Locust Gut Microbiota and Biofuel Production Research

Introduction and Research Objectives

This study was conducted with the following aims and objectives: (1) to dissect locust guts; (2) to culture anaerobic microorganisms (bacteria) from locust guts on cellulose-containing media and screen for microbes with cellulolytic activity; (3) to extract DNA from cellulolytic microorganisms; (4) to perform PCR of 16S rDNA to identify the bacteria; and (5) to clone and sequence the PCR product. Together, these objectives aim to enhance understanding of how locust guts could be used to produce cost-effective and clean biofuels for energy use.

Locusts are species found principally in desert environments. They are short-horned grasshoppers capable of forming mobile swarms that can spread across up to 20% of the earth's landmass. A typical swarm can comprise 30 million locusts and travel at 80 miles per day. A locust swarm is capable of consuming the same amount of food that several thousand people could eat in a single day.

The ability of locusts to break down plant mass rapidly motivated this study to investigate the role of locust guts in breaking down lignocelluloses, which may lead to the discovery of new microbes in the locust gut for the commercial production of biofuels (U.S. Department of Energy, 2008). By identifying microorganisms inside the locust gut, this study aims to culture those bacteria and exploit their cellulolytic activity. Shi et al. (2010) argue that the symbiotic microbiota of locust guts can be exploited for biotechnological purposes. Locust guts harbour an abundance of microorganisms that play important roles in development and resistance to pathogens; loss of these bacteria can reduce the fitness of the insect host. The roles of these microorganisms include lignocellulose digestion, methanogenesis, acetogenesis from H₂ and CO₂, nitrogen fixation, maintenance of low redox potential, and prevention of foreign microbial colonisation (Feng, Wang, Zhou et al., 2011, p. 2). Cacemier (1969) estimates that bacterial counts inside locust guts reach between 70 and 80 million in the foregut, 11 million in the midgut, and between 33 and 44 million in the hindgut. Culturing these bacteria will provide a greater understanding of the potential for these microorganisms to produce digestive enzymes capable of supporting mass biofuel production.

Metagenomic DNA can also be extracted from locust guts to evaluate their microbial communities. Appropriate DNA extraction methods are critical to achieving an unbiased evaluation of metagenomes, particularly for unculturable microorganisms. This study implements metagenomic DNA that is free of contamination, suitable for downstream processing including polymerase chain reaction (PCR), vector ligation, and enzyme digestion. Two common strategies for metagenomic DNA isolation are direct analysis methods and cell recovery methods. Commercial kits are now available that simplify cultivation-independent analysis; these include the GenElute Bacterial Genomic DNA Kit, Qiagen DNeasy Tissue Kit, QIAamp DNA Mini Kit, Wizard™ Genomic DNA Purification Kit from Promega, and PowerSoil™ DNA Isolation Kit (Shi, Syrenne, Sun et al., 2010, p. 2002). However, these kits were not designed specifically for metagenomic DNA isolation from insect guts. Traditional extraction techniques remain important for understanding the function and composition of locust gut symbionts. In this study, DNA extraction from locust guts was achieved by boiling a bacterial cell suspension in sterile distilled water in a total volume of 50 µL containing 5 µL of DNA extract, with 2 µL of 10 µmol/L used for each primer (Stokes, Holmes, Nield et al., 2001).

A further objective of this study is to apply 16S rDNA PCR to identify bacteria from the locust gut. Gene targeting of the conserved 16S rRNA gene plays an important role in the metagenomic analysis of locust gut microbial communities and has been used to understand symbiotic microbiota composition. Analysis of the 16S rRNA gene is also important for characterising the phylogenetic diversity of bacteria in the locust gut. The 16S rRNA isolated from locust guts has yielded bacterial strains classified within Brevibacterium, Corynebacterium, Stenotrophomonas, Staphylococcus, Bacillus, and Klebsiella.

Cloning and sequencing of PCR products is also important for understanding gene discovery and the microbial communities of locust guts. PCR and microbial diversity analysis together provide insights into locust gut species composition. Although PCR is highly effective for microbial diversity analysis and gene discovery, its effectiveness depends on PCR amplification efficiency, which can limit its application. Additionally, PCR can be used for both partial cloning and full-length cloning through PCR-based chromosomal walking approaches (Cowan, Meyer, Stafford et al., 2005).

Understanding the properties of cellulose, hemicellulose, and lignin is essential to understanding the metabolic potential of locust guts and to developing methods for mass biofuel production.

Cellulose, Hemicellulose, and Lignin as Biofuel Feedstocks

Cellulose is an insoluble, linear biopolymer composed of repeating β-D-glucopyranose residues linked by β-1,4 glycosidic bonds. Cellulose is not a simple sugar but a disaccharide unit known as cellobiose. It can exhibit a high degree of polymerisation, and individual glucan chains can reach lengths greater than 25,000 glucose residues. Cellulose is highly resistant to degradation; plant-derived cellulose contains large voids and highly amorphous regions. Although cellulose is produced primarily by plants, certain bacteria, algae, and animals also produce the polymer. Under aerobic conditions, cellulose is degraded into H₂O and CO₂; under anaerobic conditions, it is degraded into CH₄ and H₂. Cellulolytic species are found within the phyla Proteobacteria, Actinobacteria, Thermotogae, Bacteroidetes, Spirochaetes, Fibrobacteres, and Firmicutes, with approximately 80% found within the Firmicutes and Actinobacteria (Bergquist, Gibbs, Morris, 1999).

