Environmental effects on bacterial copper extraction from low-grade copper sulfide ores

In bacterial extraction of copper from low-grade copper sulfide ores, at least three contributions are made by Thiobacillus ferrooxidans. They are: (1) enzymatic oxidation and consequent solubilization of insoluble sulfides; (2) regeneration of ferric lixiviant for chemical oxidation and solubilization of insoluble sulfides; and (3) partial fixation of externally introduced iron in the ore. Although it is not possible at the present time to measure each of these contributions separately, it is possible to measure the combined contributions. Such measurements reveal a strong dependence of extraction efficiency on various physical, chemical, and biological factors. The following physical factors may affect the rate of bacterial copper extraction: particle-size of ore, oxygen and carbondioxide supply, oxidation-reduction potential, pH, temperature, adsorption and ion exchange capacity of ore, and surface tension effects. The following chemical factors may influence the rate of copper extraction: the mineralogy of the ore, the nature of the gangue, the distribution of the sulfide minerals in the host rock, the external supply of ferrous or ferric iron, and the availability of inorganic and organic nutrients. Finally, the following biological agents in addition to T. ferrooxidans may influence the rate of copper extraction: fungi, protozoa, Thiobacillus thiooxidans, and heterotrophic bacteria. Proper control of these various factors is essential for efficient bacterial extraction of copper from low-grade ore. It is recognized that the foregoing environmental factors also influence chemical copper extraction.     

Evaluation of Slit Sampler in Quantitative Studies of Bacterial Aerosols

Quantitative studies were conducted to evaluate the efficiency of the slit sampler in collecting airborne Serratia marcescens and Bacillus subtilis var. niger, and to compare it with the collecting efficiency of the all-glass impinger AGI-30. The slit sampler was approximately 50% less efficient than the AGI-30. This ratio remained the same whether liquid or dry cultures were disseminated when the sample was taken at 2 min of aerosol cloud life. At 30 min of aerosol cloud life, this ratio was approximately 30% for B. subtilis var. niger. S. marcescens recoveries by the slit sampler were, however, only 17% lower than the AGI-30 at 30 min of cloud age, indicating a possible interaction involving the more labile vegetative cells, aerosol age, and method of collection.     

Effects of Environmental Factors on the Survival of Airborne T-3 Coliphage

Experiments were conducted to determine the effects of storage temperatures, relative humidity, and additives on the survival of aerosolized Escherichia coli phage T-3. The aerosol stability of the coliphage, calculated as per cent recovery, was not affected by storage at 10 or -70 C for up to 4 months. However, an increase in aerosol decay rate of coliphage stored at 10 C was observed. The effect of humidities ranging from 20 to 90% relative humidity was studied, and it was observed that humidities lower than 70% relative humidity significantly reduce the survival of airborne coliphage. The effect of various compounds on the aerosol decay rate of T-3 coliphage was studied at 50 and 85% relative humidity. Addition of dextrose in 0.1 M concentrations to the disseminating fluid significantly reduced aerosol decay rate at 50% relative humidity without affecting the decay at 85%. Addition of spermine, spermidine-phosphate, thiourea, galacturonic acid, and glucosaminic acid, individually or in combination, had no effect on aerosol decay rates. The use of deuterium oxide as the suspending fluid for dissemination had no effect on aerosol stability of the coliphage.     

Imbalance of leucine flux in Lactococcus lactis and its use for the isolation of diacetyl-overproducing strains.

Diacetyl is a by-product of pyruvate metabolism in Lactococcus lactis, where pyruvate is first converted to alpha-acetolactate, which is slowly decarboxylated to diacetyl in the presence of oxygen. L. lactis usually converts alpha-acetolactate to acetoin enzymatically, by alpha-acetolactate decarboxylase encoded by the aldB gene. We took advantage of the fact that this enzyme also has a central role in the regulation of branched-chain amino acids, to select spontaneous aldB mutants in an unbalanced concentration of leucine versus those of valine and isoleucine in the medium. Industrial dairy strains of L. lactis subsp. lactis biovar diacetylactis containing point mutations and deletions of aldB were isolated and characterized. Their growth in milk was not affected, but they rapidly accumulated a large amount of alpha-acetolactate instead of acetoin from citrate in milk. Under aerated condition, strains devoid of AldB produced about 10 times more diacetyl than did the parental strains.     

