Production of 5-Aminolevulinic Acid from Monosodium Glutamate Effluent by Halotolerant Photosynthetic Bacterium (Rhodobacter capsulatus SS3)

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1 Production of -Aminolevulinic Acid from Monosodium Glutamate Effluent by Halotolerant Photosynthetic Bacterium (Rhodobacter capsulatus SS) Amornrat Chaikritsadakarn,*, Poonsuk Prasertsan, and Piyarat Boonsawang The Joint Graduate School of Energy and Environment, King Mongkut s University of Technology Thonburi, Bangkok, Thailand Department of Industrial Biotechnology, Faculty of Agro-industry, Prince of Songkla University, Songkhla, 9, Thailand Abstract: Thai people consume high quantity of monosodium glutamate in which its production process generates large amount of effluent. Monosodium glutamate effluent () has the following average values; 9,, mg/l COD,. g/l reducing sugar,.7 % v/v total nitrogen,. g/l NaCl, and. % w/w glutamic acid. The organic matter content (COD value) of was about and folds higher than those of distillery stillage (DS) (, mg/l COD), and synthetic medium () (7,7 mg/l COD), respectively. These three media were used for growth and -aminolevulinic acid (ALA) production from halotolerant photosynthetic bacteria, Rhodobacter capsulatus SS, by cultivating under aerobic-dark condition ( rpm agitation rate) at 7 C for days. Results revealed that good growth was observed in and, but not in DS, giving the dry cell weight (DCW) of.9 g/l and.7 g/l, respectively after h cultivation. However, the strain produced high concentration of ALA ( µm) in medium whereas very low amount of ALA (. µm) was found in medium. To improve ALA production, was diluted to give the COD values of,, 7,, and, mg/l and NaCl concentration was adjusted to the level present in medium. The ALA production was highest using medium followed by using with COD of, mg/l (. µm). Further increase of ALA concentration to µm in was achieved by supplementation with organic acids, glycine, and mineral. This resulted in folds higher than (. µm). Keywords : -Aminolevulinic Acid (ALA), Photosynthetic Bacteria, Monosodium Glutamate Effluent (), Glucose-salt-yeast Extract Medium ().. INTRODUCTION Agro-industry plays a significant role in the economic growth of Thailand while being the source of high polluting wastewater, for example, monosodium glutamate (MSG) effluent and distillery stillage (distillage). MSG is the sodium salt of amino acid, glutamic acid, and is widely used as flavor enhancer. It is generally produced by microorganism using sugar beets or cassava as carbon source and its production process generates large amount of effluent. MSG effluent, often warm, has high salt content and has the average values as following;, mg/l COD,. g/l glutamic acid,.7 % total nitrogen and.. Experiment at Zhangjiagang MSG Plant in China for over a year demonstrated that the MSG effluent could be treated to contain less than, mg/l COD, < mg/l ammonia nitrogen and <, mg/l sulphate. Besides MSG effluent, distillate has the following average values (mg/l); BOD 7,, COD,, reducing sugar,, solid,, nitrogen,7 and average temperature of 9 C with. (Prasertsan and Suksawat, 9). Since distillate contains many yeast cells that can be the source of vitamins, it is considered to be the nourishing source for bacterial growth. Alternative approach for treatment of these wastewater is to use as substrate for growth of particular microorganisms which can produce valuable products. Photosynthetic bacteria is proposed to be the appropriate microorganism as it produces -aminolevulinic acid (ALA), which is used as herbicide, Corresponding author: amornratchaikritsadakarn@yahoo.com insecticide, growth stimulator, etc. It was reported that ALA from Rhodobacter sphaeroides can be produced in the effluent of swine waste from an anaerobic digestor and enhanced by adding glycine ( mm) plus levulinic acid (- mm) (Sasaki et al., 99). Besides environmental aspect, ALA could also be applied