Showing papers in "Basic life sciences in 1981"

Xylanases: structure and function.

Abstract: A number of papers in this seminar are devoted to the production and characterization of cellulases. This is proper, since the use of cellulases is perhaps the most feasible method of converting cellulose to products that may be used for fuels and chemicals. However, all celluloses from hardwoods and grasses are associated with significant amounts of xylan, a hemicellulose possessing a β-l,4-linked xylose backbone, with branches containing xylose and other pentoses, hexoses, and uronic acids (Figure 1).

Thermophilic Ethanol Fermentations

Abstract: The cost and availability of petroleum and natural gas has generated interest in bioconversion processes that utilize renewable biomass resources for the production of fuels and chemical feedstocks. The bioconversion of biomass to ethanol via anaerobic fermentations offers the promise of renewable liquid fuel and renewable chemical feedstocks. The purpose of this presentation is to review some of the recent studies in my laboratory on thermophilic ethanol fermentations. The emphasis of this review will be on understanding fundamental aspects of the physiology and biochemistry of thermophilic anaerobes that may be of applied interest in developing bioconversion technology for alcohol production. Most of the findings summarized here represent material published elsewhere (1–22).

Cellulases: diversity amongst improved Trichoderma strains.

Abstract: In natural, mixed-culture fermentations of cellulose as well as in multistep conversion of cellulose to glucose, the depolymeriza-tion step seems rate limiting. Improvements in rate and yield of glucose from cellulose could greatly affect process economics (1–2). Indeed significant improvements have been achieved through pre-treatment (2–5), better strains (6–10), and process integration (11–12). As part of the Biomass Program at Cetus, we have attempted to increase the rate at which cellulose is utilized in fermentation processes by isolating organisms deregulated in the production of cellulase. For this work we have selected one of the most thoroughly studied of the cellulolytic organisms, Trichoderma reesei. This paper will focus on a few of our strains in order to illustrate their diversity both in the control of cellulase production and in the types and levels of the individual enzyme components. The data should give further support for the existence of multiple levels of control in Trichoderma. Finally, as a separate topic, cellulase productivity data will be presented for one of our best strains.

Some Aspects of Thermophilic and Extreme Thermophilic Anaerobic Microorganisms

Abstract: An interest for industrial use of thermophilic, anaerobic bacteria has clearly emerged since the 1973 oil shortages. Such bacteria are capable of converting biomass, mostly cellulose, hemicellulose and starch to desirable industrial feedstock chemicals such as acetate, ethanol, acetone, butanol, etc. (1). The thermophilic bacteria are also convenient sources of enzymes, which are more thermostable and more resistant toward denaturation when compared with corresponding enzymes from mesophilic microorganisms (2). Clearly, enzymes from thermophiles have properties which are desirable when considering industrial applications. The idea of using thermophilic microorganisms industrially is not new. For instance, several British patents since 1920 deal with fermentations of cellulose using thermophilic, aerobic, as well as anaerobic, microorganisms (3). Curiously, these efforts were to produce ethanol from renewable resources to be used as liquid fuel for combustion engines. This idea has now been rediscovered some sixty or more years after it was formulated. The cycle is complete. The problem for today is not whether we can or can not ferment biomass to desirable products but rather which are the best microorganisms to use, how can we improve them, and what is the best technology.

Cellulases of Fungi

Abstract: One of nature’s most important biological processes is the degradation of lignocellulosic materials into carbon dioxide, water and humic substances. The strong wood-degrading capability of fungi depends, in part, upon the organization of their hyphae, which gives the organisms a penetrating capacity. Different types of fungi give rise to different types of wood rot. One normally distinguishes between soft-rot, brown-rot and white-rot fungi. The blue staining fungi are also associated with wood damage. They do not, however, cause wood degradation. The morphological pattern of the attack on wood by these fungi varies. Thus, the soft-rot fungi grow in the secondary wall of the wood fiber and form cylindrical cavities with conical ends. This type of attack causes a softening of the wood surface layer which has given the name to this group of fungi.

The methanogenic bacteria, their ecology and physiology.

Abstract: A study of the methane fermentation is unavoidably concerned with a study of microbial ecology because of the obligatory interactions between two major physiological participants, the chemoheterotrophic non-methanogenic bacteria and the methanogenic bacteria. In natural anaerobic habitats containing complex organic compounds and where light, sulfate, and nitrate are limited, these two groups of bacteria are linked in the degradation of organic substrates. The ultimate formation of methane and CO2 marks the last step in a series of dissimilatory reactions by which organic compounds are completely degraded. CH4 is the most reduced form of carbon and CO2 the most oxidized form of carbon.

