Journal of Life SciencesVolume 4, Number 5, August 2010 (Serial Number 30)

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Journal of Life SciencesVolume 4, Number 5, August 2010 (Serial Number 30)

ContentsResearch Papers1 9 Study of Succinic Acid Production by Actinobacillus Succinogenes Elcio Ribeiro Borges, Ludmylla Bastos Rocha de Souza and Nei Pereira Junior Effectiveness of Some Plant Extracts on the Pupal Stage of Culex Quinquefasciatus (Diptera: Culicidae) Roqaya Mohammad Al Mehmadi 15 Isolation of Multi-Drug Resistant Paenibacillus sp. from Fertile Soil: An Imminent Menace of Spreading Resistance Pallavi B. Pednekar, Roopesh Jain, Narsinh L. Thakur and Girish B. Mahajan 20 25 30 37 Cultivation Practice of Pleurotus Fossulatus on Rice Straw Nirmalendu Das, Protik Chowdhury and Birja Pasman Identification of Genes for Powdery Mildew Resistance in Mungbean Parinya Khajudparn, Sopone Wongkaew and Piyada Tantasawat Expression of Recombinant Protein Bovine Prion pCIp264 in COS-7 Cells and Its Detection Yaozhong Ding, Yongsheng Liu, Wenqian Liu, Yanping Ma, Meng Wang, Shenghai Yang and Jie Zhang The Influence of Magnetite Nanoparticles on Human Leukocyte Activity Aneka Darov, Martina Dubnikov, Vlasta Zviov, Martina Konerack, Peter Kopansk, Hubert Gojzewski and Milan Timko 44 The Hepatoprotective Effects of Solanum Incanum on Acetaminophen-Induced Hepatotoxicity in Guinea Pigs Yahya Saleh Al-Awthan, Mohammed A. Salama and Ahmed M. Helal 49 Prolonged Production of L-DOPA Using Immobilized Aspergillus Terreus Sankar Lal Poddar and Sharmila Chattopadhyay

Reviews53 Medicinal Solid Fermentation Engineering of Chinese Traditional Medicine Fungal Yi Zhuang

Aug. 2010, Volume 4, No.5 (Serial No.30) Journal of Life Sciences, ISSN 1934-7391, USA

Study of Succinic Acid Production by Actinobacillus SuccinogenesElcio Ribeiro Borges, Ludmylla Bastos Rocha de Souza and Nei Pereira JuniorCenter of Technology, School of Chemistry, Federal University of Rio de Janeiro, Rio de Janeiro 21949-900, Brazil Received: March 29, 2010 / Accepted: June 21, 2010 / Published: August 30, 2010. Abstract: Succinic acid has recently emerged as an important chemical (commodity) because it can be used for the manufacturing of synthetic resins and biodegradable polymers and as an intermediate for chemical synthesis. Till date, succinic acid is mainly produced by chemical processes, however, due to the environmental concerns and the concepts of sustainability, researches are directed towards the production of succinic acid by microbial fermentation. The fact that carbon dioxide (CO2) is needed by the microorganisms for succinic acid production is another interesting feature. The fermentation was carried out with Actinobacillus succinogenes using a two-level fractional factorial design 25-1. The variables analyzed and their levels were: concentration of glucose, yeast extract, temperature, pH and agitation. The results show that the variables that more influenced on succinic acid production were pH, temperature and yeast extract. Key words: Organic acids, Actinobacillus succinogenes, fermentation, carbon dioxide.

1. IntroductionPresently, succinic acid is produced commercially by catalytic hydrogenation of petrochemical derived maleic acid or maleic anhydride. Due to increasing global demands for oil and the emergence of environmental consequences from excessive using fossil fuels, fermentative production of succinic acid from renewable biomass by anaerobic bacteria has become more attractive economically [1]. Utilizing renewable carbon source and greenhouse gas as substrates, bio-based succinic acid has remarkable environmental benefits [2, 3], diversifying the potential product portfolio of a biorefinery [4]. A biorefinery is a facility that integrates biomass conversion processes and equipment to produce fuels, power and chemicals from biomass. The biorefinery concept is analogous to todays petroleum refineries, which produce multiple fuels and products fromCorresponding author: Elcio Ribeiro Borges, Ph.D., research field: chemical engineering. E-mail: elcioeq@yahoo. com.br.

petroleum. Todays task is the development of useful building-block chemicals that can be produced from biomass and subsequently converted to several high-value chemicals and materials. Building-block chemicals are molecules with multiple functional groups that can be transformed into new families of usable molecules [5]. Microbial production of organic acids is a promising approach for obtaining building-block chemicals from renewable carbon sources. These processes are also favorable from a chemical and economic point of view [6]. Succinic acid has been identified as one of the top 12 chemicals derived from lignocellulosic biomass [5, 7]. The market price of petrochemically produced succinic acid is about US $ 5.9 - 8.8 kg-1 depending on its purity whereas the raw material costs based on production from maleic anhydride are about US $ 1 kg-1 succinic acid [8]. The current production of chemicals based on succinic acid accounts to about 16,000 t/y [7]. However, the market potential is estimated to be about 270,000 t/y if succinic acid replaced maleic anhydride for all uses of the latter [1, 9-11]. Because of these predictions

