Kinetic modeling of hyaluronic acid production in palmyra palm (Borassus flabellifer) based medium by Streptococcus zooepidemicus MTCC 3523

Highlights

• First report on production of high value added HA using nutritionally rich PJ.

• HA production from PJ based medium comparable to synthetic medium.

• Kinetic studies on the influence of PJ on cell growth and HA production.

• Techno-economic feasibility analysis of HA production.

Abstract

In this study, we evaluated palmyra palm sugar (PJ) formulated medium for HA production using Streptococcus zooepidemicus MTCC 3523. Concurrently, effect of various carbon and nitrogen sources, an analysis of the effect of initial sugar concentration, on HA production were examined. The PJ - soya peptone based medium was found to be the most effective, and resulted in 0.54 ± 0.08 g L^-1 of HA titer and specific growth rate of 0.5 ± 0.07 h^-1 in shake-flask experiments. Kinetic experiments were performed in a bioreactor, and the initial PJ concentrations were varied from 10 - 50 g L^-1. Maximum HA and LA titers (1.22 g L^-1 and 20. 2 g L^-1) were obtained at 30 g L^-1 PJ concentration in bioreactor. Han–Levenspiel substrate inhibition model exhibited best fit for the experimental data. The growth inhibitory PJ concentration was found to be at 90.5 g L^-1. Medium based process economic analysis proved PJ and soya peptone based medium as most economical for the production of HA. The present investigation validates the potential of PJ and soya peptone based medium for enhanced economical production of HA. This study will also provide new product dimensions and market avenues, to the marginalized palmyra palm farmers and toddy tapping community, who are unable to secure the right price for their product.

Introduction

Hyaluronic acid (HA) is a linear hetero polysaccharide with recurrent repeating β D - glucuronic acid and β N-acetylglucosamine attached by β 1-3 and β 1-4 glycosidic linkages. This natural non-sulfated glycosaminoglycan, found in connective and epithelial tissues, is part of signaling pathways and joint lubricants [1]. HA engenders extensive applications in biomedical and health care industries through its high water-holding capacity, visco-elasticity and biocompatibility. The biological activity and utility of HA is mainly dictated by its molecular weight [2]. Thus the high molecular weight HA (> 2 × 10⁶ Da) is utilized in pharmaceuticals as a filling agent in arthritis treatment, ophthalmic surgery and adhesion prevention after abdominal surgery. Whereas the low molecular weight HA (0.8 -8 × 10⁵ Da) is used in the cosmetic industry for moisturizing and wound healing [3]. HA in conjunction with chondroitin and glucosamine, is employed as a food supplement. HA bolstered by its plasticity, stability, bioavailability and lower cytotoxicity, has extensive and preferential application in drug formulations and targeted drug delivery [4].

The utility of HA is compromised by its elevated risk of viral contamination and spiraling cost of procurement. Thus HA extracted from animal sources such as rooster comb and bovine vitreous humor, poses a higher risk of viral contamination [5]. The steady increase in market demand for HA, and the large supply to demand gap has contributed to its increasing cost. Pharmaceutical grade HA accounts for less than a ton, out of total demand for bulk HA; and is marketed at USD 60,000/kg [6]. According to HA market analysis report 2016 (Transparency market research, US) the estimated global HA market was 5.36 billion USD in 2012, and is expected to reach 9.52 billion USD by 2019 [7].

In the last two decades, microbial production of HA has become the preferred alternative, reinforced by lack of viral contamination, higher product yield, cheaper production cost and highly purified product. Many wild microbial strains naturally produce HA as capsular exopolysaccharide, as a part of their self defence against invading hosts. Examples of such Strepococcus species are Streptococcus zooepidemicus, Streptococcus pyogenes, Streptococcus equisimilis, Streptococcus thermophilus and other organisms such as Pasteurella multocida and Cryptococcus neoformans [[8], [9], [10], [11], [12]]. HA production has also been assessed after incorporating key genes for HA enzymes into Bacillus subtilis, Lactococcus lactis, Escherichia coli, Corynebacterium glutamicum, and Pichia pastoris [[13], [14], [15], [16], [17], [18]]. However, the current industrial production of HA is mainly facilitated by Streptococcus species owing to its high HA titer (6 - 7 g L^-1) under optimal conditions [19]. HA production from microbial sources is primarily influenced by the cost of the raw material. Consequently, exploration of economically viable alternatives for renewable feedstocks and bioprocess technology platforms have gained momentum in industrial and academic sectors [20].

An aspect that is infrequently addressed is the Kinetic modeling of economically viable HA production from potential agricultural feedstocks [6,[21], [22], [23], [24], [25], [26], [27]]. Kinetic modeling is essential to develop a fermentation process with optimal operational conditions for the production of the metabolite of interest. In a bioprocess, the kinetic model is a set of relationships between biomass growth, substrate utilization and product formation; employed to understand microbial kinetics and to predict kinetic parameters. In large scale HA production, these kinetic variables will become the key process parameters to be monitored and controlled though some reports address kinetic modeling of the HA production using either pure sugars or renewable and economically viable feedstocks [21,[28], [29], [30], [31]]; very few reports substantiate effect of substrate inhibition on the biomass growth.

To address these issues, we employed Palmyra palm jaggery (PJ) as potential feedstock for the microbial production of HA. PJ is traditionally made by the concentrating the sap extracted from the palmyra palm trees (Borassus flabellifer). Naturally obtained PJ contains 65-85 % (w/w) sucrose and 5-15 % (w/w) reducing sugars, and is consumed directly or used for preparation of sweet confectionary items and in traditional medicine. It is rich in vitamins (Nicotinic acid - 5.24, Thaiamin - 24.0, Riboflavin - 432.0 and Vitamin C - 11.0, (all expressed as mg/100 g), and minerals (Calcium -0.06 % (w/w), Phosphorus - 0.06 % (w/w), Iron – 0.0025 (gg^-1)), vital to the growth of an organism [32]. PJ has been employed in the production of value added products such as polyhydroxybutyrate, citric acid, ethanol, D (-) lactic acid through microbial fermentation [[33], [34], [35], [36]]. PJ is a pre-treatment free feedstock, and has the potential to serve as an alternative economic substrate for microbial production of high value products such as HA. Further, utilization of PJ for the production of high value products would generate new revenue options for the marginalized palm tapping farmers. It would thus create a valuable socio economic dividend.

The aim of this study is to formulate a cost-effective medium employing palm sugar as a carbon source for the production of HA. As an immediate and significant corollary, it would yield definitive insights on biomass growth, HA production, substrate consumption and substrate inhibition in PJ based production medium. The kinetic parameters gleaned from this study would be immensely useful for effective process design leading to enhanced HA production.