to medical field in photodynamic therapy for cancer treatment. The research work aims to utilize and treat agro-industrial wastes using photosynthetic bacteria to produce valuable product especially -aminolevulenic acid (ALA) and apply ALA as bioherbicide and growth promoting factor.. MATERIAL AND METHODS. Microorganism The halotolerant photosynthetic bacterium, Rhodobacter capsulatus SS, isolated by Tangprasittiparp (999), was used in this study. The strain was maintained on agar slant of glutamate-malate (GM) medium (Lascelles, 9) and kept at the culture collection of the Department of Industrial Biotechnology, Faculty of Agro-Industry, Prince of Songkla University.. Medium.. Glucose-salt-yeast extract () medium medium contained mm glucose and. g/l yeast extract with g/l NaCl (Madmarn, ). The was adjusted to.... Cultivation medium Agro-industrial wastewater used in this study were monosodium glutamate effluent () and distillery stillage 9

2 (DS) which were obtained from Thai Fermentation Industry Co., Ltd., Rajburi province and Nateechai Co., Ltd., Suratthanee province, respectively. The supernatant (by centrifugation with, rpm for min) of both wastewaters were used and adjusted to... Analytical methods.. Dry cell weight Cells are centrifuged at rpm for min, rinsed by distilled water twice and dried at C overnight or until constant weight is obtained (Sattayasmithstid, )... Extracellular ALA Extracellular ALA is determined by colorimetric method (Sasaki et al., 97). The supernatant of the cell culture is transferred to ml of acetate buffer ( M,.7) containing µl of acetylacetone. The solution is heated in boiling water for min, then quickly cool and react with ml of Ehrlich s reagent, visualizing purple-red color in minutes. The extracellular ALA is measured with spectrophotometer (OD ).. Method.. Formulation and selection of production media from monosodium glutamate effluent () and distillery stillage (DS)... Determination of chemical composition of waste media, DS and media were analyzed for COD (APHA, AWWA, and WPCF, 9), nitrogen (AOAC, 9), reducing sugar (Nelson, 9), glutamic acid (analyze by High Performance Liquid Chromatography method at Scientific Equipment Center, Mahidol University) and NaCl (analyze by Inductively Coupled Plasma Optical Emisssion Spectrometry method at Scientific Equipment Center, Prince of Songkla University).... Comparison on growth, extracellular ALA production and COD removal of R.capsulatus SS cultivating in wastewater and synthetic medium Cultivation of R.capsulatus SS was conducted in, DS, and medium and compared the bacterial growth and extracellular ALA production.... Formulation of waste media (formulated waste media) Formulation of waste media from these two sources of agro-industrial wastes were conducted based on the same concentration of COD, nitrogen, and NaCl as those of medium with the distilled-water addition, if necessary, of molasses, NH NO and NaCl respectively... Comparison on growth and ALA production of photosynthetic bacteria cultivating in the waste and formulated waste media... Inoculum preparation One loop of Rhodobacter capsulatus SS from agar slant was inoculated into ml of medium and cultivated under aerobic-dark condition ( rpm) at 7 C for h. Turbidity is measured by spectrophotometer at nm wavelength and adjusted to. using medium before using as the inoculum.... Cultivation in formulated waste media Starter (%) of Rhodobacter capsulatus SS in medium was added into ml of flask containing, and formulated media ( and DS media) and cultivated for days under aerobic-dark condition ( rpm) in incubated shaker at 7 C. Samples ( ml) were taken every h to measure for, growth (OD ), extracellular ALA. The formulated waste medium giving the highest ALA concentration was selected for further studies.... Effect of waste source (based on equal COD and nitrogen concentration of medium), COD concentration, total nitrogen concentration, and sodium content of the medium