Determinants of tolerance to inhibitors in hardwood spent sulfite liquor in genome shuffled Pachysolen tannophilus strains.

Genome shuffling was used to obtain Pachysolen tannophilus mutants with improved tolerance to inhibitors in hardwood spent sulfite liquor (HW SSL). Genome shuffled strains (GHW301, GHW302 and GHW303) grew at higher concentrations of HW SSL (80 % v/v) compared to the HW SSL UV mutant (70 % v/v) and the wild-type (WT) strain (50 % v/v). In defined media containing acetic acid (0.70-0.90 % w/v), GHW301, GHW302 and GHW303 exhibited a shorter lag compared to the acetic acid UV mutant, while the WT did not grow. Genome shuffled strains produced more ethanol than the WT at higher concentrations of HW SSL and an aspen hydrolysate. To identify the genetic basis of inhibitor tolerance, whole genome sequencing was carried out on GHW301, GHW302 and GHW303 and compared to the WT strain. Sixty single nucleotide variations were identified that were common to all three genome shuffled strains. Of these, 40 were in gene sequences and 20 were within 5 bp-1 kb either up or downstream of protein encoding genes. Based on the mutated gene products, mutations were grouped into functional categories and affected a variety of cellular functions, demonstrating the complexity of inhibitor tolerance in yeast. Sequence analysis of UV mutants (UAA302 and UHW303) from which GHW301, GHW302 and GHW303 were derived, confirmed the success of our cross-mating based genome shuffling strategy. Whole-genome sequencing analysis allowed identification of potential gene targets for tolerance to inhibitors in lignocellulosic hydrolysates.

Genetic improvement of native xylose-fermenting yeasts for ethanol production.

Lignocellulosic substrates are the largest source of fermentable sugars for bioconversion to fuel ethanol and other valuable compounds. To improve the economics of biomass conversion, it is essential that all sugars in potential hydrolysates be converted efficiently into the desired product(s). While hexoses are fermented into ethanol and some high-value chemicals, the bioconversion of pentoses in hydrolysates remains inefficient. This remains one of the key challenges in lignocellulosic biomass conversion. Native pentose-fermenting yeasts can ferment both glucose and xylose in lignocellulosic biomass to ethanol. However, they perform poorly in the presence of hydrolysate inhibitors, exhibit low ethanol tolerance and glucose repression, and ferment pentoses less efficiently than the main hexoses glucose and mannose. This paper reviews classical and molecular strain improvement strategies applied to native pentose-fermenting yeasts for improved ethanol production from xylose and lignocellulosic substrates. We focus on Pachysolen tannophilus, Scheffersomyces (Candida) shehatae, Scheffersomyces (Pichia) stipitis, and Spathaspora passalidarum which are good ethanol producers among the native xylose-fermenting yeasts. Strains obtained thus far are not robust enough for efficient ethanol production from lignocellulosic hydrolysates and can benefit from further improvements.

Mutants of the pentose-fermenting yeast Pachysolen tannophilus tolerant to hardwood spent sulfite liquor and acetic acid.

A strain development program was initiated to improve the tolerance of the pentose-fermenting yeast Pachysolen tannophilus to inhibitors in lignocellulosic hydrolysates. Several rounds of UV mutagenesis followed by screening were used to select for mutants of P. tannophilus NRRL Y2460 with improved tolerance to hardwood spent sulfite liquor (HW SSL) and acetic acid in separate selection lines. The wild type (WT) strain grew in 50 % (v/v) HW SSL while third round HW SSL mutants (designated UHW301, UHW302 and UHW303) grew in 60 % (v/v) HW SSL, with two of these isolates (UHW302 and UHW303) being viable and growing, respectively, in 70 % (v/v) HW SSL. In defined liquid media containing acetic acid, the WT strain grew in 0.70 % (w/v) acetic acid, while third round acetic acid mutants (designated UAA301, UAA302 and UAA303) grew in 0.80 % (w/v) acetic acid, with one isolate (UAA302) growing in 0.90 % (w/v) acetic acid. Cross-tolerance of HW SSL-tolerant mutants to acetic acid and vice versa was observed with UHW303 able to grow in 0.90 % (w/v) acetic acid and UAA302 growing in 60 % (v/v) HW SSL. The UV-induced mutants retained the ability to ferment glucose and xylose to ethanol in defined media. These mutants of P. tannophilus are of considerable interest for bioconversion of the sugars in lignocellulosic hydrolysates to ethanol.

Transcriptional profiling of Saccharomyces cerevisiae T2 cells upon exposure to hardwood spent sulphite liquor: comparison to acetic acid, furfural and hydroxymethylfurfural.

