Exopolysaccharide (pullulan) production from sugarcane bagasse hydrolysate aiming to favor the development of biorefineries

https://doi.org/10.1016/j.ijbiomac.2019.01.038Get rights and content

Highlights

  • Sugarcane bagasse hydrolysate is a potential substrate for pullulan production.

  • Sugars present in the hydrolysate are converted to pullulan by yeast-like fungus.

  • Bubble column reactor showed a good performance for pullulan production.

  • Properties of pullulan from SCB are comparable to commercial food grade pullulan.

  • Pullulan production is a promising alternative in a biorefinery context.

Abstract

Pullulan is a biopolymer used in food industry produced by Aureobasidium pullulans from starch. In the present work, sugarcane bagasse (SCB) hydrolysate was evaluated as an alternative substrate in fermentation process assisted by blue LED lights. The best fermentation conditions in Erlenmeyer flasks were 25.3 °C, stirring speed of 232 rpm and yeast extract concentration of 1.88 g/L, yielding 25.19 g/L of pullulan, that corresponded to yield of 0.48 g/g and 0.28 g/(L·h) of volumetric productivity. By using a column bubble photobioreactor, similar yield values were obtained. Thermal properties of the produced pullulan as glass transition (Tg) and melting (Tm) temperatures were 38 °C and 160 °C, respectively, which were similar to the corresponding values of commercial food grade pullulan. Therefore, SCB hydrolysate is a promising substrate to produce good quality pullulan (86% of purity) with application in food industry, besides to represent a new alternative for biorefineries.

Introduction

Fossil-based polymers correspond, in general, to non-biodegradable materials, which leads to an important environmental concern. Despite of that, their global production is increasing every year, reaching approximately 322 million metric tons in 2016 (4% more than in 2015) [1]. According to CNBC (“Consumer News and Business Channel”), more than 9 billion tons of plastic have been produced worldwide since the 1950s, of which 9% was recycled, 12% was incinerated and 79% was built up in landfills or disposed indiscriminately [2].

Polymers based on renewable resources, on the other hand, are often biodegradable and biocompatible, and their applications in biomedicine (e.g. hydrogels as scaffolds for tissue engineering), as food additives and packaging and in pharmaceutical industry (drug delivery) have been extensively studied. According to European Bioplastic [3], the production of biopolymers corresponds to approximately 1% of the annual polymers production. However, an increase of 19% is expected for 2020 related to 2017, highlighting polylactic acid (PLA) and polyhydroxyalkanoates (PHAs). Also, some others polymers like dextran, xanthan gum and pullulan have interesting properties and increasing potential applications; in the case of pullulan, its high water solubility and thermal stability are often exploited [4,5].

Pullulan is a homo-polysaccharide produced by Aureobasidium pullulans, consisting of three glucose units attached by α(1 → 4) glycosidic linkages (maltotriose), which is, in its turn, attached to each other by α(1 → 6) glycosidic linkages [6]. This specific structure and the Generally Regarded As Safe (GRAS) status of pullulan allow extensive application in food industry (stabilizer, adhesive, coating or packaging material), also offering wide other potential applications, ranging from oral thin films, blood plasma substitutes and cosmetics [4,7].

In general, pullulan is produced together with melanin, a dark pigment produced by several wild strains of Aureobasidium pullulans. Pullulan purification is conducted by several treatment methods like application of activated charcoal or solvents, which increase the cost of the final product. However, some other approaches as the isolation of non-melanogenic strains of A. pullulan [8], the use of mutant strains and the use of LED lights to assist the fermentation process have been suggested [5,9]. Light is an important external factor which can regulate the metabolism, favoring the production of some biomolecules. Indeed, in several works, good performance of process assisted by blue LED-light on metabolites production was shown, e.g., Pseudomonas taetrolens [10], Cordyceps militaris [11] and Monascus ruber [12]. The effect of blue LED-light at molecular level of A. pullulan is unknown, although it was better explored for others microorganism as Monascus ruber [13,14].