The effect of COD concentration, total nitrogen concentration and sodium content of wastewater ( and DS) and formulated waste medium were studied. Cultivation was conducted as describe above and compared the bacteria growth/ ALA production on the selected medium with different concentrations of COD, nitrogen and sodium content.... Effect of organic acid, glycine, mineral, and levulinic acid (LA) added in formulated medium To improve the production of ALA from wastes, supplementation of mm glycine, mm succinic acid,. g/l propionic acid, mm MgCl, and adding mm levulinic acid (LA) at h in synthetic medium, waste and formulated waste media. Results were compared with those media without supplemementation.. RESULTS AND DISSCUSSIONS. Formulation of waste media from monosodium glutamate effluent () and distillery stillage (DS).. Determination of chemical composition of waste media Glucose-salt-yeast extract medium () was the synthetic medium for growth of Rhodobacter capsulatus SS and Rhodobacter sphaeroides ES (Madmarn, ). Comparison on the characteristics of medium with those of monosodium glutamate effluent () and distillery stillage (DS) (Table ) indicated that had higher concentrations of COD (, mg/l), reducing sugar (RS) (. ug/ml), total nitrogen (.7 %v/v), and sodium content (. g/l) than DS (, mg/l COD, 7.9 µg/ml RS,.99 % nitrogen, and.7 g/l sodium content). In addition, also contained. % w/w glutamic acid. Both wastewaters were more acidic (. and., respectively) than the medium (.). 9

3 Table Characteristics of monosodium glutamate effluent (), distillery stillage (DS) and glucose-salt-yeast extract medium () Parameter DS. ±.. ±.. ±. COD (mg/l) 7,7 ± 9., ±., ±.7 Reducing sugar (µg/ml). ±.7. ±. 7.9 ±. Total nitrogen..7 ±.99 ± (%).. Sodium content (g/l). ±. *.7 ±. * Glutamic acid -. ** - content (% w/w) * Analysed at Scientific Equipment Center, Prince of Songkla University **Analysed at Scientific Equipment Center, Mahidol University - Not determined Dry cell weight (b) Dry cell weight Extracellular ALA DS.. Comparison on growth, extracellular ALA production and COD removal of photosynthetic bacterium (Rhodobacter capsulatus SS) cultivating in wastewater and synthetic medium Cultivation of Rhodobacter capsulatus SS in, DS, and medium for extracellular ALA production was conducted (Fig. ). During cultivation in all media, the values decreased from., 7., and.7 to.,., and., respectively. The strain grew better in medium than in and DS, giving the dry cell weights of.,., and. g/l, respectively. Extracellular ALA production was highest (. µm) in medium while only small amount (. µm) was produced in and not produced in DS. was a better source than DS as it contained higher organic matter, in term of COD values especially glutamic acid. The consumption of the organic substances resulting in the COD removal 9. % in which was higher than that in DS (. %) but lower than in (.7 %) (Table ). Between these two sources of wastewater, was selected for further studies. (c) Extracellular ALA Fig. Effect of different medium on (a), dry cell weight (b), and extracellular ALA production (c) during cultivation of Rhodobacter capsulatus SS under aerobic-dark condition on a shaker ( rpm) at 7 C for days. Table COD removal of,, and DS DS.. COD DS removal - Initial 7,7,, - Final,, 9, % COD removal (a) DS.. Formulation of waste media (formulated waste media) The two sources of agro-industrial wastewaters were formulated for production of ALA based on the same concentration of COD, nitrogen, and NaCl as those of medium in six formula were given as following : A : ( folds with COD value of 7, mg/l, COD of medium was 7,7 mg/l B : (. folds with total nitrogen value of.7 %v/v, total nitrogen of medium was. %v/v C : (add. g/l NaCl) with sodium content value of g/l, sodium content of medium was g/l D : DS (.9 folds with COD value of 7, mg/l, COD of medium was 7,7 mg/l E : DS (. folds with total nitrogen value of 9

