (formerly Clostridium thermoaceticum) was separately prepared by dissolving solid nutrients

ATCC 39073 was purchased from the American Type to hydrolyzed nipa sap under N2 atmosphere. Culture Collection (Manassas, VA, USA) in freeze- As total sugar concentration in the fermentation -dried state. The procedures for revival of the micro- broth decreased, the medium containing substrate organism and subsequent inoculum preparation were and nutrients was fed stepwise by a peristaltic pump. conducted as described by Nakamura et al. [23]. Total sugar concentration in the broth was increased by around 10 g/L after each feeding. The nutrient con- centrations added for each feeding time, the ferment-

508 D. VAN NGUYEN et al.: FED-BATCH FERMENTATION OF NIPA SAP… Chem. Ind. Chem. Eng. Q. 23 (4) 507−514 (2017) ation temperature, stirring rate and pH control were pounds were detected. In fact, sucrose is regarded as similar to those for batch fermentation. Sampling was the primary sugar in original nipa sap [25]. During conducted just before and after feeding in order to collection and storage process, hexoses and lactic determine sugar and product concentrations. acid can be spontaneously formed by hydrolysis and fermentation [26]. Analyses

The concentrations of sucrose, glucose, fructose Table 1. Chemical composition of nipa sap before and after were determined by HPLC (Shimadzu, Kyoto, Japan) enzymatic hydrolysis; results are reported with standard devi- equipped with a RI detector and a Shodex sugar KS- ation: ≤ 0.2 g/L for sugars, ≤ 0.3 g/L for lactic acid, ≤ 0.4 g/L for 801 column (Showa Denko, Kanagawa, Japan). The ash and ≤ 1.3 g/L for total chemical compositions column was maintained at 80 °C and eluted with Milli- Concentration, g/L Chemical composition -Q water at a flow rate of 1 mL/min. The limit of det- Before hydrolysis After hydrolysis ection for this method is between 2.7 and 4.5 mg/L Sucrose 82.1 0.9 [24]. Ash content in the concentrated nipa sap was Glucose 29.8 72.2 determined by incineration at 600 °C for 2 h. Fructose 38.3 81.3 To analyze acid concentrations, an Aminex Lactic acid 1.6 1.6 HPX-87H column (Bio-rad, Hercules, CA, USA) was Ash 6.0 6.0 used at 45 °C with 5 mM aqueous H2SO4 solution as Total chemical 157.8 162.0 mobile phase, and the flow rate was set at 0.6 composition mL/min. Prior to injection into HPLC, samples were fil- tered through a 0.45 µm membrane to remove micro- In this process, after enzymatic treatment with organisms. The limit of detection of HPLC using this invertase, 99% of sucrose in nipa sap was hydro- column varied from 1.2 to 5.0 mg/L [24]. lyzed, increasing the concentrations of glucose and Growth of M. thermoacetica was determined by fructose to 72.2 and 81.3 g/L, respectively. The measuring the optical density (OD) using Shimazu obtained hydrolyzed nipa sap was used for acetic UV-Vis spectrophotometer (Kyoto, Japan) at 660 nm acid fermentation by M. thermoacetica. with water as reference. In order to evaluate the performance of the ferm- Batch fermentation entation techniques, conversion efficiency and acetic The capacity of batch fermentation to provide acid yield were respectively calculated according to high concentration of acetic acid was first explored Eqs. (1) and (2), while productivity was estimated with using glucose and fructose mixtures (1:1) as carbon Eq. (3): sources. Conversion efficiency () % = Figure 1a shows a comparison of acetic acid production from batch fermentations of 18.2, 28.9 and Acetic acid produced ()g /L (1) = 100 48.0 g/L of total sugar mixtures by M. thermoacetica. Total sugars added ()g /L In general, lag periods of about 24 h were observed, regardless of the substrate concentration. Acetic acid yield= Acetic acid production from 18.2 g/L sugars was Acetic acid produced () g = (2) faster than that from 28.9 and 48.0 g/L sugars Total sugars consumed () g between 24-72 h of fermentation. This might be exp- lained by the fact that high sugar concentration can Acetic acid productivity = cause slow initial cell growth for M. thermoacetica Acetic acid produced ()g /L (3) = which might lead to low acetic acid production rate in Fermentation time (h) the first 30 h of fermentation, as demonstrated by Witjitra et al. [27]. RESULTS AND DISCUSSION At 96 h fermentation, acetic acid concentrations from fermentations of 18.2, 28.9 and 48.0 g/L sugars Chemical composition and hydrolysis of nipa sap were similar to be 15.9, 13.8, 16.1 g/L, respectively. Table 1 shows the chemical composition of nipa Then, the fermentation of 18.2 g/L sugars almost sap before and after hydrolysis by invertase. The raw stopped because no more substrate remained in the nipa sap contained 82.1 g/L of sucrose, 29.8 g/L of broth. In contrast, the fermentations of 28.9 and 48.0 glucose and 38.3 g/L fructose. In addition, 1.6 g/L of g/L continued for final acetic acid concentrations of lactic acid and 6.0 g/L of inorganics as minor com- 24.3 and 25.9 g/L, respectively.

