Grant St. Julian

Biomass Conversion: Fermentation Chemicals and Fuels

Recent events clearly establish that petroleum can no longer be relied upon as a stable, economical raw material for energy and industrial chemicals. Plant biomass is currently being evaluated as a desirable alternative raw material to petroleum because of renewability and abundance. The most abundant form of biomass on the planet earth is lignocellulose which is composed of cellulose, hemicellulose, and lignin. An estimated 4 X 10(9) tons per year of cellulose alone is readily available for conversion to energy or feedstuffs. This article explores the current state of research on the transformation of cellulose, hemi-cellulose, and lignin by various microorganisms and the subsequent production of fuels and chemicals. Current research activities are covered including technologies available for the utilization of biomass, chemicals from fermentation processes, conversion of biomass to sugar, direct bioconversion to liquid fuels.

Glycerol accumulation while recycling thin stillage in corn fermentations to ethanol

SummaryBy succesive recycling of the thin stillage in mashing and fermenting fresh corn, the glycerol content in each fermentation increased by about 0.4% and accumulated to a high of 2.1% in the beer of the fifth recycle. Glycerol concentration declined after the fifth recycle. The original fermentation contained 0.8% glycerol.

Growth pattern of Bacillus popilliae in Japanese beetle larvae.

Abstract Induction of milky disease in 50% of Japanese beetle (Popillia japonica) larvae by feeding requires about 109 spores of Bacillus popilliae per gram of soil. The infectious process occurs in four phases: (1) An initial incubation phase of about 2 days during which there is no evidence of infection in the hemolymph. (2) A vegetative phase of poliferation in the hemolymph which lasts until day 5 when prespores occur and a few spores first are observed. (3) An intermediate phase between day 5 and day 10 characterized by concomitant vegetative growth, prespore formation, and sporulation; maximum vegetative populations of about 109 cells per ml hemolymph occur during this phase but the number of spores exceed that of vegetative cells by the end of the phase. (4) Thereafter, a sporulation phase which terminates by day 14 to day 21 with typical milkness and death of larvae; vegetative populations steadily decline and large numbers of spores accumulate during this phase. Milky larvae contain an average of 5 × 1010 spores per ml hemolymph. Throughout the process microscopic evidence indicates many vegetative cells die without forming spores; dead cells disappear from the hemolymph by some unknown lytic or phagocytic process. Thus the massive spore populations which characterize milky disease result from accumulation of spores during a prolonged period of simultaneous vegetative growth and sporulation rather than from an extended period of exclusively vegetative growth followed by sporulation of most cells.

BACTERIA, SPIROCHETES, AND RICKETTSIA AS INSECTICIDES

Most bacteria pathogenic to insects are classified in the families Pseudomonadaceae, Enterobacteriaceae, Lactobacillaceae, Micrococcaceae, and Bacilliaceae; spirochetes and rickettsia are in the families Spirochaetaceae and Rickettsiaceae, respectively. Except for Bacilliaceae, these families contain nonsporulating microorganisms. Most spore-forming bacteria pathogenic to insects belong to the family Bacilliaceae. The identification of microorganisms associated with insects is inadequate because of inaccurate descriptions. A comprehensive evaluation of bacterial classification and identification can be found on the sixth and seventh editions of Bergey’s Manual of Determinative Bacter i~ logy .~ ,~ However illogical the existing scheme may be, we feel strongly that it should be adhered to by the insect pathologist, and the use of generalized names without reference to the specific causative disease agent should not continue. The incorrect hypothesis among insect pathologists that insects harbor special microorganisms is probably the main cause of misnamed bacterial species. Often, microorganisms from diseased insects are named for the insect from which they are isolated and are incriminated as pathogens. This practice should be discouraged. The procedures outlined in Koch’s canons for demonstrating that a disease is caused by a microorganism are as follows: finding the specific microorganism in all cases of the disease; isolating it in “pure culture” (description of the pure culture isolate should be by established taxonomic procedures) ; inoculating (or feeding) the isolate into the host insect and experimentally producing the original disease; and finally, reisolating the microorganism from the experimentally diseased insect and demonstrating it to be the same as the pure culture isolate previously inoculated. In this communication, we shall, when possible, ignore unauthenticated bacterial species claimed to be insecticidal. Bucher7z9 classified bacterial insect pathogens as either obligate, facultative, or potential. We shall discuss, according to Bucher’s categorization, nonsporulating bacterial pathogens, including true bacteria, spirochetes, and rickettsiae and sporulating bacterial pathogens, including “crystalliferous” types.

Infection of Popillia japonica larvae with heat-activated spores of Bacillus popilliae☆

Abstract Maximum outgrowth of spores of Bacillus popilliae resulted after they were heated at 50°C, but outgrowth diminished as the temperature was increased to 80°C. Although outgrowth was limited and often variable, infectivity was predictably influenced by heat activation of spores. The greatest number of Popillia japonica larvae (92.6%) were infected by injection of one million spores per larva heated at 50°C. By contrast, maximum infection from the same number of unheated spores was only 38.9%. Infectivity of spores heated at 60°C and at 70°C generally was intermediate between these values; heating at 80°C afforded minimal infectivity. Apparently infectivity depends on the efficacy of spore outgrowth which, in turn, is influenced by appropriate heat treatment.

