UMYU Journal of Microbiology Research

E-ISSN: 2814 – 1822; P-ISSN: 2616 – 0668

ORIGINAL RESEARCH ARTICLE

Bio-Prospecting Xylose-Utilizing, Exopolysaccharide (EPS)-Producing Bacteria and EPS Quantification through Submerged Fermentation using Xylose as the Major Carbon Source

¹*Ukponobong Antia, ¹Nsikak Stephen, ¹Victoria Umoh, ¹Maria Bassey, ²Itorobong Udo, and ³Adebare J. Adeleke.

¹Department of Microbiology, Akwa Ibom State University, Mkpat Enin, 524106, Nigeria

²Department of Microbiology, University of Uyo, Uyo, 520101, Nigeria

³Department of Microbiology, Modibbo Adama University, Yola, 640230, Nigeria

Abstract

Many microorganisms are capable of producing Exo-polysaccharides (EPS) while utilizing simple sugars and hexoses. These EPS found applications in various fields, such as agricultural biotechnology, pharmaceuticals, textiles, and food industries. However, there is a lack of studies on EPS-elaborating bacteria that can utilize pentoses like xylose. Therefore, the utilization of alternative carbon sources for EPS production has become a focus of recent research. This study aimed to prospect bacteria that can utilize xylose for EPS production. Samples from agricultural soil, dump sites, saline soil, cement-contaminated soil, fresh cow milk, cow dung, and yogurt were serially diluted and cultured in a salt-based medium with xylose as the primary carbon source. Slimy and mucoid colonies were selected as potential EPS-producing isolates and identified morphologically and biochemically using the VITEK 2 Automated identification system. The quantification of EPS production by these isolates was conducted through submerged fermentation with xylose as the sole carbon source. The mean heterotrophic bacterial count of xylose-utilizing bacteria ranged from 2.1x10⁶ CFU to 3.5x10⁸ CFU per gram of the samples analyzed. The slimy and mucoid colonies were identified as members of the genera Staphylococcus, Enterobacter, Kocuria, Klebsiella, Enterococcus, Serratia, and Burkholderia. The quantities of EPS produced by the isolates ranged from 0.04 g/L to 2.0 g/L, with E. cloacae D1, E. cloacae D2, K. oxytoca D2, and K. oxytoca G1 elaborating the highest amount of EPS. Bacterial isolates capable of utilizing xylose for EPS production were obtained from various sources, showing potential for further optimization.

Key words: Bioprospecting, exopolysaccharides, lignocellulosic biomass, submerged fermentation, xylose.

INTRODUCTION

Exopolysaccharides (EPS) are long-chain polysaccharides secreted by microorganisms to survive in harsh environments (Netrusov, 2023). They are the major components of microbial biofilms, known to protect the producing microorganisms against adverse effects of the host immune system, pH, temperature, and antibiotics (Nguyen et al., 2020). EPS may be soluble or insoluble polymers and are commonly observed to accumulate around the cells (Moscovici, 2015). Exopolysaccharides are high molecular weight polymers of dextran, gellan, xanthan, alginate, levan, cellulose, pullulan, curdlan, hyaluronic acid, and succinoglycan (Mishra and Jha, 2013). The monomers making up these polymers include D-glucose, D-mannose, L-rhamnose, L-fucose, D-galacturonic acid, D-glucuronic acid, D-mannuronic acid, N-Acetyl-D-glucosamine, N-Acetyl-D-galactosamine, and some non-carbohydrate constituents such as carboxyl, sulfate, phosphate, and pyruvate substituents (Fretias et al., 2011). Many bacteria have been reported to produce EPS, including Acetobacter, Rhizobium, Alcaligenes, Pseudomonas, Enterobacter, Xanthomonas, Klebsiella, Bacillus, and a host of Lactic Acid Bacteria (Netrusov et al., 2023).

Lactic acid bacteria have been described as very versatile producers of exopolysaccharides applicable in the production of milk and milk products such as cheese, yogurt, cultured cream, and milk-based desserts (Mende et al., 2016). Due to their unique characteristics, microbial exopolysaccharides have found applications in various fields such as agricultural biotechnology, pharmaceuticals, food industries, detergents, beverages, textiles, cancer treatments, culture media formulation, paint, paper, and petroleum industries (Suryawanshi et al., 2022).

The production of EPS by microbes is believed by many researchers to be affected by the medium composition; likewise, carbon and nitrogen sources are important parameters in their biosynthesis (Netrusov et al., 2023). Several sugars have been used in the production of EPS including sucrose, glucose, lactose, and maltose, as well as sugar alcohols such as mannitol, sorbitol, and other substrates such as whey, starch, sugar concentrates, methanol, and alkenes (Kumar et al., 2007).