4 .7 %v/v, total nitrogen of medium was. %v/v F : DS (add 9. g/l NaCl) with sodium content value of g/l, sodium content of medium was g/l.. Factors affecting ALA production from waste and formulated waste media of photosynthetic bacterium cultivating The effects of COD concentration, total nitrogen concentration, and sodium content of,, and DS were studied (Fig.,, ). Cultivation of R. capsulatus SS in medium as well as formulated and DS based on the same concentration of COD (formula A and D, respectively), nitrogen (formula B and E, respectively), and NaCl (formula C and F, respectively) as those of medium. The values of, formula C, D, E, and F decreased from.7, 7.,.7,., and. to.,.,.9,., and.9, respectively whereas the values of formula A and B increased from.99 and 7. to. and., respectively (Fig. a). Dry cell weight of formulated and DS..... A D B E.. Effect of waste source based on equal COD and nitrogen concentration of medium From the result, Formula A gave higher concentrations of dry cell weights (.97 g/l) and extracellular ALA (. µm) than formula D (. g/l and µm, respectively) (Fig. ) and R. capsulatus SS grew better in formula B than in formula E, giving higher concentrations of dry cell weights (.7 g/l) and extracellular ALA (.7 µm) than formula E (. g/l, µm, respectively) (Fig. ). Thus the best waste source was formula A; which based on the same COD concentration of medium since it gave highest dry cell weight and ALA production (.97 g/l and. µm, respectively). Extracellular ALA of formulated and DS A D B E Fig. Dry cell weight and extracellular ALA production from R. capsulatus SS cultivating in waste based on equal COD (formula A, D) and nitrogen concentration (formula B, E) of medium... Effect of COD concentration R. capsulatus SS grew better in formula A than. Formula A gave higher concentrations of dry cell weights (.97 g/l) and extracellular ALA (. µm) than (. g/l and µm, respectively) (Fig. ). A B C D E F Dry cell weight of and formula A Fig. from cultivation of Rhodobacter capsulatus SS in and formulated (A-C) and DS (D-F) under aerobic-dark condition on a shaker ( rpm) at 7 C for days.... MSG E A 9

5 Extracellular ALA of and formula A A.. Effect of NaCl concentration Both and formula C did not produce extracellular ALA (Fig. ). Extracellular ALA production from R. capsulatus SS was highest ( M) in (C/N ratio = 9,7), followed by (formula A, C/N ratio = 7,77) (. M) and (formula B, C/N ratio =,) (.7 M), respectively (Fig. c). Thus the optimum C/N ratio for R. capsulatus SS cultivation in formulated waste media was between 9,7 to,. Dry cell weight of and formula C Fig. Effect of COD concentration of and formula A from Rhodobacter capsulatus SS cultivation under aerobic-dark condition on a shaker ( rpm) at 7 C for days... Effect of nitrogen concentration R. capsulatus SS grew better in formula B than. Formula B giving higher concentrations of dry cell weights (.7 g/l) and extracellular ALA (.7 µm) than (. g/l, (M, respectively) (Fig. ).... C Dry cell weight of and formula B Extracellular ALA of and formula C... B.... C Extracellular ALA of and formula B Fig. Effect of sodium content in and formula C from Rhodobacter capsulatus SS cultivation under aerobic-dark condition on a shaker ( rpm) at 7 C for days. B.. Effect of supplementation with nutrient in formulated Since formulated waste media gave very low ALA production, supplementation with nutrients, as given below, were studied and results were illustrated in Fig. 7,, 9. Fig. Effect of nitrogen concentration of and formula B from Rhodobacter capsulatus SS cultivation under aerobic-dark condition on a shaker ( rpm) at 7 C for days. 9