Regulatory Controls in Relation to Overproduction of Fungal Cellulases

Abstract: A growing trend in the development of viable processes for the conversion of renewable cellulosic biomass to glucose is the use of microbial cellulases as biological catalysts. Unfortunately, the cost of the cellulase enzymes has been prohibitive for large scale industrial application in saccharification of cellulose. The high cost of cellulase is due largely to the low yield and to the low specific activity of enzymes from the available microbial strains. Improvement of the cellulolytic microbial strains can be considerably enhanced through selective screening programs. However, the rationale for selection and the chance of isolation of more useful strains are hampered by our lack of understanding of mechanisms controlling the synthesis and secretion of cellulase. Successful genetic cloning and expression of the cellulase genes from Tviohodevma or any other cellulolytic microorganism is similarly dependent upon a sound basic knowledge of the control mechanisms. Tviohodevma genetics is an unexplored abyss. Although Tviohodevma is reported to have a sexual stage, in the Hypoovea (1,2) mating types are generally unavailable. Thus, traditional methods of delineating genetic linkages are elusive.

Feasible improvements of the butanol production by Clostridium acetobutylicum.

Abstract: Butanol as a fermentation product was discovered by Pasteur (7) in 1862 and the formation of acetone by a “Rottebacillus” was described by Schardinger (9) in 1905. Later a fermentation process for the production of acetone and butanol from carbohydrates was patented (4). Thereafter, Weizmann (13) isolated Clostridium acetobutylicum which was especially suitable for the production of these solvents from corn starch. A number of factories were operated on the basis of this fermentation in various countries. With the increasing availability of low-cost petrochemical raw materials and the growth of the chemical industries the fermentation process became uneconomical in industrialized countries and was discontinued. At present only a few plants are in operation, mostly in agricultural countries. Excellent reviews on the development of this process and on its operation have been published (2, 8, 10).

Formation of hydrocarbons by bacteria and algae.

Abstract: The chemical investigation of biologically synthesized hydrocarbons did not begin early in the history of the systematic study of fats. All the neutral or highly non-polar lipids were included in a category of compounds designated as waxes. The waxes were monoesters of fatty acids and long chain alcohols, hydrocarbons, long chain alcohols, and high molecular weight compounds. Systematic investigations into the derivation and chemical nature of the constituents of waxes was started in 1942 by the American Petroleum Institute Project 43 which was designed to determine a) the part played by microorganisms in the formation of petroleum, b) the type hydrocarbons synthesized as animal and plant products to the extent and variety necessary to be able to form crude oil and c) whether radioactive and thermal sources of energy can transform organic matter into petroleum. The rationale for this project was apparently based on a number of factors. In 18 99 it was proposed that complex organisms, such as trees, fish and animal fats could be a direct source of the hydrocarbons in petroleum (1). In 1906, the isoprenoid hydrocarbon squalene was isolated as the major constituent of shark liver oil (2–4). Diatom nobs in tertiary opal shales were reported in 1926 (5).

Metabolic compromises involved in the growth of microorganisms in nutrient-limited (chemostat) environments.

Abstract: Biochemical research, particularly over the past 50 years or so, has revealed ever more clearly the underlying unity of living processes. And this possibly has obscured to some extent the fact that there are nevertheless important physiological differences between microbial cells and, say, the cells of higher animals. One of the most fundamental of these, and one which undoubtedly has considerable evolutionary significance, is evident in the ways in which the different cells accommodate to environmental change. Clearly, the cells of higher animals have evolved to spend the whole of their existence in a closely regulated environment, and this is a condition of life for them. But microbial cells are markedly different. They generally are exposed to environments that fluctuate extensively (and often rapidly) and, being free-living creatures, they do not possess the capacity to regulate their surroundings. Instead, they respond to environmental change by changing themselves — structurally and functionally — and seemingly have acquired in the course of evolution a whole armoury of sophisticated control mechanisms whereby to effect such change.

