Expected Output: 1 Journal Papers and 1-2 Invention Disclosure

Research plan

Mythreyi chandoor 4/12/2008

Project: Studying the lignocellulosic degradation in soil and characterization of the microcosm responsible for the lignin digestion. Design of a Bioreactor based on this natural process for enhancing the lignocellulosic degradation and Biofuel Production.

Expected output: 1 journal papers and 1-2 invention disclosure.

Literature review (Background):

Soil organisms have long been recognized as the agents of nutrient transformations, and therefore play a key role in soil fertility and ecosystem functioning. In order to determine pools and fluxes of nutrients, knowledge of the quantity and activity of soil microbial biomass is needed. Size of the soil microbial biomass pool has implications on microbial growth efficiencies, immobilization of carbon, nitrogen, and other nutrients, rates of turnover of soil microbial biomass, microbial energetics, and modeling of soil organic matter dynamics. Measurement of soil microbial biomass, while always of interest to soil microbiologists, became an ever increasing topic of scientific investigations with the development of relatively simple and rapid, yet integrative protocols, specifically chloroform fumigation-incubation (measures the flush of CO2 or inorganic nitrogen during a 10-day incubation by surviving organisms following killing of most organisms with exposure to chloroform), substrate-induced respiration (measures the immediate response in respiration of CO2 to an easily decomposable substrate such as glucose), and adenosine triphosphate (measures the ATP content of soil organisms as an indicator of potential metabolic activity). These methods were the first to holistically quantify the entire soil microbial population as a single entity. Previously, labor-intensive methods were used to quantify soil microbial biomass, including plate counting and direct observation under a microscope. Since the development of these holistic methods in the 1970s, numerous other biochemical approaches have been developed to either improve the characterization of the more active portion of the microbial community, to reduce analysis time, or to quantify other nutrient

Components within the biomass, e.g., arginine ammonification, chloroform fumigation-extraction, rehydration-extraction, microwave irradiation-extraction, hot water extraction, and rehydration-mineralization

Soil is composed of different biotic and abiotic systems which help in degrading the organic matter present in the soil .The Biotic system consists of large microbial population which is also called as microcosm which have the potential to degrade the tough plant cell wall materials such as lignin, cellulose and hemicelluloses. Lignin is the most abundant aromatic polymer in nature. It is synthesized by higher plants, reaching levels of 20–30% of the dry weight of woody tissue. Although white-rot fungi were long recognized as efficient lignin-degrading microbes, research on their enzymology and genetics is still going on. The impetus for increased research interest can be traced to the discovery of “ligninases” and potential commercial applications in the pulp and paper industry and in the degradation of xenobiotics.

Research on lignin biodegradation has accelerated greatly during the past 20 years, mainly because of the substantial potential applications of bioligninolytic systems in pulping, bleaching, converting lignin’s to useful products and treating of agricultural wastes using bacteria. Isolation and identification of environmental friendly microorganism for lignin degradation becomes an essential, because the focus is on the efficiency of the lignin degradation.

Soil biology represents a diverse group of organisms that reside during at least a part of their life cycle in the soil. These organisms vary widely in size from macrofauna > 10 mm in length (earthworms, spiders, beetles, mice, moles, etc.) to micro and mesofauna <10 mm in length (protozoa, nematodes, etc.) to microscropic forms of bacteria, fungi, and algae. Soil organisms can be primary producers of organic materials (e.g., phototrophic algae and bacteria), but more commonly are heterotrophic consumers of preformed organic materials. These heterotrophic organisms are essential in the cycling of nutrients and transfer of energy following the senescence of plant materials. Soil organisms also play major roles in soil formation and soil structural development by forming biotic pores, transforming soil minerals and organic matter into stable aggregates and catalyzing mineral weathering processes.

Decomposition of organic material:

All heterotrophic organisms are involved in the consumption and breakdown of organic materials. Decomposition of organic matter is an integral part of the global carbon cycle, where photosynthesis and respiration are dominant fluxes Soil mesofauna and macrofauna often fragment plant and animal residues, which increases the surface area and exposes internal constituents to soil microfauna and microflora for further attack. Larger soil organisms, therefore, stimulate soil microbial activity and also distribute smaller organisms within soil as a result of their generally greater range of mobility. In addition, larger soil organisms such as earthworms, ants, and beetles physically move organic substrates from the soil surface to within the soil, which can enhance decomposition by placement in a more favorable zone for microbial attack because of less extreme moisture and temperature variations.

Factors affecting decomposition in soil are environmental conditions (i.e., temperature, aeration, and moisture, supply of nutrients, pH, and redox potential) and chemistry of the substrate (i.e., distribution of easily decomposable and resistant fractions, particle size and surface area, presence of microbial inhibitors, and content of non-carbonaceous nutrients such as nitrogen and phosphorus). In general, water and oxygen need to be balanced for most rapid decomposition However, halogenated compounds such as vinyl chloride and various solvents decompose more rapidly when exposed to alternating aerobic and anaerobic environments.

When inorganic nitrogen is low in soil and residues with high carbon-to-nitrogen ratio are incorporated in soil, microbial activity is limited by the availability of nitrogen for growth of soil organisms. Incomplete decomposition of organic matter can lead to accumulation of resistant compounds in soil, collectively termed humus. Rate of decomposition of primary plant constituents follows the order: water soluble components > cellulose, hemicellulose, and fats > lignin and waxes. Cellulose is a carbohydrate composed of a linear chain of ß 1-4 linked glucose units. Cellulose decomposition occurs under both aerobic and anaerobic conditions. Aerobically, various fungi and some facultatively anaerobic bacteria convert cellulose to carbon dioxide, water, and cell biomass with the aid of three types of enzymes, i.e., C1, Cx, and ß glucosidase. Hemicellulose is a polysaccharide composed of pentoses, hexoses, and/or uronic acids. A variety of fungi and bacteria produce both endoenzymes (which cleave bonds within the polymer) and exoenzymes (which cleave monomers and dimers from the end of the polymer).