Among these substrates, glucose and sucrose have been extensively used for the production of exopolysaccharides (Wu et al., 2021). The importance of effectively utilizing carbon sources is underscored by the fact that the carbon source accounts for 30% of the running cost of EPS production (Onilude et al., 2012; Oner, 2013). In an attempt to reduce overhead costs due to the high cost of carbon sources, researchers are currently experimenting on EPS production using lignocellulosic wastes such as sugarcane straw (Abdeshahian et al., 2020), corn cob (Jesus et al.,2014), rice hull (Wang et al., 2014), and rice straw (Jazini et al., 2017) for EPS production. The results obtained so far indicate the selective utilization of glucose over xylose in these biomasses, potentially leading to the wastage of the hemicellulose fraction of these biomasses. Bioprospecting for microorganisms that are better producers of biomolecules is an essential aspect of research that should be constantly considered by researchers (Antia et al., 2018; George et al., 2023); hence, this research was carried out to bio-prospect bacterial isolates that can utilize xylose as the main carbon source for EPS production.

METHODS AND MATERIALS

Isolates and EPS production on solid media

Various samples from different sources including samples from agricultural, saline, cement-contaminated, and dump site soils, as well as fresh cow milk, cow dung, and yogurt were aseptically obtained and transported to the microbiology laboratory in Akwa Ibom State University (AKSU).

Serial dilutions of the samples were carried out sequentially up to 10-7, and bacterial isolates were obtained using the pour plate technique on a salt-based medium with the following composition (g/l): 0.15 KH2PO4, 0.5g/L K2HP04, 0.1g/L NaCl, 0.2g/L MgSO4, yeast extract 0.8%, 18 agar-agar, and 5% xylose (Ventorino et al., 2019). After 96 hours of incubation at 30 ℃, bacterial isolates displaying ropy or slimy colonies were chosen for further analysis to determine EPS production through submerged fermentation using a method adapted from Ventorino et al. (2019).

Identification of bacterial isolates using VITEX 2 Compact system

The bacterial isolates underwent cultural and morphological examination before being analyzed using the VITEK 2 compact identification system (bioMerieux). Fresh cultures of the isolates, aged 18–24 hours, were prepared and inoculated into the VITEK system. The results obtained were collected and automatically compared against an extensive microbial profile database to generate a tentative identity for the organisms.

Synthesis of EPS by the selected bacterial isolates

The ability to form slimy colonies in a salt-based medium supplemented with 5% w/v xylose as a carbon source was used as the criterion for selecting isolates for further evaluation of their ability to produce EPS in submerged fermentation (Ventorino et al., 2019). The bacterial cells were pre-cultured in a 10 mL nutrient broth supplemented with 2% w/v xylose and incubated for 18 – 24 hours at 30 ℃. The concentration of cells in the broth was standardized using a haemocytometer. One (1) mL of the nutrient culture containing about 1\(\times\)0^6 cells/mL in the nutrient broth was transferred into a broth of the same composition as the salt-based medium described above, without the agar.

After 48 hours of incubation at 30 ℃, the EPS produced was quantified following the method outlined by Palomba et al. (2012). In summary, the culture underwent centrifugation at 5000 rpm for 10 minutes, and the EPS-containing supernatant was treated with 2 volumes of chilled 98% v/v ethanol before being left to incubate overnight at 4 ℃. Subsequent centrifugation at 5000 rpm for 10 minutes allowed for the recovery of pellets, which were then reconstituted in 1 mL of distilled water, dried in an oven, and weighed to determine the amount of EPS. The EPS quantity was expressed as polymer dry mass per liter (PDM, mg/L) of the initial wet medium.

The weight of EPS produced was calculated as the weight of the filter paper with the precipitate minus the weight of the filter paper. The resulting value was then converted to mg/L following standard conversion procedures.

RESULTS AND DISCUSSION

Bacterial isolation, enumeration and screening for EPS producers in xylose containing medium

The mean heterotrophic plate count of bacterial isolates capable of utilizing xylose for EPS production, obtained from the analyzed samples, ranged from 2.1x10^6 CFU/g in the agricultural soil sample to 3.5x10^8 CFU/mL in the yogurt sample (Table 1). This count was measured after 96 hours of incubation, reflecting the organisms’ requirement to synthesize enzymes for xylose utilization as the sole carbon source in the medium.

