Isolation and characterization of novel enzymatic activities from gut metagenomes to support lignocellulose breakdown

The agricultural sector produces a large amount of organic waste as by-products (crop remains, foliage, seed pods, straw, etc.). Currently, these materials are not properly treated and their uncontrolled disposal can lead to many problems. In many countries crop remains are burned on the field, sometimes causing severe air pollution as well as damage health. This contributes to climate change and could impact the climate further to the point of irreversible damage. To realize a sustainable agriculture, organic wastes should not be disposed or burned but used as a cheap source for biomaterials. In line with the philosophy of the bio-based economy, agricultural waste is recycled and used as raw material in the chemical industry, replacing fossil fuels. However, the process of converting agricultural waste into useful products is not efficient and can be improved further for optimization. One of the dominant components of agricultural waste is lignocellulose, a complex biomaterial that is difficult to handle. To break down this complex structure, large amounts of energy or chemicals for treatment are required. This thesis aims to explore novel natural biological catalysts that can help to degrade lignocellulose and deliver useful compounds for the bio-based chemical industry. To discover such novel catalysts, the digestive systems of a number of different animals: goats, springtails, isopods and termites were investigated. Animal guts are mini ecosystems that contain many unknown interesting bacteria. These microorganisms are adapted to the host and might have interesting properties that can be explored. By looking at these organisms and their catalysts it is possible to identify and mine novel genes that can be used to breakdown biomass. This process creates substrates that can be used for many other procedures. In this thesis my main focus was on enzymes that can break down carbohydrates. The various bonds in complex carbohydrate molecules are cleaved by different enzymes. Every bacterium has a suit of carbohydrate-active enzymes, called CAZymes. Using metagenomics and bioinformatic tools to explore the genomes of microbial communities in search of novel CAZymes. Unlike traditional culturing methods, metagenomics is aimed at the whole genome of the communities involved, that is, all bacteria jointly. In addition, genes encoding antibiotic resistance, and the production of secondary metabolites were investigated since these two gene categories greatly contribute to the survival of bacteria in complex microbial communities. The work on this thesis shows the possibilities of using bioinformatic tools to investigate the microbiome communities and mine for interesting enzymes from metagenomes. The bacterial community appears to be very diverse and different between hosts. However, at the enzymatic level there is a core group of carbohydrate enzymes and antibiotic resistances. Some of the studied enzymes show the potential for bio-applications. The method could be further tested on different animal metagenomes. With the expansion of sequencing and bioinformatic tools as well as advances in computing and machine learning, it is possible to use and understand more about the natural environment. These tools could help to mine enzymes with interesting properties. Together with enzyme characterization, the bioinformatic tools could be improved further. When the whole process is streamlined and part of a pipeline, a large number of enzymes can be identified and tested to find optimal conditions for their action. These enzymes can be combined together to create cocktails, which can efficiently breakdown biomass. Since the enzymes are natural and the products are used as substrates, little energy and resources are wasted. Beneficial enzymes and bacteria are also preserved and promoted. By creating a recycling plant, agricultural waste can turn into new substrates and products, which in turn can help to improve the environment.