509 D. VAN NGUYEN et al.: FED-BATCH FERMENTATION OF NIPA SAP… Chem. Ind. Chem. Eng. Q. 23 (4) 507−514 (2017)

reased. Many reports indicated that high concentra- tion of the product might inhibit acetic acid production as well as cell growth [27-30]. Although Wang et al. [30] demonstrated that the maximum acetic acid toler- ance for M. thermoacetica is around 48 g/L, other stu- dies showed that acetic acid concentration as low as 10 g/L can cause a reduction in rates of both cell growth and acetic acid production [28]. Therefore, not only the substrate concentration, but also the format- ion of acetic acid in higher concentration could be a possible reason for the decrease in fermentation rate. Figure 1b compares the conversion efficiencies of the different sugar concentrations in function of time. Increasing substrate concentration sharply dec- reased conversion efficiency of sugars to acetic acid. This shows that high substrate concentration could not be effectively converted to acetic acid in batch fermentation. Fed-batch fermentation has potential to solve this problem. Choice of substrate concentration and feeding time for fed-batch fermentation Table 2 summarizes the results from batch fer- mentation of different substrate concentrations. Although 28.9 and 48.0 g/L provided higher acetic acid concentration than 18.2 g/L, their sugar con- Figure 1. Comparison of :a) sugar concentration (filled sumption could not reach 100%. In the fermentation symbols), acetic acid concentration (open symbols) and b) of 48.0 g/L sugars, 31% of the sugars still remained conversion efficiency during batch fermentations of glucose + after 240 h, 25.9 g/L of acetic acid was produced, fructose (1:1) in various concentrations by Moorella thermo- corresponding to a low conversion efficiency of 54%. acetica; square: 18.2 g/L; round: 28.9 g/L; triangle: 48.0 g/L of The conversion rate of sugars to acetic acid is total sugars (glucose + fructose). high with the lowest substrate concentration, as evi- denced by the data in Figure 1a and b. In more detail, As shown in Figure 1a, acetic acid production similar conversion efficiencies of 90 and 84% were rates for both fermentations of 28.9 and 48.0 g/L dec- achieved respectively for 18.2 and 28.9 g/L after 168 h reased gradually when acetic acid concentration inc- of fermentation. However, high efficiency of 87% was

Table 2. Summary of batch and fed-batch fermentations of standard sugars and hydrolyzed nipa sap to acetic acid by Moorella thermoacetica; Results are reported with standard deviation ≤ 0.1 g/L for sugars and acetic acid

Substrate feeding, g/L Acetic acid Ferment– Time Sugar con- Substrate Concen– Average pro- Conversion Yield ation mode Initial Total h sumption, % tration, g/L ductivity, g/(L/h) efficiency, % g/g Hydrolyzed nipa sapa Batch 10.1 10.1 119 100 9.9 0.08 98 0.98 Glucose + fructose Batch 18.2 18.2 96 100 15.9 0.17 87 0.87 – – 168 100 16.3 - 90 0.90 28.9 28.9 168 97 24.3 0.15 84 0.87 48.0 48.0 168 56 21.9 - 46 0.81 – – 240 69 25.9 0.11 54 0.79 Hydrolyzed nipa sap Fed-batch 19.3 49.0 (low 408 100 42.3 0.10 86 0.86 feeding rate) 22.2 49.0 (high 240 100 42.6 0.18 87 0.87

feeding rate) aNguyen et al. [19]

