Screening the gut of dung beetles and dung beetle larvae for hemi-cellulolytic fungi and enzymes for application in the biofuel industry

Abstract

Biofuel production from lignocellulose material is an attractive alternative to fossil fuel. The use of lignocellulose material for biofuel production is imperative because of the numerous advantages that it offers. Biofuel is environmentally friendly and in developing countries such as South Africa, it has the potential to reduce the use of imported fuel and create jobs. Currently, several constraints are affecting the implementation of biofuel. One of the constraints is the cost-effectiveness and the efficiency of the enzymes involved in the enzymatic degradation of lignocellulose polymers to monomers, which can further be fermented to bioethanol. The potential way to reduce enzymatic degradation cost could be by supplementing the fungal enzymes with accessory enzymes such as endo-xylanase. The enzyme production cost is also dependent on the carbon source used. Lignocellulose materials that are regarded as waste must be assed for their use as enzyme inducer carbon sources and as biomass for biofuel production. This is a potential route that will reduce enzyme and biofuel production costs. Biofuel production cost can further be reduced by finding a yeast that can ferment xylose and ferment in the presence of inhibitors released during lignocellulose pretreatment. This study sought to tackle the enzymatic hydrolysis constraints and also search for xylose-fermenting yeast by exploring the gut microbiota of dung beetle. The gut of the dung beetle has recently received great attention since it is proposed to be a bioreactor for lignocellulolytic microorganisms that can be used in biofuel applications. This is because dung beetles feed on the dung of herbivorous animals and the dung is composed of 80% undigested plant material. In this study the guts of four Scarabaeidae dung beetles Kheper nigroanaeus Boheman, Heteronitis castelnaui, Pachylomerus femoralis, Anachalcos convexus and dung beetle larvae, Euoniticellus intermedius were screened for hemicellulolytic fungi and xylose-fermenting yeast. Hundred and thirty-two yeast isolates and two-hundred and twenty-two filamentous fungi were isolated and identified using ITS and D1/D2 regions. The yeast isolates were assigned to 8 genera and 18 species, Trichosporon was the most dominant genus while Candida tropicalis was the most dominant specie. Some of the yeast isolates were identified as uncultured fungi. This yeast must be characterised to be certain if they are novel species. The fungal isolates were assigned to 12 genera and 25 species, Aspergillus was the most dominant genus while Hypocrea lixii was the most dominant specie. The yeast isolated could assimilate xylose and could grow at a maximum temperature of 40 °C. Furthermore, these yeast isolates could also grow in the presence of 3 g/L acetic acid. Most of the fungal isolates had xylanolytic activity. The phylogenetic analysis revealed close genetic relatedness between isolates from the different dung beetle species and dung beetle larvae. The profile of the fungal genera was similar in the different dung beetles. Both guts and the larvae had Aspergillus, Hypocrea, Trichoderma, Talaromyces and Penicillium. The filamentous fungi that showed good xylanolytic activity were further screened for their ability to produce xylanase enzyme using thatch grass as an inductive carbon source. Thatch grass was selected in this study since it is in-house plant-based biomass. Thatch grass is abundantly available in South Africa; it is used for animal grazing but the more it grows it loses its nutritional content. Once it reaches this stage, it is no longer used and most of it is burnt. The fire from burning grass contains higher levels of nitrogen-containing chemicals that pollute the environment. Its compositional analysis (cellulose 46%, hemicellulose 27% and lignin 10%) also attributed to its selection as potential inductive carbon and attractive lignocellulose biomass for biofuel production. The higher xylanase activity of 283.43, 270 and 287.03 nkat/ml were observed from Aspergillus fumigatus L1XYL9 (Euoniticellus intermedius larvae), Hypocrea lixii AB2A3 and Neosartotya sp AB2XYL20 (Anachalcos convexus), respectively. This was achieved when acid pretreated thatch grass was used as an inductive carbon source. Aspergillus fumigatus L1XYL9 (Euoniticellus intermedius larvae), Hypocrea lixii AB2A3 and Neosartotya sp AB2XYL20 (Anachalcos convexus) showed xylanase activity of 393,22, 313,06 and 200 nkat/ml when grown on synthetic xylan. Neosartotya sp AB2XYL20 showed higher xylanase activity on thatch grass. The suitable production process for xylanase enzyme on acid pretreated thatch grass was assessed by conducting a comparative study on solid-state and submerged fermentation using L1XYL9 (Euoniticellus intermedius larvae), Hypocrea lixii AB2A3 and Neosartotya sp AB2XYL20 (Anachalcos convexus) as the best xylanase producer on acid pretreated thatch grass. The strain showed better xylanase activity when submerged fermentation was used. In this study, Hypocrea lixii AB2A3 was selected for further studies since it was the most dominant species and also showed good xylanase activity. Thatch grass was pretreated differently to evaluate the suitable chemical for pretreating thatch grass. Thatch grass was pretreated with dilute sulphuric acid 1.2% and maintained the pH of 5.5 by using sodium hydroxide while another batch was pretreated the same way and was washed with distilled water till pH of 5.5. The other batch was then pretreated with ammonium solution and was also washed with distilled water to maintain a pH of 5.5. The above-mentioned pretreated thatch grass was tested as an inductive carbon source as well as untreated thatch grass. The xylanase activity was determined to assess a good inductive carbon. All the thatch grass pretreated and washed with distilled water showed very low xylanase activity. The untreated thatch grass resulted in lower xylanase activity as compared to xylanase activity achieved when pretreated thatch grass was used. Parameters such as agitation speed and initial inoculum size were also assessed during xylanase production by Hypocrea lixii AB2A3 on acid pretreated thatch grass. Xylanase activity increased from 525 nkat/ml (Inoculum size 2×106 spore/ml and agitation speed 150 rpm) to 584.8 nkat/ml (Inoculum size 2×106 spore/ml and agitation speed 200 rpm). The crude xylanase from Hypocrea lixii AB2A3 was used to hydrolyse acid pretreated thatch grass. This resultant in xylose yield of 138 mg/g of substrate and glucose yield of 49 mg/g of substrate. Crude xylanase was mixed with commercial celluclast™. This enzyme mixture resulted in a xylose yield of 128 mg/g substrate and a glucose yield of 549 mg/g of substrate. The results obtained in this study show that indeed gut of the dung beetles and dung beetle larvae are a rich source of microorganisms that can play an important role in biofuel application and remediating the environment by degrading plant-based biomass regarded as waste into valuable products. It is imperative to evaluate the gut microbiota of dung beetles from different regions in South Africa for their application in the biofuel industry to reinforce its implementation. Thatch grass is a potential inductive carbon and lignocellulose biomass for biofuel production.