End-Product Tolerance and Ethanol

Abstract: Fossil fuels, and in particular oil and its derivatives, are the source of the vast proportion of fuels and chemicals used today in developed countries of the World. However, fossil fuels have two insurmountable drawbacks which restrict their production and utilization. The first of these, all too well known to inhabitants of both developed and underdeveloped countries of the World, is that the sources of most of the more desirable fossil fuels are approaching exhaustion, so that they are fast becoming a precious and expensive commodity. Secondly, their continued and increasing use has brought with it environmental pollution problems.

Toward elucidating the mechanism of action of the ligninolytic systems in Basidiomycetes.

Abstract: The fact that this lecture is scheduled between lectures on xylanases and amylases perhaps illustrates the common misconception that the ligninolytic system bears a close biochemical similarity to other common biopolymer-degrading microbial systems. It does not. It is a most unusual system which has not yet been defined biochemically. However, recent studies, which I shall review here, have resulted in progress toward this end.

The role of proline in osmoregulation in Salmonella typhimurium and Escherichia coli.

Abstract: A variety of strategies have evolved for the regulation of the internal osmotic strength of organisms, but the details of these osmoregulatory mechanisms are poorly understood. In bacteria, internal osmolarity is maintained mainly by the accumulation of amino acids (3,10,16) and inorganic ions (4,9), such that it exceeds the osmolarity of the growth medium. A discovery made a quarter of a century ago by J. H. B. Christian suggested that proline has a special role in osmoregulation: in media of inhibitory osmolarity the growth and respiration rates of Salmonella orianenburg were stimulated specifically by the addition of proline (5,6). Using this observation as motivation, we have selected proline overproducing mutants of Salmonella typhimurium and found that some, as a result, have acquired an increased growth rate in media of inhibitory osmolarity (8; L. Csonka, manuscript in preparation). We found that in these proline over-producing mutants, the intracellular proline levels were regulated such that they increased with increasing osmotic stress. Here, we present experimental results which suggest that in the over-producing mutants, and in wild type strain, the proline permeases play an important role during osmotic stress for the regulation of the intracellular proline levels.

Anaerobic fermentations of cellulose to methane

Abstract: The microbial populations responsible for the anaerobic degradation of cellulosic biopolymers appear to be taxonomically diverse and variable, but the basic pattern of these complex fermentations is similar wherever they occur. This in turn suggests that the common denominator of these microbial populations is overall physiology rather than taxonomy. For example, if one compares the microorganisms found in mesophilic and thermophilic fermentations of cellulose to CO2 and CH4, individual isolates from these fermentations will, by and large, be taxonomically distinct, but physiologic counterparts in terms of the overall reactions catalyzed can be readily identified in each fermentation. This is perhaps implicit in the general concept of a “food chain”; however, within this single constraint, it allows for extensive diversity in terms of pH, temperature, products, product composition, substrates, inhibitors, product and substrate tolerance and nutrition. This diversity is currently the object of a considerable research effort which should define the environmental parameters for the degradation of complex cellulosic biopolymers and lead to the isolation of new types of bacteria.

Amylases: Enzymatic Mechanisms

Abstract: Starch is a major storage form for carbohydrates in nearly all green plants. Utilization of starch by the plants or by other organisms requires initial solubilization of the starch and conversion to dextrins and oligosaccharides by amylases. Various organisms have developed different strategies for starch breakdown, but all involve an initial attack by an amylase, perhaps concomitantly with another enzyme such as a dextrinase or debranching enzyme, followed by a glucosidase which converts the dextrins or oligosaccharides to glucose.

Structural studies of ribulose 1,5-bisphosphate carboxylase/oxygenase.

Abstract: From studies of three crystal forms of ribulose bisphosphate (RuBP) carboxylase from Nicotiana tabacum (called I, II, and III), we have determined the subunit organization of RuBP carboxylase in increasing detail. Combined x-ray diffraction and electron microscope data from these crystals show that there must be some multiple of eight polypeptide chains in the molecule and that the polypeptides are arranged around a fourfold axis of symmetry. At low resolution the eight copies of each polypeptide are equivalent. In more formal terms, the RuBP carboxylase molecule is characterized by point group symmetry D4 (422, see Figure 1 below). The molecule has a square cross section, about 11 nm on an edge, and a cylindrical channel about 2 nm in diameter which runs along the fourfold axis perpendicular to the square cross section. Four large sub-units are arranged in a ring perpendicular to the fourfold axis, and two such rings are eclipsed, forming a two-level structure that extends about 10 nm along the fourfold axis.