510 D. VAN NGUYEN et al.: FED-BATCH FERMENTATION OF NIPA SAP… Chem. Ind. Chem. Eng. Q. 23 (4) 507−514 (2017) obtained much faster at 96 h for 18.2 g/L. Fur- one (named high feeding rate) would add the nutri- thermore, average productivities for the fermentations ents and substrates during the exponential and stat- of 18.2, 28.9 and 48.0 g/L sugars were 0.17, 0.15 and ionary phases between 24 and 72 h fermentation. 0.11 g/(L/h), respectively as seen in Table 2. Rapid These strategies were compared for fed-batch ferm- conversion and high productivity are desirable to red- entation of nipa sap using M. thermoacetica. uce energy consumption during fed-batch ferment- Fed-batch fermentation ation. For these reasons, sugar concentrations near 18.2 g/L would be ideal for fed-batch fermentation. Fed-batch fermentation of hydrolyzed nipa sap To determine the proper feeding time, batch fer- was performed at an initial substrate concentration of mentation of 18.2 g/L sugars was studied in detail. around 20 g/L. Total sugars after feeding were regul- Figure 2 shows the fermentation kinetics of 18.2 g/L ated to remain below this concentration. According to sugars. A lag phase was observed in the first 24 h Wang et al. [30], the maximum acetic acid tolerance when very little acetic acid was produced at low cell of M. thermoacetica was 0.8 M (48 g/L). Therefore, to density. In the next 24 h, M. thermoacetica grew consume all sugars and to achieve high conversion rapidly and reached a maximum optical density of 2.0. efficiency, total sugars were added until 50 g/L only. Subsequently, the cell density remained at maximum Figure 3 shows substrate consumption and the in the stationary growth phase between 48-72 h fer- obtained acetic acid concentration at (a) low and (b) mentation. Meanwhile, acetic acid was produced sig- high feeding rates during fed-batch fermentation of nificantly, with rapid substrate consumption. At 96 h, hydrolyzed nipa sap. For both experiments, the sec- no sugars remained and acetic acid concentration ond feeding was implemented at 72 h but in the case reached 15.9 g/L. These results demonstrated that of (a) low feeding rate, the third and fourth feedings fast acetic acid production occurred with increasing were conducted when most substrates were con- and high cell density. sumed with sugar concentration below 2.0 g/L.

Figure 2. Batch fermentation profile of 9.0 g/L glucose + 9.2 g/L fructose by Moorella thermoacetica.

From 72 h to 96 h, the optical density of cells in the death phase showed 2.5-fold decrease from 2.0 to 0.8 when 1.5 g/L of sugars remained in the fermenter. As discussed in several reports [27,31-33], the lack of substrates and nutrients can cause cell death. There- fore, both substrate and nutrients would be added to the fermentation medium to maintain the activity of microorganism in fed-batch fermentation. Based on these fermentation kinetics, two stra- Figure 3. Effect of feeding rate on fed-batch fermentation of tegies are applicable for the feeding time. The first hydrolyzed nipa sap by Moorella thermoacetica. one (qualified as low feeding rate) would wait for most Short arrows indicate the feeding times. of the substrates to be consumed, while the second

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As shown in Figure 3a, total 49.0 g/L sugars in could remain during extended fermentation with many hydrolyzed nipa sap were fermented completely in feeding cycles. low feeding rate and acetic acid concentration reached 42.3 g/L at 408 h. This corresponds to a conversion efficiency of 86%. However, when little sugar remained in the fermentation medium from 120 to 144 h and from 216 to 264 h, substrate consumption rate decal- erated. Even after the third and fourth feedings, sub- strate consumption rate remained slow for nearly 24 h, resulting into a prolonged fermentation time of 408 h. To increase the speed of fed-batch fermentation, high feeding rate was explored. Figure 3b presents the results from fed-batch fermentation of hydrolyzed nipa sap with high feeding rate. The time intervals for the third and fourth feed- ings were reduced to be lower than 72 h. As a result, substrate consumption and acetic acid production maintained their high rates until total substrates were consumed completely at 240 h. From total of 49.0 g/L sugars in nipa sap, 42.6 g/L acetic acid was obtained with a conversion efficiency of 87%. This conversion efficiency could not reach 100% because part of the sugars might have been used for cell growth [27-29]. High feeding rate and low feeding rate showed similar final acetic acid concentration and conversion efficiency. However, fed-batch fermentation with high feeding rate reduced the total fermentation time and therefore improved 1.7-fold acetic acid productivity from 0.10 to 0.18 g/L/h, as compared to that with low Figure 4. Comparison of: a) acetic acid yield and b) acetic acid feeding rate. productivity of each feeding cycle in fed-batch fermentations of Comparison of fermentation performance during hydrolyzed nipa sap by Moorella thermoacetica with low and high feeding rates. each feeding cycle Figure 4 compares acetic acid yield (a) and ace- Figure 4b compares acetic acid productivities for tic acid productivity (b) of each feeding cycle in fed- each feeding cycle in fed-batch fermentations. High batch fermentations of hydrolyzed nipa sap. In the and low feeding rates showed similar productivities in first 3 feedings, the yield obtained with high feeding the first batch cycle. In later batch cycles, high feed- rate was lesser than the one with low feeding rate ing rate expressed much superior productivity due to because the microorganisms might not have enough its shorter fermentation time. Additionally, the lack of time to finish the metabolism until the end-product, substrates and nutrients during low feeding rate with and part of the consumed sugars might be in forms of extended fermentation time could have caused cell intracellular intermediate compounds. Nevertheless, death and reduced acetic acid production [27,31-33]. th in the 4 feeding, metabolism could have been com- Thus, the fast feeding of substrate and nutrients might pleted and high feeding rate slightly surpassed low have maintained viable cells and bioconversion of feeding rate in terms of yield. sugars into acetic acid. Either for low or high feeding rate, the yield was In general, both fed-batch fermentations with lower in the first feeding as compared with the sub- low and high feeding rates showed a similar trend of sequent ones. This may be due to the fact that the acetic acid productivities. Compared with the first microorganisms actively grew in the first feeding and batch, higher acetic acid productivities were obtained made more cells than in the next ones [34]. Acetic in the second batch because the first batch might acid yield for all feedings were above 0.68 g/g sugar. have had lag and exponential phases while the sec- This reveals that high acetic acid yield from nipa sap ond feeding might have been conducted in stationary phase with already high cell density. For this reason,

