Sweet sorghum juice as an alternative carbon source and adaptive evolution of Lactobacillus brevis NIE9.3.3 in sweet sorghum juice and biodiesel derived crude glycerol to improve 1, 3 propanediol production

Highlights

• Sweet sorghum juice a potential carbon source for 1, 3-PDO production.

• Strain adaptation improved 1, 3-PDO production by Lactobacillus brevis NIE9.3.3.

• Maximum 1, 3-PDO titer is 38.4 g/L.

Abstract

Sweet sorghum juice (SSJ) is considered as an ideal complement for carbon supplement in ethanol fermentation for its ease of cultivation. Extraction of fermentable sugars from the sweet sorghum is very simple in comparison to lignocellulosic biomass. Hence sweet sorghum is a suitable candidate as a feedstock. In the present study, batch fermentations were carried out using Lactobacillus brevis NIE9.3.3, a facultative anaerobe, isolated through onsite enrichment technique to produce 1,3-propanediol and other co-metabolites, in glucose-glycerol co-fermentation. To make the process more sustainable, the glucose supplemented in the production media was replaced with SSJ. The supplementation of 40 g/L sorghum juice and 40 g/L crude glycerol in the production media resulted in the titre of 25.9 g/L 1, 3-PDO with a volumetric yield of 0.64 g _1,3-PDO/g glycerol. Adaptation of the microorganisms and cultivation under controlled conditions of temperature and substrate concentrations followed by selection was carried out, that is, adaptive evolution. Among the adaptively evolved strains, PD 20.100 has displayed better performance and increased the titres up to 38.4 g/L with a volumetric yield of 0.64 g _1,3-PDO/g _glycerol. The industrial applicability of the fermentation process was checked in pilot scale and the production yield was comparable with that of flask scale. The utilization of agricultural and biodiesel industrial waste for the production of 1,3-PDO by a non-pathogenic organism and the strain improvement through ALE for better utilization and conversion of substrates indicates the novelty of this work.

Introduction

The conversion and utilization of renewable feedstock for the production of valuable materials is an important research area in recent years because of the decreasing supply of non-renewable resources, increasing global energy demand and negative environmental impact [24]. Glycerol is a by-product of biodiesel industries where 1 ton of raw glycerol is produced for every 10 tons of biodiesel. The crude glycerol produced from the biodiesel industry contains impurities such as alcohols, salts, heavy metals and water [8]. Sweet sorghum juice is a well-established carbon source for the production of ethanol. SSJ contains an adequate amount of sugar content which can be readily utilized by the microbes for fermentation. Sweet sorghum, from which SSJ was derived, is considered as an eligible option as a feedstock for fermentation processes due to the high content of sugars and cultivation conditions. It can also withstand the driest seasons which make it highly drought-resistant [22], [26]. The management of by-products from the biodiesel industry and unexpended agro-residues are better options as the carbon source in microbial growth media for the production of metabolites such as citric acid, succinic acid, propionic acid and 1,3-PDO [9]. Among these chemicals, 1, 3-propanediol has attracted more attention worldwide due to its vast application.

1,3-PDO has numerous applications in polymers, cosmetics, foods and adhesives [2]. The major application of 1,3-PDO is the synthesis of polytrimethyleneterephthalate (PTT), a new group of polyester having application particularly in textile and fibre industries due to its superior stretching and stretch recovery characteristics [10]. The chemical synthesis of 1,3-PDO has many disadvantages in that it requires high pressure, high temperature and catalysts. So as an alternative to chemical synthesis, biological methods can be adopted. DuPont is producing 135 g/L of 1,3- PDO using a genetically modified E. coli strain using corn sugar as the sole carbon source [14]. Bioconversion of glycerol to 1,3-PDO involves a coupled oxido-reductive process, yielding more reducing equivalents and energy molecules like ATP for growth and secondary metabolite production [25]. The production of 1,3-PDO from glycerol is a reduction process that consumes 1 mol NADH per mol of 1,3-PDO. In the reductive pathway, the first enzyme, adenosylcobalamin dependent glycerol dehydratase, GDHt (E.C.4.2.1.3.0), removes a water molecule from glycerol to form 3-HPA (an intermediate in 1,3-propanediol synthesis) by a radical mechanism [23]. 3-HPA is further reduced to 1,3-PDO by NADH+ H+ -dependent enzyme 1, 3-propanediol dehydrogenase (1,3-propanediol-oxidoreductase) [1]. The conversion of glycerol to dihydroxyacetone is catalyzed by NAD+ -dependent glycerol dehydrogenase in the oxidative pathway. The phosphorylation of the latter product is catalysed by the enzyme dihydroxyacetone kinase and finally glycolysis [3], [31]. The by-product formation is different based on the organisms and culture conditions. The change in NADH/NAD+ ratio can influence the metabolite distribution as mediated by the oxidation state of the carbon used [20]. Lactobacillus sp. which are non-pathogenic, having probiotic applications in food and pharmaceutical industries are found to be native producers of 1,3-PDO. Among them, L. reuteri , L. panis and L. diolivorans were reported for 1,3-PDO production, whereas L. diolivorans was considered being industrially relevant strain producing high titres of 85 g/L 1,3-PDO [17].

Various physical and chemical factors affect product formation during fermentation [6]. The microbial processes have certain disadvantages such as high cost, low yields, productivity and tedious downstream processing. The cost of the process depends on the cost of the organic and inorganic supplements in the production media, cost of operation and efficiency of bioconversion. Selection of the cheap raw material and its efficient utilization are the critical factors in the economization of the product [13]. To increase the efficiency of the process, adaptive laboratory evolution can opt which reduces the inherent intolerance of the biocatalytic cell towards the contaminants in the feedstock [5]. It is performed by allowing the microbial cells to grow at a specific condition for the long term and selecting the strain which fulfils the criteria the best. It has been practised widely for the improvement of secondary metabolites with major industrial applications [19].

In this study, the microbial production of 1,3-PDO by a non-pathogenic strain, L. brevis N1E9.3.3 using SSJ as a carbon source along with crude glycerol was evaluated. Based on the analysis, pH optimization studies have been done to increase the bioconversion and yield. Adaptive laboratory evolution (ALE) was adopted to improve the substrate uptake by the microbial cell for better production of metabolites. For the best results, pH studies were done on the selected ALE strain. Batch fermentation was carried out in Schott bottles under anaerobic condition with varying concentrations of glucose and crude glycerol to check the production of 1,3-PDO and other co-metabolites. The comparative analysis between wild type and evolved strains were done. To check the reproducibility of results from 100 mL flasks to pilot scale, batch fermentation has been carried out in a bioreactor. This study validates the efficiency of L. brevis N1E9.3.3 to utilize SSJ as the carbon source to form end products such as 1,3-propanediol, lactic acid and acetic acid. The improved efficiency of ALE strain for the better 1,3-PDO yield was also evaluated.