Fermentation of d-Xylose and l-Arabinose to Ethanol by Erwinia chrysanthemi.

Erwinia spp. are gram-negative facultative anaerobes within the family Enterobacteriacae which possess several desirable traits for the conversion of pentose sugars to ethanol, such as the ability to ferment a broad range of carbohydrates and the ease with which they can be genetically modified. Twenty-eight strains of Erwinia carotovora and E. chrysanthemi were screened for the ability to ferment d-xylose to ethanol. E. chrysanthemi B374 was chosen for further study on the basis of its superior (4%) ethanol tolerance. We have characterized the fermentation of d-xylose and l-arabinose by the wild type and mutants which bear plasmids containing the pyruvate decarboxylase gene from Zymomonas mobilis. Expression of the gene markedly increased the yields of ethanol (from 0.7 up to 1.45 mol/mol of xylose) and decreased the yields of formate, acetate, and lactate. However, the cells with pyruvate decarboxylase grew only one-fourth as fast as the wild type and tolerated only 2% ethanol. Alcohol tolerance was stimulated by the addition of yeast extract to the growth medium. Xylose catabolism was characterized by a high saturation constant K(s) (4.5 mM).

Fermentation of d-Xylose to Ethanol by Genetically Modified Klebsiella planticola.

d-Xylose is a plentiful pentose sugar derived from agricultural or forest residues. Enteric bacteria such as Klebsiella spp. ferment d-xylose to form mixed acids and butanediol in addition to ethanol. Thus the ethanol yield is normally low. Zymomonas spp. and most yeasts are unable to ferment xylose, but they do ferment hexose sugars to ethanol in high yield because they contain pyruvate decarboxylase (EC 4.1.1.1), a key enzyme that is absent from enteric bacteria. This report describes the fermentation of d-xylose by Klebsiella planticola ATCC 33531 bearing multicopy plasmids containing the pdc gene inserted from Zymomonas mobilis. Expression of the gene markedly increased the yield of ethanol to 1.3 mol/mol of xylose, or 25.1 g/liter. Concurrently, there were significant decreases in the yields of formate, acetate, lactate, and butanediol. Transconjugant Klebsiella spp. grew almost as fast as the wild type and tolerated up to 4% ethanol. The plasmid was retained by the cells during at least one batch culture, even in the absence of selective pressure by antibiotics to maintain the plasmid. Ethanol production was 31.6 g/liter from 79.6 g of mixed substrate per liter chosen to simulate hydrolyzed hemicellulose. The physiology of the wild-type of K. planticola is described in more detail than in the original report of its isolation.

Activated sludge treatment of synthetic wastewater containing pentachlorophenol.

Activated sludge treatment of a pentachlorophenol (PCP)-containing synthetic waste was examined. With a waste containing some sugars, and 40-120 mg/L PCP, laboratory activated sludge required about seven days for acclimation. However, the prior addition of a quasipure culture of PCP-metabolizing Arthrobacter resulted in immediate acclimation. Even with acclimated sludge, however, the system was upset for two days by a simple step change from 40 to 120 mg/L of PCP. The stability of the system to such a shock load was considerably improved when a chemostat culture of the PCP-metabolizing Arthrobacter was fed slowly into the mixed liquor. Kinetic models were developed to describe the dynamic response of the system in terms of growth parameters, hydraulic detention time, sludge age, and bleed-in rate from the chemostat.

Microbial treatment of soil to remove pentachlorophenol.

Direct inoculation of bacteria capable of degrading pentachlorophenol (PCP) into PCP-contaminated soil was investigated as a prophylactic measure to reduce the hazards of runoffs when spills occur or when wooden poles freshly treated with PCP-containing preservatives are located near streams and lakes. In laboratory tests at 30 degrees C, the direct addition of 10 PCP-utilizing Arthrobacter cells per g of dry soil reduced the half-life of the pesticide from 2 weeks to <1 day. Soil inoculation also was shown to be an effective way to increase the PCP disappearance rate in a test conducted in an outdoor shed.

Isolation and characterization of a pentachlorophenol-degrading bacterium.

With a new enrichment protocol, pentachlorophenol (PCP)-degrading bacteria were isolated from soil, water, and sewage. When characterized, all isolates were related and shared characteristics of the genus Arthrobacter. Growth rates for strain NC were determined for a number of substrates, including PCP and 2,4,6-trichlorophenol. Changes in PCP concentration affected growth rate and length of the lag phase but not cell yield. Increasing the pH from 6.8 to 7.8 decreased the length of the lag phase for growth on PCP. Cessation of growth, upon incremental addition of PCP, was found to be related to a decrease in pH rather than to a buildup of a toxic metabolite. Degradation of PCP by strain NC was shown to be complete.

Kinetics of biodegradation of p-nitrobenzoate and inhibition by benzoate in a pseudomonad.

The degradation of p-nitrobenzoate (p-NBA) by domestic sewage was inhibited by benzoate, and a model for this behavior was found in a soil isolate. The isolate, a pseudomonad, utilized p-NBA and benzoate by separate adaptive enzyme pathways. In oxygen uptake experiments, the degradation of p-NBA was competitively inhibited by benzoate, but the degradation of benzoate was not affected by the presence of p-NBA. 4-Nitrocatechol was not implicated in the inhibition. p-Hydroxybenzoate, which is the p-NBA degradation pathway, also had a decreased rate od degradation when benzoate was present. The growth rate of the isolate on the aromatic substrates and on glucose autoclaved in the medium was 0.3 h-1. When glucose was autoclaved separately, the growth rate was less, about 0.2 h-1. The apparent Km in oxygen uptake experiments was 25 micrometer for p-NBA and benzoate and 5 micrometer for p-hydroxybenzoate.

The products of cellulose.

The recommendation of our panel was that the following were specific areas of promising research: 1) A more complete technology assessment in broad terms. 2) Additional fundamental studies on microbial conversion to methane. 3) Conversion of wood or crop residues to feed for ruminants by the simplest possible methods. 4) More complete characterization of the quality of syrups produced by enzymatic or acid hydrolysis. 5) Continued study of effective use of the lignin and hemicellulose by-products.

Growth rates of a pseudomonad on 2,4-dichlorophenoxyacetic acid and 2,4-dichlorophenol.

The growth of a pseudomonad on 2,4-D (2,4-dichlorophenoxyacetic acid) and 2,4-DCP (2,4-dichlorophenol) was studied in batch and continuous culture. The optimum growth rate using 2,4-D was 0.14/h at 25 C in a pH range from 6.2 to 6.9. Highest specific growth rate using 2,4-DCP was 0.12/h at 25 C in a pH range from 7.1 to 7.8. Growth was strongly inhibited by 2,4-DCP above a concentration of 25 mg/liter whereas no appreciable inhibition was observed with 2,4-D at concentrations up to 2,000 mg per liter. Growth on 2,4-DCP was described by Monod kinetics at subinhibitory concentrations but the inhibition by 2,4-DCP exhibited an unusual linear response to substrate concentration, and did not fit a model based on noncompetitive inhibition. The lag phase of batch cultures was found to depend on both 2,4-DCP concentration and prior adaptation of the inoculum. A study such as this on the kinetics of growth on related substrates may be useful as a method of finding the rate-limiting step in a metabolic sequence.