512 D. VAN NGUYEN et al.: FED-BATCH FERMENTATION OF NIPA SAP… Chem. Ind. Chem. Eng. Q. 23 (4) 507−514 (2017) acetic acid production occurred rapidly in the second REFERENCES batch. However, acetic acid productivities declined after the second feeding cycle. Since acetic acid can [1] N. Yoneda, S. Kusano, M. Yasui, P. Pujado, S. Wilcher, Appl. Catal. A: Gen. 221 (2001) 253-265 inhibit cell growth and acetic acid production [30,34], high acetic acid concentration accumulated from the [2] E. Lück, G.-W. von Rymon Lipinski, in Ullmann's Encyc- lopedia of Industrial Chemistry, Wiley-VCH Verlag GmbH fermentation might have caused lower acetic acid & Co. KGaA, Weinheim, 2000, pp. 671-692 productivities in the third and fourth feeding cycles. [3] M. Cheryan, in Encyclopedia of Microbiology, M. Comparison with traditional acetic acid production Schaechter, Ed., Academic Press, Oxford, 2009, pp. 144- –149 In the traditional vinegar production from nipa [4] D.F. Vitaliano, J. Pol. Anal. Manag 11 (1992) 397-418 sap in the Philippines, around 4.5–5.5% acetic acid [5] H. Cheung, R.S. Tanke, G.P. Torrence, in Ullmann's was produced from 15–22% nipa sap [18]. The pre- Encyclopedia of Industrial Chemistry, Wiley-VCH Verlag sent method achieved a similar acetic acid concen- GmbH & Co. KGaA, Weinheim, 2000, pp. 209-238 tration of 42.6 g/L. In addition, this study showed 87% [6] V. Béligon, L. Poughon, G. Christophe, A. Lebert, C. Lar- conversion efficiency which is 3.2-fold higher than the roche, P. Fontanille, Bioresour. Technol. 192 (2015) 582- one from the traditional method to be 27% [18]. In –591 other words, from 1 kg of nipa sap, the current pro- [7] S. Saka, H. Miyafuji, Y. Kohara, H. Kawamoto, (Shiro cess can provide 115 mL of acetic acid while only 36 Saka), US Patent 8409832 (2013) mL can be obtained from the traditional vinegar pro- [8] S. Saka, H. Kawamoto, M. Hisashi, Y. Kazuyoshi, M. duction. Consequently, this technology is ultra-effi- Shozo, N. Yousuke, S. Yutaka, N. Kenichi, N. Phaibo- cient to convert sugars in nipa sap to acetic acid. onsilpa, T. Shigeo, (Shiro Saka), Japan Patent 2010239913 (2010) CONCLUSIONS [9] H. Kawamoto, T. Fujii, Y. Ito, S. Saka, J. Jpn. Inst. Energy 95 (2016) 162-166 In this study, the conversion of high substrate [10] Y. Ito, H. Kawamoto, S. Saka, Fuel 178 (2016) 118-123 concentrations to acetic acid by M. thermoacetica [11] L. Hamilton, D. Murphy, Econ. Bot. 42 (1988) 206-213 was explored and showed that increasing sugar con- [12] P. Tamunaidu, N. Matsui, Y. Okimori, S. Saka, Biomass centration caused low conversion efficiency in batch Bioenerg. 52 (2013) 96-102 fermentation. Therefore, fed-batch fermentation tech- [13] A.E.A. Päivöke, Agric., Ecosyst. Environ. 13 (1985) 59-72 nique was investigated. As a result, fed-batch ferm- [14] P. Tamunaidu, S. Saka, in Zero-Carbon Energy Kyoto entation of total 49.0 g/L sugars in nipa sap could 2011, Springer, 2012, pp. 121-126 produce 42.6 g/L of acetic acid, which is similar to the [15] D. Rajagopal, S.E. Sexton, D. Roland-Holst, D. Zilber- vinegar production process but with much higher ace- man, Environ. Res. Lett. 2 (2007) 044004 tic acid yield of 0.87 g/g sugar. Thus, fed-batch ferm- [16] S. Udofia, E. Udo, Global J. Agric. Sci. 4 (2005) 33-40 entation of the underutilized nipa sap by M. thermo- [17] A. Paivoke, M.R. Adams, D.R. Twiddy, Process Biochem. acetica may be a promising route for efficient acetic 19 (1984) 84-87 acid production. [18] P. Lim-Castillo, in Authenticity in the Kitchen: Proceed- ings of the Oxford Symposium on Food and Cookery, R. Acknowledgments Hosking Ed., Oxford Symposium, UK, 2006, pp. 295-306 This work was supported by the Japan Science [19] D.V. Nguyen, P. Sethapokin, H. Rabemanolontsoa, E. Minami, H. Kawamoto, S. Saka, Int. J. Green Technol. 2 and Technology Agency (JST) under the Advanced (2016) 1-12 Low Carbon Technology Research and Development [20] H.L. Drake, A.S. Gößner, S.L. Daniel, Ann. N.Y. Acad. Program (ALCA), for which the authors are extremely Sci. 1125 (2008) 100-128 grateful. The first author would like to acknowledge [21] M.C. Reed, A. Lieb, H.F. Nijhout, Bioessays 32 (2010) the financial support received from JICA under the 422-429 AUN/SEED-Net Project for his PhD study. Further- [22] J. Hong, Biotechnol. Bioeng. 28 (1986) 1421-1431 more, the authors are thankful to Dr. Pramila Tamu- [23] Y. Nakamura, H. Miyafuji, H. Kawamoto, S. Saka, J. naidu from Malaysia-Japan International Institute of Wood Sci. 57 (2011) 331-337 Technology, Universiti Teknologi Malaysia for pro- [24] H. Rabemanolontsoa, K. Yoshimizu, S. Saka, J. Chem. viding the nipa sap samples used in this study. Technol. Biotechnol. 91 (2016) 1040-1047 [25] J. Van Die, P.M.L. Tammes, in Transport in Plants I: Phloem Transport, M.H. Zimmermann, J.A. Milburn Eds., Springer-Verlag, Berlin, 1975, pp. 196-222