Decomposition products of hemicellulose include carbon dioxide, water, cell biomass, and a variety of small carbohydrates. Chitin is an amino sugar found in fungal cell walls and insect skeletons. Chitin is decomposed by various fungi and bacteria with the aid of chitinase enzymes into N-acetylglucosamine and chitosan. Lignin is an aromatic compound composed of repeating benzene rings that are branched and complex. The aromatic structure of lignin makes it difficult to decompose. Only a few fungi and bacteria have the capability to decompose lignin, requiring first depolymerization into smaller aromatic acids and alcohols, side chain removal and methoxyl group oxidation, and finally ring opening.

Research has been done in the fields of soil microbial characterization, but the relation between the microcosm involved in mulch degradation, specifically lignin degradation is still going on.

Wheat (Triticum aestivum L.) straw residues contained in fiberglass screen envelopes were exposed to field conditions at Bozeman and Huntley, Mont. Samples from above-soil, on-soil, and buried exposures were taken periodically during an 18-month period to measure weight losses and changes in N and P contents of the residues. Percentage losses were inversely related to residue amounts. After 18 months, on-soil residue losses averaged 31% at Bozeman and 40% at Huntley. The 50% decomposition stage for buried straw occurred in 3 months at Huntley and 6 months at Bozeman, and after 18 months, losses of buried straw averaged 93% at Bozeman and 98% at Huntley. Compared to the original straw, both N and P percentages increased as much as six fold in the buried samples during decomposition. For buried straw, maximum percent N was 1.18 at Bozeman and 1.46 at Huntley. Similar values for maximum percent P were 0.132 at Bozeman and 0.147 at Huntley.

This proposal mainly focuses on the microcosm and its environment which enhances and degrades lignocellulosic content in Biomass.

Targets:

The major problem in the wood degradation process is a lack of understanding the efficient mechanism which can lead to a total lignin degradation .Several studies have already been done for the characterization of the microbial community and their enzyme complex degrading these substrates naturally and in the lab scale. However, the efficient mechanism is still yet to be discovered. Therefore, the study has been designed under the following objectives: (1) Finding out the microbial mixture responsible for the digestion of dry grasses and wood chips; (2) Finding out the environmental conditions such as varied temperature, pH, moisture content and other mineral components which enhances the dry grass digestion and; (3) Incorporation of engineering methods to mimic soil microbial pathways in processing dry grass and wood chip digestion into a bio-reactor focusing on lignincellulose digestion.

Anticipated Problems statements:

The isolation and characterization of the specific microorganism responsible for lignocellulosic degradation from the whole set of microcosm might be effected by mixed culture or overgrowth of the mixed population. The calculation of exact percentage of degradation in mulch might be bit difficult as the soil is not homogeneous matter and separation of total mulch from soil is a tough task.

Experimental plan:

This is a tabular form of the different sections of the experiment to be carried out

Materials: Hungate tubes; 40 pint jars (wide mouthed bottles, analytic chemicals, molecular biology reagents etc).

Equipments: DNA and protein electrophoresis devices, 30°C incubator, -80°C freezer, florescent microscope etc.

Methodology (details only for the first subproject)

These are basically divided into 5 steps.

Soil analysis:

Two types of soil have been selected: Palouse soil & Lind soil. The soil layer which is taken is about few cm depths in order to avoid the top most layer of soil which is affected by soil erosion

1. Predetermination of soil characteristics: Predetermination of soil characteristics such as pH,percent,moisture,concentrationofmineralssuchassulfur,nitrogen,magnesium,potassium,sodium,and composition mixture of sand clay and silt. Soil analysis and predetermination of soil characteristics is done by Department of soil
2. Mulch preparation: The mulch mainly contains wood chips, wheat straw, dry grass. First pretreatment is initially done and then equal amounts of these three components are mixed and put in separate bags and weighed. Incubation period of the mulch will be 4 months. At an interval of 4 weeks the weight of each bag is checked and the amount of degradation is calculated by using methods such as NDF (neutral detergent fiber) ADF (acid detergent fiber) ADL (acid detergent lignin). For every incubation four jars will be used. Therefore total number of jars will be 40
3. Initial soil sample analyses for microcosm studies: These studies will be based on basic as well as selective media for bacteria and fungi. Selection of isolated colonies for further plating them in poly dyes agar plates.
4. Designing a bioreactor: Analyzing these facts based on the data collected and understanding it in a way to design a bioreactor for lignocelluloses digestion.

Anticipated results

The first subproject is to find out the microcosm and its environment needed for lignocellulosic degradation. At least one paper is expected to be published and an invention is expected to be made on the new microbial population which has a significant property to degrade the lignocellulosic biomass in soil environment. The time will be 6 months.

The second subproject is to improve the micro organism enzymatic ability or to construct a mutation platform on particular isolated micro organism in order to enhance the efficiency. At least two papers will be published but the job seems more difficult.

The third subproject bases on the first two ones and is expected to improve the strain for industrial using. It will cost at least four years from now.

Time line

Only for the first subproject

Needs for facility, equipments, other assistance and funding

Potential collaborators

Prof. Dr. Shulin Chen and the members of ethanol group.

Ann Kennedy, Department of soil sciences

Other collaborators may be needed.

Reference:

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