Pullulan is still an expensive product, usually produced from hydrolysed from starch with prices ranging from US$30 to US$40/kg for food grade pullulan [15]. Therefore, the use of different low-cost sources such as potato starch [[16], [17], [18]], sugarcane bagasse [5], rice hull [19], sweet potato [20] and peat [21] hydrolysates have been successfully considered to make its production cost competitive. Among these possibilities, sugarcane bagasse (SCB) is a potential biomass and the SCB hydrolysate has not been extensively explored for this propose. This lignocellulosic material has been studied as raw material to produce energy, biofuels and a number of different compounds and, in this context, pullulan produced from SCB can be an interesting alternative to enhance the economic viability of biorefineries.

SCB is a massive agroindustrial by-product in Brazil, mainly burnt off for energy and steam generation in sugar-alcohol industries. In the last decades, the carbohydrate fraction of SCB has been extensively studied for second generation ethanol production. Although some industrial facilities are already dedicated to the production of cellulosic ethanol, considering the complexity of the lignocellulosic materials and the intrinsic techno-economic problems, the process is not yet a mature and consolidated technology. Therefore, to improve the financial liability of current biorefineries, obtaining diverse products apparently seems an acceptable strategy.

In a previous work, the effects of different wavelengths of LED lights on low melanin containing pullulan production using pure glucose as carbon source were evaluated [5]. Additionally, an experiment using hydrolysate of sugarcane bagasse was included in that work, but only with preliminary results. Considering the potential of sugarcane bagasse hydrolysate for pullulan production specifically in the context of lignocellulosic biorefineries, in the current work SCB hydrolysate was used as carbon source for pullulan production and the fermentative parameters were studied in the process assisted by blue LED light. The optimized conditions were also used in a bubble column photobioreactor. This reactor configuration was chosen due to its simple design and operation, and excellent heat and mass transfer [22]. Finally, pullulan obtained from the column bubble reactor was characterized and compared to food grade pullulan in respect to its physical and thermal properties.

Section snippets

Preparation of SCB hydrolysate

Sugarcane bagasse (SCB) was kindly supplied by Usina Vale Onda Verde (Onda Verde-SP, Brazil). It was dried, milled and classified through standard Tyler sieves. The biomass used in this study had the following composition: 40.35% of cellulose, 27.94% of hemicellulose, 26.26% of lignin and 5.45% of other minor compounds (extractive and ash). SCB with average particle size between 1.16 and 5.66 mm was then pretreated with 0.3 M NaOH at 70 °C for 4 h [23].

After pretreatment, the biomass was

Effect of variables on pullulan production

The influence of important variables on pullulan production by Aureobasidium pullulans using enzymatic hydrolysate-based medium was evaluated and the results are presented in Table 1. The highest pullulan production (25.19 g/L) was observed in run 14 (25 °C, 240 rpm and 1.75 g/L of YE) followed by 21.6 g/L and 20.07 g/L observed in run 12 (30 °C, 240 rpm and 1.75 g/L of YE) and run 2 (30 °C, 200 rpm and 1.75 YE), respectively. On the other hand, lower pullulan productions (0.70–1.60 g/L) were

Conclusion

SCB, a readily collectable feedstock, presents great potential for pullulan production. Under the stablished conditions, the pullulan production was considerably improved, with temperature as the most influential fermentation variable. The yield obtained using a bubble column reactor was similar with that one obtained in the flasks. Thermal properties of produced pullulan from SCB hydrolysate are quite comparable to commercial food grade pullulan. Therefore, the SCB hydrolysate is a promising

Acknowledgements

The authors also gratefully acknowledge to São Paulo Research Foundation (FAPESP) – Brazil (grants #2017/11086-4, #2016/23758-4 and #2016/10636-8) by financial support. The authors also gratefully acknowledge to Dr. Fernando Carlos Pagnocca from Center for Study of Social Insects (CEIS/UNESP) for the donation of the strain Aureobasidium pullulans LB 83.

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