6 Sample X Sample Y * G. M. H. N. I. (. folds O. (. folds J. ( folds P. ( folds K. ( folds Q. ( folds L. (add NaCl. g/l) R. (add NaCl. g/l) * The supplementation in sample B contains mm glycine, mm succinic acid,. g/l propionic acid, mm MgCl, and added mm levulinic acid (LA) at h (Sattayasmithstid, ). I and O; (. folds with COD value of, mg/l and had total nitrogen about.7 % that it was adjusted total nitrogen as similar as total nitrogen in (total nitrogen =. %) J and P; ( folds with COD value of 7, mg/l, COD of medium was 7,7 mg/l K and Q; ( folds with COD value of, mg/l L and R; (added NaCl. g/l) with sodium content of g/l, sodium content of medium was g/l... of R. capsulatus SS cultivation in and formulated under aerobic-dark condition on a shaker ( rpm) at 7 C for days. In Fig. 7 the of this cultivation was between and. and of formulated was higher than. Fig. 7(Y), of (nutrients supplementation) and (nutrients supplemena\tation) was rapidly decreased at h cultivation due to the addition of mm levulinic acid (LA) ; in after of and increased to normal (about.) since LA inhibited ALA dehydratase at that time.... Dry cell weight of R. capsulatus SS cultivation in and formulated.... Dry cell weight of formula G and M (a) G M. of sample Dry cell weight of formula H and N folds folds 7 time (h) folds (adjust sodium content).... H N (X). LA of sample (b) time (h). folds folds folds (adjust sodium content) (Y) Fig. 7 changes of R. capsulatus SS cultivation in each media (X) and media with the nutrients supplementation (Y) 9

7 Dry cell weight of formula I and O Dry cell weight of formula L and R.... I O.... L R (c) (f) Dry cell weight of formula J and P (d) Dry cell weight of formula K and Q J P K Q Fig. Dry cell weight of R. capsulatus SS cultivation in (a) synthetic media (formula G and M), (b) waste media (formula H and N), and (c, d, e, f) formulated waste media with (formula I-L) and without nutrients supplementation (formula O-R) under aerobic-dark condition on a shaker ( rpm) at 7 C for days. Dry cell weight values were between and g/l, with DCW in formula G (.9 g/l) was higher than formula K(diluted folds;.9 g/l) and dry cell weight of formula H (.7 g/l) was higher than formula J and I (diluted folds,. folds in.9 g/l and. g/l, respectively) since may be contained appropriate nutrient than the formulated in dilution folds and. folds, respectively. Figure ; DCW of formula K ( folds diluted;.9 g/l) was higher than another formulated waste media (formula I, J, L, and O-R), as well as nutrients supplementation stimulated growth of R. capsulatus SS and induced ALA quantity (Sattayasmithstid, ). It showed that ( folds diluted) with organic acid, glycine, and mineral addition was the production media for extracellular ALA formation (Fig. 9) with the lag phase in the first h., log phase was - h., and stationary phase was -7 h. On the other hand, formula G, H, I, J gave dry cell weight of.9 g/l and.7 g/l,. g/l and.9 g/l, respectively) which less than formula M, N, O, P (. g/l,. g/l,. g/l, and. g/l, respectively) since the essential organic compounds were used to produce ALA rather than to activate R. capsulatus SS growth (ALA production rate; q p value (h - ) was higher than bacterial growth rate; dry cell weight (g/l)). Whereas Fig. and 9; nutrients supplementation in formula Q were important for ALA production than R. capsulatus SS growth that it could be produced extracellular ALA in high values (.7 µm) but they gave dry cell weight in low values (.7 g/l). And dry cell weight in formula R (. g/l) was lower than formula L (. g/l) that may be sodium chloride addition inhibited nutrients supplementation for R. capsulatus SS growth. (e) 9

8 ... Extracellular -aminolevulinic acid of R. capsulatus SS cultivation in and formulated Extracellular ALA of formula G and M (a) G M Extracellular ALA of formula J and P (d) J P Extracellular ALA of formula H and N Extracellular ALA of formula K and Q H N K Q (b) (e) Extracellular ALA of formula I and O Extracellular ALA of formula L and R I O L R (c) (f) Fig. 9 Extracellular ALA of R. capsulatus SS cultivation in (a) synthetic media (formula G and M), (b) waste media (formula H and N), and (c, d, e, f) formulated waste media with 97

