Optimization of Carbon and Nitrogen Sources for Extracellular Polymeric Substances Production by Chryseobacterium indologenes MUT.2

1. Background
    Bacterial Extracellular Polymeric Substance (EPS) are environmental friendly polymeric materials that are used in a wide range of industrial applications (food, textile, cosmetics, pharmaceuticals, etc.) (1). Traditional EPS derived from other natural sources (i.e. plants, algae, and animals) fail to perform in some applications,  in contrast,  bacterial EPS may demonstrate new and improved properties. Furthermore, compared to synthesis of EPS by higher plants and algae, their microbial production is more productive and less resource-intensive. Moreover, microbial production enables the control of process conditions to obtain higher yields and desired properties (2). However, due to their high production costs, only a few bacterial EPS are commercially available (e.g. xanthan, gellan gum, hyaluronic acid). To make the process more cost-effective, improvements in the product yield is necessary, which, could be achieved through optimization of the culture condition, development of the high yield strains (e.g. through genetic manipulation), or application of the low-cost substrates (3). Nutritional and environmental conditions strongly affect microbial EPS’ synthesis and composition (4). Carbon and nitrogen sources generally play a significant role, because these nutrients are directly linked to the cell proliferation and metabolite biosynthesis (5, 6). Also, the nature and concentration of the carbon source can regulate the secondary metabolism through phenomena such as catabolic repression (7).
    A number of statistical experimental design combined with the response surface methodology (RSM), such as factorial design, uniform design, Central Composite Design (CCD), and Box-Behnek Design (BBD) were applied for optimization of the fermentation conditions (1). M. Khani et al. (1) have used CCD for  optimization of the operating parameters for EPS production by C. indologenes MUT.2. But, as far as we know, there is limited knowledge regarding nutritional requirement for EPS production by C. indologenes MUT.2. In addition, there hasn’t been any report with respect to the medium optimization improvement for EPS production. C. indologenes MUT.2 was isolated from garden soil and hot spring water samples were collected from Arak city in Iran in order to investigate the corrosion inhibition by EPS. The EPS produced by the isolated C. indologenes MUT.2 was found to have high anti-corrosive properties (8). The electrochemical behavior of this new type of anti-corrosive EPS has been examined using electrochemical techniques and surface analytical examinations (FT-IR, EIS) (8). Both carbon and nitrogen sources were optimized in the present study by employing statistical method based on the single-factor experiments in order to improve the EPS production by C. indologenes MUT.2.

2. Objectives
    In this study, for the first time, the effects of carbon and nitrogen sources were studied in order to improve the EPS production by submerged cultivation of C. indologenes MUT.2. The obtained information is considered to be fundamental and useful for development of the C. indologenes MUT.2 cultivation process, as well as an efficient production of the EPS on a laboratory scale stirred tank bioreactor.

3. Materials and Methods

3.1. Microorganism and Inoculum Preparation
    The mesophile microorganism C. indologenes MUT.2 was kindly provided by the Biological Science and Technology Department, Malek Ashtar University, Tehran, Iran. The strain was maintained on nutrient agar at 4°C and sub-cultured monthly. The strain was grown and maintained in LB-agar with the following composition: 5.0 g.L-1sodium chloride, 5.0 g.L-1yeast extract, 10.0 g.L-1 tryptone, and 15.0 g.L-1 agar. During the preparation, the pH of the media was adjusted at 7.0 and then sterilized (121ºC/15min). Following to the 24 h of growth at 30ºC, the culture was maintained at 4ºC (9).
    Inoculum cultures were prepared in the LB-broth containing the following components as % W/W-final concentration: 0.5 sodium chloride, 1.0 tryptone, and 0.5 yeast extract (10). The pH of the medium was adjusted to the 7.0 before autoclaving (121ºC/15 min). C. indologenes MUT.2 cells were inoculated in 250-mL Erlenmeyer flasks containing 150 mL of LB-broth at 30±2ºC for 24 h. The flasks were placed in an orbital shaker (Tecnal model. TE-424, Tehran, Iran) at 170 rpm. Cell growth was monitored spectrophotometrically (PerkinElmer model Lambda 20) by measuring the optical density at 600 nm after 24 h of incubation until the cell count has reached 107 CFU.mL-1.