2

Study of Succinic Acid Production by Actinobacillus Succinogenes

and the rising oil, price interest in succinic acid production by fermentation processes is increasing. Succinic acid, also known as amber acid or butanedioic acid, is a dicarboxylic acid with the molecular formula C4H6O4. Succinic acid can be used as a precursor for the production of many industrial chemicals fermentation for use in the agricultural, food and pharmaceutical industries (for example, as surfacetants, detergents, adipic acid, 1,4 butanediol, tetrahydrofuran, N-methyl pyrrolidinone, 2-pyrrolidinone, succinate salts, gamma-butyrolactone, various green solvents, biodegradable polymers such as polybutyrate succinate (PBS), and ingredients to stimulate animal and plant growth) [2, 10, 12, 13]. Various working groups are dealing with the fermentation of succinic acid with strains of Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens [3, 14, 15], recombinant Escherichia coli strains and Corynebacterium glutamicum [16]. A. succinogenes shows a distinctive ability to produce a relatively large amount of succinic acid from a broad range of carbon sources such as arabinose, cellobiose, fructose, galactose, glucose, lactose, maltose, mannitol, mannose, sorbitol, sucrose, xylose or salicin under anaerobic conditions [17]. Actinobacillus succinogenes is an anaerobic, gram-negative bacterium that naturally produces high concentrations of succinate as a fermentation end product in addition to formate, acetate, and ethanol. Its CO2 concentration has been shown to regulate the levels of key enzymes of the PEP carboxykinase pathway in A. succinogenes at high CO2 levels. PEP carboxykinase levels rise, whereas alcohol dehydrogenase and lactate dehydrogenase are not detectable. Consequently, CO2 functions as an electron acceptor and alters the flux of PEP, which metabolises to pyruvate and lactate / ethanol at low CO2 levels but makes succinate at high CO2 levels [18]. Usually, culture medium is important to the yield of any fermentation products. The physiological and nutritional factors such as initial sugar concentration,

complex nitrogen sources, carbonate ion concentrations, pH and temperature of the growth medium are reported to be the most critical factors affecting both cell growth and succinic acid production [19, 20]. The pH is an important factor that affects both growth and growth-associated production of molecules. Succinic acid production is a CO2 fixing process [21] and the pH of the medium affects the solubility and the availability of the CO2, it is therefore most critical factor affecting succinic acid production [22, 23]. The carbon and nitrogen sources generally play a significant role because these nutrients are directly linked with cell proliferation and metabolite biosynthesis [24]. The utilization of cheap carbon sources instead of glucose is important for the cost-efficient production of succinic acid. However, these influential factors have not been studied systematically. The fermentation cost of bio-based succinic acid is however a key aspect for its competition with oil-based succinic acid. Therefore, it is necessary to optimize the culture conditions of A. succinogenes to improve succinic acid production. Thus, being the objective of this work is to verify the effect of process variables for succinic acid production from glucose by Actinobacillus succinogenese strain CIP 106512.

2. Experiment2.1 Microrganism and Culture Medium An Actinobacillus succinogenes strain (CIP 106512) was obtained from the Pauster Institute. The culture stock was maintained in Trypticase Soy Agar (TSA) slants at 4 . Inoculum (20 mL prepared in trypticasein soy broth (TSB) sterile medium, transferred to flask (150 mL)) was incubated at 150 rpm and 37 in an orbital shaking incubator (Marconi, Piracicaba, Brazil) during 16 h, which was the time necessary for the microorganisms to enter a logarithmic phase of growth. Then the inoculum was seeded in a medium whose composition was (in g/Ll) glucose 20.0, yeast extract 10.0, NaHCO3 10.0, NaCl 1.0, MgSO4 0.05, K2HPO4 6.8, and NaH2PO4 15.5.

Study of Succinic Acid Production by Actinobacillus Succinogenes

3

2- Manometer 4- Milipor membrane

CO2

3- Rotameter 0.05 vvm

1 - CO2 cylinder

6- Orbital shaking incubator Fig. 1 Schematic circuit of the gas distribution in the fermentation.