The colonies of the isolates were mostly smooth, medium, slimy, and mucoid, in accordance with Mu’Minah et al. (2015) who reported that EPS-producing bacteria are characterized by mucoid colonies. The isolates obtained from cow dung, agricultural soil, fresh cow milk, dump site, and yogurt were found to have slimier colonies compared to isolates from other samples, and hence they were purified, identified, and used for further analysis.

Table 1: The Mean Heterotrophic Count of Xylose Utilizing Bacterial Isolates Obtained from various samples

Identification of the screened bacterial isolates using VITEK 2.0 Compact System.

Twelve (12) of the isolates showing thick slimy colonies were characterized morphologically, and selected for identification based on VITEK 2.0 Compact identification system. Table 2 shows the percentage probability of similarity of the obtained isolates with those in the VITEX database. The organisms belonged to the genera: Staphylococcus, Enterobacter, Kocuria, Klebsiella, Enterococcus, Serratia and Burkholderia. Seven (7) out of the twelve isolates were members of the gram positive group while the remaining 5 were gram negative isolates. Torres et al. (2012) and other researchers have reported on EPS production by Enterobacter and Klebsiella, respectively. However, only few reports on EPS production using a pentose sugar as the carbon source have been made. The abundance of lignocellulosic hemicelluloses with their enormous amount of pentoses makes it very important to begin to pay attention to this biomass fraction to avoid its continuous wastage (Antia et al., 2023). The VITEX 2 system by Bioérieux is relatively a new system for rapid bacteria identification and previous studies confirmed that this system gives rapid and reliable identification results with pure bacterial cultures within a short period (Ling et al., 2003). Details of the Biochemical reactions as obtained from the VITEX analyses are presented in Table 3 and 4.

Table 2: Microscopic, Cultural Characteristics and Identification of the Isolates Based on VITEK 2.0 Similarity Index/Probability of Accurate Identification

Table 3: VITEK 2 Testing for Gram positive organisms (Staphylococcus heamolyticus,Staphylococcus warneri,Kocuria kristinae,Enterococcus casseliflavus)

Table 4: VITEK 2 Testing for gram negative organisms (Enterobacter cloacae, Burkholderia cepacia, Serratia marcescens, Klebsiella oxytoca)

Quantification of EPS Production by the Identified Bacterial Isolates.

Previous studies showed that the quantities and composition of microbial EPS depends on species of organism and the cultural conditions used (Donot et al., 2012). The utilization of alternative carbon sources for microbial production of exopolysaccharide (EPS) has gained significant attention in recent years. Though several studies had shown that many microorganisms are good producers of EPS, not all EPS producers are capable of utilizing xylose for EPS production (Zhao et al., 2020).

In our study, the amount of EPS produced varied among the isolates obtained. When utilizing xylose as the sole carbon source, the isolates were capable of producing EPS in a range from 0.04 g/L to 2.0 g/L (Table 5). These quantities align with those reported for bacterial isolates cultured in the presence of glucose (Vijayabaskar et al., 2011; AMAO et al., 2019). Enterobacter cloacae isolated from cow milk and cow dung, along with klebsiella oxytoca from cow dung and yogurt, exhibited the highest EPS production at 2.0 g/L, followed by Burkholderia cepacia from yogurt and Staphylococcus heamolyticus from cow dung producing 1.8 g/L of EPS.

However, some authors have reported higher EPS values under different culture conditions; the variations in EPS production could be attributed to the specific bacterial isolates and growth environments. In our research, we focused on EPS production by utilizing xylose as the primary carbon source among the isolates. Cultivating xylose-utilizing bacteria in the presence of other readily accessible sugars might enhance the EPS yield of these isolates (Antia et al., 2019).

Table 5: Weight of Extracted EPS produced by the obtained isolates in a submerged fermentation using xylose as the sole carbon source.

CONCLUSION

This study presented compelling evidence of substantial EPS production by a variety of bacteria when exclusively utilizing xylose as the carbon source. Enterobacter cloacae and Klebsiella oxytoca stood out as top producers, yielding 2.0 g/L of EPS. The vitek 2.0 identification method proved to be more efficient in identifying the isolates compared to traditional biochemical techniques. By employing optimization techniques and exploring pentose sugars, there is potential to enhance EPS production when utilizing plant lignocellulosic biomass as the carbon source.

ACKNOWLEDGEMENT

The authors wish to thank the management of Akwa Ibom State University and the Laboratory staff of the Department of Microbiology of the same institution for their support towards the successful completion of this research.

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