Mechanisms of Yeast Genetics

Abstract: Saccharomyces cerewisiae is a lower eukaryote ideal for many current biological studies. It shares certain properties with higher eukaryotes: a nucleus containing multiple chromosomes packed in chromatin structures, and specialized organelles such as vacuoles and mitochondria. In addition, the organization of yeast structural genes is similar to that of higher eukaryotic cells: most functionally related genes are scattered on different chromosomes rather than linked together in operons (21). Yet, while yeast are more complex than bacteria, they still share many of the technical advantages which permitted rapid progress in the genetic and biochemical studies of prokaryotic organisms. Some of the properties which make yeast particularly amenable to study are their short generation time, the existence of both stable haploids and diploids, and the ease of replica plating and mutant isolation. Furthermore, the sophisticated classical genetics of yeast allows one to fully exploit recent technological advances in genetic engineering.

Basic Biology of Microbial Fermentation

Abstract: The great majority of what we know about microbial fermentations was discovered before 1950 (See review by Wood (1)). Since then microbial and biochemical science has passed on to questions of structure and function of macromolecules, organelles and cells thought to be more intriguing or deserving of attention. Now for economic and strategic reasons, attention is once again focused on microbial fermentations for their potential to produce useful and energy-containing products from starch, waste materials or currently unusable biomass. Further, strategists in this area have a kind of tacit belief that, somewhere in the tremendous explosion of biological and chemical knowledge since 1950, there are missing pieces of information and techniques, that when capitalized upon, will propel microbial fermentations from their current state of near obliyion back to center stage, being caught up in a new wave of bioinnovation. In such a scenario the task will be to couple the new knowledge with the old science and technology of fermentation. In such a new endeavor those with the old knowledge need to know what opportunities the new knowledge makes possible, and the practitioners of new science need to know a fair amount about the old science rather than attempting to “reinvent the wheel”.

The biochemical genetics of glycolysis in microbes.

Abstract: Mutants for most reactions of glycolysis have been described both in Escherichia coli and in Sacchavomyces cevevisiae The pathway between glucose and pyruvate has three irreversible and seven reversible reactions (Figure 1), and most of the intermediates are needed in biosynthesis. Thus, one might expect mutants in an irreversible step to be impaired in growth on glucose but unimpaired gluconeogenically, and mutants in a reversible step to require supplementation even for gluconeogenic growth. To a first approximation this pattern is found but there are deviations (Table I). For example, E, coli mutants blocked between triose-P and phosphoenol-pyruvate do require supplementation (e.g., by glycerol) for growth on lactate (1,2) but phosphoglucose isomerase mutants grow without supplementation by glucose (3) — probably because glucose-6-P and its products are not essential for growth of this organism. Aldolase mutants also do not require supplementation for gluconeogenic growth, and the explanation is unknown; it might relate to other aldolases (see ref. 4). The growth on glucose of a (double) pyruvate kinase mutant occurs because phosphoenolpyruvate is used by the phosphotransferase (PTS) reaction intiating glucose metabolism and such a mutant fails to grown on non-PTS sugars (5).

Conversion of Reductases to Dehydrogenases by Regulatory Mutations

Abstract: Nicotinamide adenine dinucleotide (NAD+)-linked oxidoreductases catalyze reactions that are generally in favor o f NAD+ f ormation at neutral pH. However, in vivo enzymes of this kind can function as either dehydrogenases or reductases. We have a case in which an enzyme that normally acts to reduce L-lactaldehyde is converted by a series of mutations that affect gene expression to an enzyme that acts to oxidize L-l, 2-propanediol. In Escherichia coli both aerobic and anaerobic utilization of L-fucose requires the expression of an inducible trunk pathway mediated by fucose permease (1), fucose isomerase (2), fuculose kinase (3), and fuculose 1-phosphate aldolase (4). The aldolase cleaves the six carbon substrate into dihydroxy-acetone phosphate and lactaldehyde (Figure 1). Anaerobically, lactaldehyde is completely reduced to propanediol by L-l, 2-propanediol: NAD+ 1-oxidoreductase (propanediol oxidoredutase), an enzyme with a molecular weight of 76,000 consisting of two electro-phoretically indistinguishable subunits (5). For each mole of fucose fermented, one mole of propanediol is secreted into the medium (6). The sacrifice of one half of the carbon skeleton of fucose in this way permits the further metabolism of dihydroxyacetone phosphate without an exogenous hydrogen acceptor.