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DUNG VAN NGUYEN DOLIVNA FERMENTACIJA SOKA NIPA PALME U HARIFARA SIRĆETNU KISELINU POMOĆU Moorella RABEMANOLONTSOA thermoacetica (f. Clostridium thermoaceticum) SHIRO SAKA

Department of Socio-Environ- Razvijen je efikasan proces za konverziju soka nipa palme u sirćetnu kiselinu. Ovaj biljni mental Energy Science, Graduate sok je hidrolizovan invertazom do glukoze i fruktozu. Šaržna fermentacija glukoze i fruk- School of Energy Science, Kyoto toze bila je neadekvatna pri povećanoj koncentraciji supstrata. Nasuprot tome, dolivna University, Yoshida-honmachi, fermentacija hidrolizovanog biljnog soka sa velikom brzinom dolivanja drastično je pove- Sakyo-ku, Kyoto, Japan ćala koncentraciju sirćetne kiseline i produktivnost na 42,6 g/l i 0,18 g/(l h), redom. Svi šećeri hidrolizovanog biljnog soka su potrošeni, sa prinosom sirćetne kiseline od 0,87 g/(g šećera). Sve u svemu, hidrolizovani sok nipa palme efikasno je fermentisan u sir- NAUČNI RAD ćetnu kiselinu, koja je vredna hemikalija i potencijalni biorafinerijski intermedijer.

Ključne reči: sok nipa palme, dolivna fermentacija, Moorella thermoacetica, sirćetna kiselina, biorefinerija.

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