3.2. Culture Medium
    The initial basal medium used contained glucose (20 g.L-1), yeast extracts (5 g.L-1), K2HPO4 (6 g.L-1), NaH2PO4 (7 g.L-1), NH4CL (0.7 g.L-1), and MgSO4 (0.5 g.L-1). The medium constituents and glucose were sterilized separately by autoclaving at 121°C for 15 min, and were mixed thoroughly before inoculation. The chemicals used in this study were of reagent grade.

3.3. Carbon and Nitrogen Source Screening
    For evaluating the effect of carbon and nitrogen sources on fermentation performance, cultures were conducted in 500 mL shake flasks filled with 300 mL of the medium. Then, the bacterial pre-culture medium (6% , V/V) was inoculated in the flasks and incubated for 96 h. The flasks were held at 30ºC on a rotary shaker at 170 rpm.

3.4. Fermentation Conditions
    The fermentation medium was inoculated with 6% (V/V) of the seed culture and then cultivated at 30ºC in a 3 liter stirring tank bioreactor (Bioflow III, New Brunswick Scientific Co., New Brunswick, NJ, USA). Unless otherwise specified, fermentation were conducted under the following condition: temperature at 30ºC, aeration rate 1.0 vvm, agitation speed 250/min, the initial pH 7.0, and the working volume 2 l.

3.5. Analysis of Biomass
    The bacterial dry cell biomass was collected by centrifuging 1 mL culture samples at 11000 g for 10 min (Sigma 1-16K, No.12134). The centrifuged cell pellet was washed twice with distilled water and dried at 80ºC in an oven for 24 h to obtain cell dry weight (CDW). CDW was calculated using a gravimetric method and was expressed as g.L-1.

3.6. Analysis of EPS
    Mycelial pellets were separated by centrifugation (Sigma 6-16K, No.12256) at 16000 g for 10 min and the supernatant was mixed with three volumes of the chilled absolute ethanol to precipitate the EPS. The sample solutions were stored at 4ºC for 24 h to enhance the precipitation. Finally, the EPS were recovered by centrifugation at 9000 g for 15 min and dried at room temperature. EPS production was calculated using a gravimetric method and was expressed as g.L-1.

4. Results

4.1. Effect of Carbon Sources
    The following set of experiments were done in order to investigate the effects of the various types of carbon sources on the cell growth of C. indologenes MUT.2 and production of EPS. In all cases, nitrogen was provided as yeast extract at the same concentration that was used in the basal medium, where each carbon source was added to the basal medium at 20 g.L-1 instead of glucose. (Figure 1) shows the time profile of the biomass concentration (A) and EPS production (B) for the cultures carried out with all different types of the tested carbon sources as well as the culture without addition of the any type of carbon source as the control. The results obtained from shake-flask experiments revealed that, the change in the employed carbon source has affected both the amount of the produced biomass and the EPS production. Although galactose, maltose, and lactose gave good mycelial growth, they led to low EPS yields. The preferred carbon sources; sucrose and glucose, generally resulted in the highest EPS production of  8.32 and 6.37 g.L-1, respectively as shown in (Table 1).
 
4.2. Effect of the Nitrogen Source
    To investigate the effect of the nitrogen source on EPS production and mycelial growth, eight different nitrogen sources (N source) were examined (Figure 2). These N-sources were added on the basis of an equivalent N-content. Although yeast extract and glycine were favorable for the mycelial growth of the C. indologenes, the maximum EPS production of 8.87 g.L-1 was achieved when glutamic acid (5 g.L-1) was employed (Figure 2B). Improvement in the cell growth, EPS production, and EPS/CDW ratio through application of the different nitrogen sources are summarized in (Table 2).

4.3. Cultivation of C. indologenes MUT.2 in Shake-Flasks with the Optimized Medium
    Using all optimized medium components, trials in a series of the shake-flasks was done using 20 g.L-1 sucrose as the carbon source and 5 g.L-1 glutamic acid as nitrogen source. The time course of the cell growth, EPS production, and pH values are shown in (Figure 3). The maximal EPS production was achieved as: 8.9 g.L-1 after 3.5 days of the fermentation, while, the maximum mycelial yield was about 1.96 g.L-1 achievable after 3.5 days. This indicates a 53% increase in the EPS production using the optimized medium (8.9 g.L-1) compared to EPS production using the basal medium (5.8 g.L-1), as previously described. In addition, similar increase in the biomass concentration was achieved using the optimized medium.