cAMP and Regulation of Carbohydrate Metabolism

Abstract: Cyclic nucleotides including cAMP and cGMP are found in many procaryotes. Table I provides a representative selection of bacteria from which cAMP has been identified and quantitated. With only a few exceptions, the mechanism of action of these nucleotides and the physiological consequences of variations in the intracellular levels are not known, cGMP has also been found in a variety of bacteria (Table II), it is usually present in amounts at least an order of magnitude smaller than those observed for cAMP. Intracellular concentrations of cGMP of 3–35 nM have been reported for Escherichia coli (3). 30 nM cGMP is equivalent to 18 molecules per cell if the volume of a cell is assumed to be 10-15 1. It has been proposed that cGMP in E. coli is an artifact of adenylate cyclase activity (70). However, a guanylate cyclase activity distinct from adenylate cyclase by several criteria has been purified and partially characterized from E. coli (40). In animal cells, it was originally proposed that cAMP and cGMP act in opposition, the “Yin Yang Effect.” This hypothesis has received less support as more evidence has accumulated (59).

Molecular genetics and microbial fermentations.

Abstract: The interaction of the field of molecular genetics, especially the part of it termed recombinant DNA technology, with the business of large-scale microbial fermentations is a subject much in the news these days. From the reaction of Wall Street to the recent and prospective public offerings of genetic engineering companies, one would think these companies were in the business of synthesizing the Philosopher’s Stone rather than developing methods for getting very large vats of smelly microorganisms to produce bio-chemicals more effectively.

Fermentation of plant polysaccharides: role of biochemical genetics.

Abstract: The application of biochemical genetics to the improvement of industrially important microorganisms has long been focused primarily on medical and agricultural products. Over the past few years, several novel ideas for genetic modification have been evoked for microbial systems. These advances in both cellular and molecular genetics and the advent of specific techniques in both in vitro and in vivo gene transfer for prokaryotic and eukaryotic organisms now give rise to applications in biotechnology for conversion of agricultural renewable resources to fuels and chemical feedstocks. These techniques include the use of directed mutasynthesis, cell and protoplast fusion, DNA transformation, and recombinant DNA technology. New and significant advances have been demonstrated in cellulosic conversions, enzymatic hydrolysis, alcohol production and tolerance, and substrate preparation relative to biological conversions. This biotechnology is now a prime candidate for transformation of fermentation genes necessary for plant polysaccharide conversions.

Microbial Adaptations to Stress: Some Lessons to be Learned from Aerobes

Abstract: There are microbial adaptations to “stress” or unusual conditions that would seem to be of obvious importance for optimizing fermentative capacities for the production of fuels. Three of these areas are the specific topics of talks in this session. There are two other environmental constraints upon microbial viability and metabolism that have been the subjects of investigations in our laboratory. These are the problem of pH—both extremely acidic and extremely alkaline—and the problem of the controls regulating aerobic vs. a more fermentative mode of metabolism. While all these studies have been conducted in aerobes, they touch upon concerns with respect to the issues at hand.

Hydrogen Formation by the Biophotolysis of Water Via Glycolate and Formate

Abstract: I am substituting for Professor H G Wood who was scheduled to chair this session but because of last minute commitments was unable to attend this meeting Knowing his interests in the theme of this symposium I am sure he would have opened the session with a few remarks about some of the interesting research he is doing in the field of microbial metabolism With your permission I will utilize some of his time to report some results we have obtained on the biophotolysis of water with the formation of hydrogen

Microbial enzymes attacking plant polymeric materials.

Abstract: This symposium is good evidence of the current interest in utilization of renewable resources via fermentation. For substrates, various forms of plant biomass are available in large quantities or may be produced on energy farms. Solar energy is fixed by photosynthesis of green plants initially as small molecules, but these are rarely available in high concentrations or large quantities. Most of the excess over immediate metabolic requirements are rapidly converted into more complex molecules which are utilized by the plants as reserve foods or as structural materials. Most of these are polymers that are awkward to use for chemical or biological processes because they are chemically and physically complex and are attacked only by limited groups of specialized organisms that do not normally produce high levels of fermentations products of commercial interst. Therefore, we would like to convert these polymers back to the more usable monomers. An exception to the above is sucrose, a non-polymeric plant reserve food which is a simple soluble molecule, readily separated from plant juices in a high degree of purity. Not surprisingly, sucrose molasses is the basis for many industrial fermentations, notably the production of ethanol by yeasts.