"Cellulose is the most abundant biopolymer in the world. Consolidated bioprocessing (CBP) is a system in which cellulase production, substrate hydrolysis, and fermentation are accomplished in a single process step by cellulolytic microorganisms" (Carere, Sparling, Cicek et al., 2008, pp. 1342–1343). Discarded cellulose biomass can be derived from agricultural, forestry, and municipal sources, providing feedstocks for biofuel synthesis. The annual global production of cellulose is estimated at approximately 7.5 × 10¹⁰ tons, generated through photosynthetic processes. Cellulose is also found in plant cell walls alongside lignin, hemicellulose, and xylan.

Biofuels can be produced from different sources, including food crops and cellulosic substrates. Food crops such as sugar beets and sugarcane provide approximately 60% of total global bioethanol production. Other crops, including wheat, cereal crops, and corn, can be converted to glucose through fermentation.

"The term hemicellulose refers to a group of homo- and heteropolymers consisting largely of anhydro-β-(1-4)-D-xylopyranose, mannopyranose, glucopyranose, and galactopyranose main chains with a number of substituents" (Jeffries, 1994, p. 234). Hemicellulose is typically found in association with cellulose and is primarily present in plant cell walls, including as a component of softwoods (glucomannan) and hardwood hemicelluloses (Ratledge, 1994).

Hemicelluloses are important heteropolymers, including arabinoxylans, present alongside cellulose in plant and wood cell walls. Varieties of hemicellulose include pentoses such as xylose and arabinose, and hexoses including glucose, mannose, galactose, rhamnose, and corresponding uronic acids. The major industrial application of hemicellulose is its conversion into biofuels, though this application has not yet been exploited at mass scale. Jeffries (1994) notes that hemicellulose composition depends on plant species, age, stage of growth, and environmental conditions.

Lignin is a chemical compound commonly derived from wood, cells, fibres, and vessels of lignified plant elements. After cellulose, lignin is the most abundant renewable organic material, with between 40 and 50 million tons produced annually. Lignin is composed of building blocks including coniferyl alcohol, p-coumaryl alcohol, and sinapyl alcohol. Its inter-monomer linkages differ among hardwood, softwood, and grass lignins. Lignin degradation can provide insights into enzymatic mechanisms and pulp bleaching reactions. Despite its global abundance, lignin is poorly degraded by most microorganisms due to its complex structure.

The Role of Locust Gut Microbiota in Biofuel Production

Given the large global production of cellulose, hemicellulose, and lignin, and their importance as biofuel feedstocks, locusts — through their gut microbiota — may provide a means of breaking down the complex cellulolytic structures, enabling large-scale biofuel production.

Predictions of a 54% increase in worldwide energy consumption between 2001 and 2025 have driven considerable interest in bioenergy production to meet future needs. Energy derived from biofuels is produced from the metabolism of living organisms. Scientists and environmentalists have identified biofuels as one of the most promising alternatives to petroleum and fossil fuels. Biofuels are derived from biomass materials initially in solid form and later converted to liquid or gaseous fuels for storage and use (Groom, Gray, Townsend, 2008).

Cellulose, hemicellulose, and lignin are abundant on earth and can be converted to biofuels; however, large-scale production remains limited in many countries. A significant challenge for commercial biofuel production is the cost of breaking down fibrous plant material. The destructive capability of locusts offers a potential solution: their gut microbiota could enable breakdown of tough cellulosic material. This study explores the locust gut wall to identify metagenomes and microbial communities that are capable of digesting lignocelluloses. Identifying these microbes will enhance understanding of cellulose degradation, and the resulting sequence data will be useful for identifying enzymes suited to commercial biofuel production.

Rinke et al. (2011) note that a key challenge in biochemistry is developing processes to break down lignocellulosic biomass, including cellulose, hemicellulose, and lignin. The locust gut has been identified as a system possessing mechanisms capable of performing this breakdown. "The desert locust, Schistocerca gregaria, possesses an abundant gut microbiota consisting predominantly of Enterobacteriaceae" (Rinke et al., 2011, p. 2689). The locust gut wall contains microbes capable of rapidly digesting cellulose, and cellulolytic activity identified in the locust gut enhances lignocellulose digestion.

Kurtböke and French (2008) also reveal that termite guts are significant contributors to the biodegradation and biorecycling of lignocelluloses, relevant to the biofuels industry. Approximately 2,000 species of termites exist globally, and analysis of termite guts has shown production of cellulase, ligninase, xylanase, and keratinase in considerable quantities. The authors therefore argue that an efficient biorecycling system using termite guts could contribute to biofuel production.

This study offers several contributions to the field. First, it enhances understanding of locust gut composition and the microorganisms present within it. Willis et al. (2010) argue that locusts, compared with other insects, exhibit abundant enzymatic activity against tough cellulosic substrates. The digestive fluids of locust guts are particularly important for breaking down lignocellulosic biomass. Second, the study advances understanding of strategies to reduce the high costs associated with breaking down tough cellulose; utilising locusts for this purpose will lower costs and improve cost-effectiveness in biofuel production. Third, the study contributes to the body of knowledge on using locust guts for mass biofuel production. Given the continuous rise in global petroleum prices, biofuels could serve as an alternative transportation and industrial fuel, potentially helping to reduce petroleum prices worldwide.