2.2 Fermentation Assays Batch fermentations were carried out in 500 mL flask which containing 200 mL medium with CO2 as the gas phase (Fig. 1). Media were inoculated with 10%.(v / v) of inoculum in the same medium and incubated at 150 rpm and 37 for 24-48 h. A separately autoclaved solution of carbohydrate was added aseptically to the medium after autoclaving. 2.3 Fractional Factorial Design The fermentation was carried out using a two-level fractional factorial design 25-1. The variables analyzed and their levels were (Table 1): concentrations of glucose (20-40 gL-1), yeast extract (5-11 gL-1), temperature (32-42 ), pH (6-8) and agitation (100-300 rpm). The statistical significance was checked using analysis of variance (ANOVA). The values of succinic acid concentration obtained and statistical significance were analyzed by one-way ANOVA. The statistical Fractional Factorial Design (STATISTIC 6.0) software was used to investigate conditions to obtain high succinic acid concentration.Table 1 Factors and levels used in the experimental design. Parameters A- pH B- Temperature () C- Yeast extract (g / L) D- Glucose (g / L) E- Agitation (rpm) -1 6 32 5 20.0 100 Real levels 0 +1 7 8 37 42 8 11 30.0 40.0 200 300

2.4 Analytical Methods After growth, the cells necessary for each fermentation assay were separated from media by centrifugation at 8,000 rpm for 20 minutes, and their concentration was determined by measuring the optical density of a diluted sample at 600 nm (SPECTRUMLAB 22 PC), using a standard curve of absorbance against dry cell mass. The residual glucose concentration was measured in the cell-free supernatant by the glucose oxidaseperoxidase method using an enzymatic kit (Laborclin, Pinhais, Brazil). Succinic acid concentration produced was estimated on high performance liquid chromategraphy (HPLC). The sample (20 L) was injected into a column (C18, 250 mm length 4.6 mm internal diameter column, 9 m; StrodsII Peek) at a flow rate of 0.9 mL/min. Degassed and filtered sulfuric acid (0.01 N) was used as the mobile phase [25]. Succinic acid yield was defined as the amount succinic acid produced from 1 g glucose expressed in percentage.

3. Results and DiscussionThe experiments were performed using the statistical methodology of response surfaces (RSM) and a Pareto chart, which are statistical models widely used to study the aggregate effect of multiple variables and to seek optimal conditions for a multivariable system [26]. Two-level fractional factorial design 25-1 leading to a total number of 19 runs was generated (16 independent

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Study of Succinic Acid Production by Actinobacillus Succinogenes

Table 2 Fractional factorial design 25-1 investigating effects of pH, temperature, yeast extract, agitation and glucose concentration on final succinic acid concentration, yield and volumetric productivity. Parameters Experiment number A 8 8 7 B 42 42 37 C 11 11 7 D E (24 h) YP / S QP (g / Lh) (g / g) 0.55 0.33 0.50 0.38 0.60 0.30 Measured response Ef (%) 50.60 92.20 46.30 (48 h) QP YP / S (g / Lh) (g / g) 0.28 0.34 0.24 0.19 0.58 0.31 Ef (%) 52.30 89.23 48.30

AS 13.2 11.98 9.05

AS 13.60 11.6 9.43

1 9 PC

300 40 100 20 200 30

PC: Center points (average of the values); A: pH; B: Temperature (); C: Yeast extract (g/L); D: Agitation; E: Initial glucose concentration (g/L); AS: Succinic acid (g/L); QP: Productivity (g/Lh); YP/S: Product yield in relation to the substrate; Ef: Efficiency.

runs and 3 repetitions of the central point - Data not shown). Final succinic acid concentration, volumetric productivity and yield were the response variables in all experiments. Table 2 presents some of the final results, more relevant to the fermentation process, which presented larger values for final succinic acid concentration (experiments 1 and 9, besides the results obtained with the average of the 3 central points). Analysis of these results showed that the concentration of succinic acid varied between 1.7 gL-1 and 13.6 gL-1, and that the largest concentrations, above 8.75 gL-1, resulted from a pH adjustment to 7.0 (Data not shown). Van der Werf et al. [18] reported that cell growth was affected adversely at low pH, possibly reflecting higher maintenance requirements at lower pH values. The largest concentration of succinic acid was obtained with the sample cultivated in experiment number 1, with the following conditions: initial glucose concentration of 40 gL-1, yeast extract of 11.0.gL-1, temperature of 42 , pH of 8.0 and speed agitation of 300 rpm, during 48 hours of fermentation. However, the efficiency of the fermentation process depends on the initial glucose concentration (experiment number 9, with 20 gL-1 of glucose, presents the greatest efficiency). According to the analysis of variances (ANOVA -Data not shown), the best model for succinic acid concentration was the Reduced Quadratic Model and the values of R2 (0.992), pure error (0.056), which

show that the model is in accordance with the experimental data. Based on the effects of parameters using the Pareto chart (Fig. 2), pH (parameter A) appears as the most important parameter, followed by the parameter B (temperature), parameter E (glucose), parameter C (yeast extract), followed by the interactions between the parameters, and finally agitation (parameter D), which bore a lesser influence as independent variables on succinic acid concentration. The parameter A showed a positive effect, considering minimal confidence range of 90% (P