9 (formula I-L) and without nutrients supplementation (formula O-R) under aerobic-dark condition on a shaker ( rpm) at 7 C for days. From Fig. 9, ALA production of this cultivation was between and µm in formulated (formula M) that ALA production in formula M was higher than formula G and formula H-L, N-R (formulated in.,, folds. Fig. 9; ALA production of formula M ( µm) was higher than formula Q (supplementation & diluted folds; µm) and ALA in formula M ( µm) was higher than formula G ( µm) in. folds. To improve ALA production, was diluted to give the COD values of,, 7,, and, mg/l to the level present in medium (7, mg/l COD) and formulated was achieved by supplementation with organic acids, glycine, and mineral; that formulated with nutrients supplementation (formula Q, P, O) ( folds, folds,. folds produced ALA µm, µm, µm, respectively) could produce ALA higher than formulated (formula K, J, I) ( folds, folds,. folds produced ALA 9 µm, µm, µm, respectively). Thus this supplementation could give optimized formulated. For comparison on the ALA production of formula Q ( folds diluted with nutrients supplementation;, mg/l COD) with the another formulated (with supplementation) (formula O, P, R), formula Q will be selected for production media because it gave the highest ALA concentration and highest dry cell weight in µm and.7 g/l, respectively (Fig. (e), 9(e)) that may be its organic matter content was appreciated for bacterium growth and ALA formation. Formula M, O, P, Q (.,.,. and.7 µm, respectively) produced ALA higher than formula G, I, J, K (.,.,. and. µm, respectively) thus nutrients supplementation induced ALA production and nutrients supplementation did not affected for ALA production in (waste media) that may be some organic matter in suppressed nutrients or inhibited ALA synthase in, then dilution of could be the way to increase ALA production. From above, C/N ratio of media was found to be between 9,7 to, (Fig. 7c) and the selected production medium for this research work was formula Q ( folds, which gave C/N ratio of 9,. ACKNOWLEDGEMENT This research work was supported by The Joint Graduate School of Energy and Environment (JGSEE). REFERENCES [] Ano, A., Funahashi, H., Nakao, K., and Nishizawa, Y. (999) Effect of glycine on - aminolevulinic acid biosynthesis in heterrophic culture of Chlorella regularis YA-. J. Biosci. Bioeng., pp. 7-. [] Madmarn, W. () Increase Yield of - Aminolevulinic Acid from Photosynthetic Bacterial Mutant Strain and Its Application in Sex-reverse Nile Tilapia (Oreochromis niloticus Linn.) (M. Sc. dissertation). Prince of Songkla University, Thailand, pp. -. [] Prasertsan, P. and Suksawat, P. (9) Production of Single Cell Protein from Distillery Stillage. J. Prince of Songkla University., (), pp. -. [] Sattayasmithstid, S. () Optimization for production of -aminolevulinic acid from halotolerant photosynthetic bacteria Rhodobacter capsulatus SS (M. Sc. dissertation). Prince of Songkla University, Thailand, pp. 7-. [] Tangprasittaparp, A. and Prasertsan, P. () -Aminolevulinic Acid from Photosynthetic Bacteria and Its Application. J. Sci. Technol. Prince of Songkla University, Thailand., (), pp CONCLUSIONS. Monosodium glutamate effluent () was selected for cultivation of R. capsulatus SS, and extracellular ALA production (. µm).. Supplementation of (Media Y) with mm succinic acid,. g/l propionic acid, mm glycine, mm MgCl and mm levulinic acid (Media Y) gave higher ALA concentration than formula (Media X) because glycine and succinic acid are the substrate for ALA production; propionic acid is a cofactor; MgCl is a trace element; and LA is an inhibitor of ALA dehydratase.. Addition of mm levulinic acid (LA) increased ALA production as LA is an inhibitor of ALA dehydratase.. The C/N ratio range for R. capsulatus SS cultivation was between 9,7 to, and the C/N ratio for R. capsulatus SS in formulated ( folds was 9,.. Formulated ( folds diluted and nutrients supplementation; formula Q) was the production media since its contains much appropriate organic matter more than the. 9