Active site of the replication protein of the rolling circle plasmid pC194.

Mutation analysis of the rolling circle (RC) replication initiator protein RepA of plasmid pC194 was targeted to tyrosine and acidic amino acids (glutamate and aspartate) which are well conserved among numerous related plasmids. The effect of mutations was examined by an in vivo activity test. Mutations of one tyrosine and two glutamate residues were found to greatly impair or abolish activity, without affecting affinity for the origin, as deduced from in vitro gel mobility assays. We conclude that all three amino acids have a catalytic role. Tyrosine residues were found previously in active sites of different RC plasmid Rep proteins and topoisomerases, but not in association with acidic residues, which are a hallmark of the active sites of DNA hydrolyzing enzymes, such as the exo- and endonucleases. We propose that the active site of RepA contains two different catalytic centers, corresponding to a tyrosine and a glutamate. The former may be involved in the formation of the covalent DNA-protein intermediate at the initiation step of RC replication, and the latter may catalyze the release of the protein from the intermediate at the termination step.     

Adult emergence phenology in checkerspot butterflies: the effects of macroclimate,topoclimate, and population history

The prediction of adult emergence times in insect populations can be greatly complicated by microclimatic gradients, especially in circumstances where distributions of juveniles along those gradients vary from year to year. To investigate adult emergence patterns in topographically heterogeneous habitats, we built a model of postdiapause development of the Bay checkerspot butterfly, Euphydryas editha bayensis. The model uses slope-specific insolation as the rate-controlling variable, and accounts for both solar exposure of the habitat and cloud cover. Instar-specific larval mass gains per unit of insolation were determined from mark-recapture experiments. A small correction for daily low temperatures was used to calibrate the model to five years of field data on larval mass. The model predicted mean mass of 90% of larval samples within 4 clear days over a 70–120 day growing season. The magnitude of spatial variation in emergence times across habitat slopes is greater than annual variation in emergence times due to yearly weather conditions. Historical variation (yearly shifts in larval distributions across slopes) is an important determinant of mean population emergence dates. All of these factors need to be considered in understanding adult emergence phenology in this butterfly and in other insects inhabiting heterogeneous thermal environments. Such an understanding can be useful in managing insect populations for both pest control and conservation.     

High-efficiency gene inactivation and replacement system for gram-positive bacteria.

A system for high-efficiency single- and double-crossover homologous integration in gram-positive bacteria has been developed, with Lactococcus lactis as a model system. The system is based on a thermosensitive broad-host-range rolling-circle plasmid, pG+host5, which contains a pBR322 replicon for propagation in Escherichia coli at 37 degrees C. A nested set of L. lactis chromosomal fragments cloned onto pG+host5 were used to show that the single-crossover integration frequency was logarithmically proportional to the length of homology for DNA fragments between 0.35 and 2.5 kb. Using random chromosomal 1-kb fragments, we showed that homologous integration can occur along the entire chromosome. We made use of the reported stimulatory effect of rolling-circle replication on intramolecular recombination to develop a protocol for gene replacement. Cultures were first maintained at 37 degrees C to select for a bacterial population enriched for plasmid integrants; activation of the integrated rolling-circle plasmid by a temperature shift to 28 degrees C resulted in efficient plasmid excision by homologous recombination and replacement of a chromosomal gene by the plasmid-carried modified copy. More than 50% of cells underwent replacement recombination when selection was applied for the replacing gene. Between 1 and 40% of cells underwent replacement recombination when no selection was applied. Chromosomal insertions and deletions were obtained in this way. These results show that gene replacement can be obtained at an extremely high efficiency by making use of the thermosensitive rolling-circle nature of the delivery vector. This procedure is applicable to numerous gram-positive bacteria.