Microscopic observations of germination and septum formation in pycnidiospores of Botryodiplodia theobromae

Abstract A population of aseptate pycnidiospores of the fungus Botryodiplodia theobromae can be induced to germinate or to form septa delimiting two cells; this developmental process is dependent upon nutritional and environmental factors. Transmission electron microscope investigations indicate that during germination of the aseptate spore, a new inner wall layer is synthesized de novo at the site of germ tube emergence. Formation of the septum also involves the de novo synthesis of an inner wall layer which comprises the majority of the septum and completely surrounds the spore. The wall of the germ tube emerging from the septate spore is a direct extension of this inner layer deposited during the formation of the septum. Although the early stages of spore germination may involve localized enzymatic degradation of the internal layers of the spore wall, transmission and scanning electron micrographs of germinating spores show that the outer wall layers are physically fractured by the emerging germ tube. It is suggested that spore germination and septum formation are initially similar processes regarding cell wall genesis but that some mechanism responsive to environmental and nutritional conditions determines the course of development.

Milky disease development in field-infected Japanese beetle larvae.

Abstract By correlated visual and microscopical examination, milky disease of field-infected, third-instar Japanese beetle larvae is categorized into four phases. The phases are described as sequential disease symptoms I through IV. All four phases persist simultaneously throughout experimental incubation. Larvae die during all phases of the disease; however, the largest percentage of death is at phase II and III of the infectious process. At phase II, 90% of the total population of cell types in the infected hemolymph are vegetative cells; in phase III 65–76% are vegetative cells with 17–28% spores. The massive spore population (95% of population) that characterizes milky disease is designated phase IV; less than 30% of larvae reach this phase of the disease.

Stored Bacillus popilliae spores and their infectivity against Popillia japonica larvae

Abstract Bacillus popilliae spores were stored for about 7 years under three separate conditions: frozen in sterile distilled water, smeared on glass microscope slides, and stored in loam soil at room temperature. In separate experiments, each of the 7-year-old preparations was fed to Popilla japonica larvae at concentrations of 10 3 , 10 5 , 10 7 , and 10 9 spores/g of soil. A significant decrease in the percentage of larvae infected occurred in all of the aged spore tests. B. popilliae spores stored in soil, for the extended period, produced 3% larval infection only at the 10 9 spores concentration; similar results were obtained from frozen spores. When P. japonica larvae were fed spores stored dried on slides, about 20% of the larvae developed milky disease. When aged frozen spores were artificially injected into larvae, 12% became infected at concentrations of 1 × 10 6 spores/larvae; dried spores at the same concentration infected about 38% of the insect larvae. We conclude from these data that aged B. popilliae spores are significantly less infective against P. japonica larvae than young spores.

Biochemistry of milky disease: Radiorespirometry of pyruvate, acetate, succinate, and glutamate oxidation by healthy and diseased Japanese beetle larvae

Abstract Oxidation of pyruvate, acetate, succinate, and glutamate was compared in healthy third-instar Japanese beetle ( Popillia japonica ) larvae and in those infected with the milky disease bacterium, Bacillus popilliae . Both healthy and infected larvae oxidize these compounds via the tricarboxylic acid cycle. However, oxidation of all compounds, except succinate, is higher in healthy than in infected larvae. The oxidation rate of all four compounds varies throughout the infectious process. As the disease progresses, oxidation of pyruvate and glutamate is greatest in phase I when there are few observable bacterial cells within the larval hemolymph. Oxidation of acetate and succinate increases in phase II, characterized by rapid vegetative growth of the bacteria. The oxidation rate of acetate and succinate decreases upon further disease development (phases III and IV), when there is a greater demand for energy compounds and for biosynthetic intermediates by the bacterial cells in transition from vegetative growth to sporulation.

Rate of oxygen uptake of healthy and diseased larvae of the Japanese beetle

Abstract Conventional Warburg vessels were used to obtain oxygen uptake data on healthy and milky diseased larvae of the Japanese beetle ( Popillia japonica ). The mean Q O 2 O 2 live larvae (LL) and 95% confidence limits were as follows: healthy third-instar larvae, 0.392 ± 0.047; milky third-instar larvae, 0.312 ± 0.050; refrigerated third-instar larvae, 0.156 ± 0.048; and early pupae, 0.332 ± 0.058. Repeated determination of Q O 2 O 2 LL on single larvae over a 6-hr period resulted in steadily decreasing values. No significant correlation existed between oxygen uptake and the dissolved oxygen content of the hemolymph. Injury to larvae caused by insertion of a polarograph needle for dissolved oxygen determination decreased Q O 2 O 2 LL values. After 1 day the rate of oxygen uptake was seen to return to the initial level. Regression analysis failed to establish a correlation between the concentration of microorganisms in diseased thirdinstar larvae and Q O 2 O 2 .