4.4. Fermentation in the Stirring Tank Bioreactor
    Although shake flasks are the most frequently used bioreactors in biotechnology for an initial process development, very little is known regarding their characteristics from an engineering point of view (11, 12). There are several reports in the literature regarding the scaling-up of processes from the shake flasks to the stirred tank bioreactors (13, 14). Therefore, the bioreactor fermentation process was developed on the basis of data obtained from shake flasks cultivation experiments. The production of EPS was studied in a 3 liter bioreactor using the basal and the optimized culture medium.
      Figure 4 shows the time courses of mycelial growth and EPS production by C. indologenes MUT.2 in a 3 liter stirred tank fermenter with the basal medium and the optimized culture medium. In the basal medium, the EPS concentration reached a maximum level of 5.84 g.L-1 after 3 days, while, maximum mycelial concentration was 1.59 g.L-1 after 3 days (Figure 4A). In an optimized culture medium as shown in (Figure 4B), the mycelial growth was continuously increased towards the end of fermentation and its final mycelial concentration has indicated an amount of almost 1.98 g.L-1 at day 3.5. The initial pH of the fermentation broth slowly decreased from 7.00 to 5.02. The EPS production reached 10.15 g.L-1 after 3.5 days of the fermentation, which is 1.7 times higher than that of fermentation in the basal medium. Optimization of the operating parameters (e.g. agitation, aeration, and dissolved oxygen tension) in the bioreactor fermentation deserves further investigation, which is being carried out in our laboratory.

5. Discussion
    The results obtained in the present study are in agreement with the general concept expressed in the early studies (15, 16) which have reported the highest exopolysaccharides yield using fructose, sucrose, and glucose as carbon sources. Recently, Zhang et al. (17), studied the production of gellan exopolysaccharide by growing strains of Sphingomonas paucimobilis QHZJUJW in the medium containing various carbon sources such as glucose, sucrose, maltose, malt extract powder, and vegetable oil. They found that the highest EPS production of the strain in the sucrose containing medium. Qiang et al. (18) have used RSM for EPS production by Klebsiella sp. H-207, isolated from activated sludge. A maximum EPS yield of about 15.05 g.L-1 was achieved under optimized conditions in terms of both the medium composition and culture condition, which consisted of sucrose 31.93 g.L-1, KNO3 2.17 g.L-1 and K2HPO4 5.47 g.L-1, a seed age of 13 h, with an inoculum size of 10.6% and an incubation temperature of 28.9°C. Screening of the most significant fermentation parameters affecting levan production was done by the statiscal designs for the microbial strain Pseudomonas fluorescens NCIM 2059. Six nutritional variables (i.e. sucrose, casein peptone, NH4Cl, KH2PO4, MgSO4, and NaNO3) were studied to define an optimal medium. A significant increase in the levan yield from 5.27 up to 15.42 g.L-1 under these conditions was found (19). In a recent study, a novel bacterium strain BM39, associated with Pantoea sp., was isolated from sediments of Tyrrhenian Sea. The strain was selected for its ability to produce a very high levels of the glucan EPS. Kinetic studies of EPS production by the strain BM39 cultivated in the shaken cultures using a medium containing 80 g.L-1 of sucrose (EMS medium) and 80.0 g.L-1 of fructose (EMF medium) as carbon sources, was shown an EPS production the amounts of which was estimated as 11.82±1.06 g.L-1 and 11.05±1.17 g.L-1, respectively, while the productivity was 0.39±0.04 g.L-1.h and 0.37±0.04 g.L-1.h-1 (20). Bounaix et al. (21) have clearly demonstrated a high biodiversity of EPS produced by sourdough LAB strains with sucrose substrate (21).
    In this work, culture medium for production of EPS by C. indologenes MUT.2 was optimized. Compared with the basal culture medium in shake-flasks and stirred tank bioreactor, the use of optimized culture medium has resulted in a 53% and 73% increase in EPS production, respectively. No reports are currently available in the literature regarding the optimization of the extracellular polymeric substance production by C. indologenes MUT.2 in the submerged culture. Our study provides an important information for the large-scale production of the bioactive natural products such as EPS by C. indologenes MUT.2, which can be used as an functional anti-corrosive.