Global gene expression was analyzed in Saccharomyces cerevisiae T2 cells grown in the presence of hardwood spent sulphite liquor (HW SSL) and each of the three main inhibitors in HW SSL, acetic acid, hydroxymethyfurfural (HMF) and furfural, using a S. cerevisiae DNA oligonucleotide microarray. The objective was to compare the gene expression profiles of T2 cells in response to the individual inhibitors against that elicited in response to HW SSL. Acetic acid mainly affected the expression of genes related to the uptake systems of the yeast as well as energy generation and metabolism. Furfural and HMF mainly affected the transcription of genes involved in the redox balance of the cell. On the other hand, the effect of HW SSL on S. cerevisiae T2 cells was distinct and considerably more diverse as compared to the effect of individual inhibitors found in lignocellulosic hydrolysates. This is not surprising as HW SSL contains a complex mixture of inhibitors which may act synergistically. HW SSL elicited significant changes in expression of genes involved in diverse and multiple effects on several aspects of the cellular structure and function. A notable response to HW SSL was decreased expression of the ribosomal protein genes in T2 cells. In addition, HW SSL decreased the expression of genes functioning in the synthesis and transport of proteins as well as metabolism of carbohydrates, lipids, vitamins and vacuolar proteins. Furthermore, the expression of genes involved in multidrug resistance, iron transport and pheromone response was increased, suggesting that T2 cells grown in the presence of HW SSL may have activated pheromone response and/or activated pleiotropic drug response. Some of the largest changes in gene expression were observed in the presence of HW SSL and the affected genes are involved in mating, iron transport, stress response and phospholipid metabolism. A total of 59 out of the 400 genes differentially expressed in the presence of HW SSL, acetic acid, HMF and furfural, belonged to the category of poorly characterized genes. The results indicate that transcriptional responses to individual lignocellulosic inhibitors gave a different picture and may not be representative of how the cells would respond to the presence of all the inhibitors in lignocellulosic hydrolysates such as HW SSL.

Optimization, validation, and application of a real-time PCR protocol for quantification of viable bacterial cells in municipal sewage sludge and biosolids using reporter genes and Escherichia coli.

Biosolids result from treatment of sewage sludge to meet jurisdictional standards, including pathogen reduction. Once government regulations are met, materials can be applied to agricultural lands. Culture-based methods are used to enumerate pathogen indicator microorganisms but may underestimate cell densities, which is partly due to bacteria existing in a viable but non-culturable physiological state. Viable indicators can also be quantified by realtime polymerase chain reaction (qPCR) used with propidium monoazide (PMA), a dye that inhibits amplification of DNA found extracellularly or in dead cells. The objectives of this study were to test an optimized PMA-qPCR method for viable pathogen detection in wastewater solids and to validate it by comparing results to data obtained by conventional plating. Reporter genes from genetically marked Pseudomonas sp. UG14Lr and Agrobacterium tumefaciens 542 cells were spiked into samples of primary sludge, and anaerobically digested and Lystek-treated biosolids as cell-free DNA, dead cells, viable cells, and mixtures of live and dead cells, followed by DNA extraction with and without PMA, and qPCR. The protocol was then used for Escherichia coli quantification in the three matrices, and results compared to plate counts. PMA-qPCR selectively detected viable cells, while inhibiting signals from cell-free DNA and DNA found in membrane-compromised cells. PMA-qPCR detected 0.5-1 log unit more viable E. coli cells in both primary solids and dewatered biosolids than plate counts. No viable E. coli was found in Lystek-treated biosolids. These data suggest PMA-qPCR may more accurately estimate pathogen cell numbers than traditional culture methods.

Bacterial genomes: habitat specificity and uncharted organisms.

The capability and speed in generating genomic data have increased profoundly since the release of the draft human genome in 2000. Additionally, sequencing costs have continued to plummet as the next generation of highly efficient sequencing technologies (next-generation sequencing) became available and commercial facilities promote market competition. However, new challenges have emerged as researchers attempt to efficiently process the massive amounts of sequence data being generated. First, the described genome sequences are unequally distributed among the branches of bacterial life and, second, bacterial pan-genomes are often not considered when setting aims for sequencing projects. Here, we propose that scientists should be concerned with attaining an improved equal representation of most of the bacterial tree of life organisms, at the genomic level. Moreover, they should take into account the natural variation that is often observed within bacterial species and the role of the often changing surrounding environment and natural selection pressures, which is central to bacterial speciation and genome evolution. Not only will such efforts contribute to our overall understanding of the microbial diversity extant in ecosystems as well as the structuring of the extant genomes, but they will also facilitate the development of better methods for (meta)genome annotation.

Effect of nitrate and glucose addition on denitrification and nitric oxide reductase (cnorB) gene abundance and mRNA levels in Pseudomonas mandelii inoculated into anoxic soil.

The effect of glucose addition (0 and 500 µg C g(-1) soil) and nitrate (NO(3)) addition (0, 10, 50 and 500 µg NO(3)-N g(-1) soil) on nitric oxide reductase (cnorB) gene abundance and mRNA levels, and cumulative denitrification were quantified over 48 h in anoxic soils inoculated with Pseudomonas mandelii. Addition of glucose-C significantly increased cnorB(p) (P. mandelii and related species) mRNA levels and abundance compared with soil with no glucose added, averaged over time and NO(3) addition treatments. Without glucose addition, cnorB(p) mRNA levels were higher when 500 µg NO(3)-N g(-1) soil was added compared with other NO(3) additions. In treatments with glucose added, addition of 50 µg NO(3)-N g(-1) soil resulted in higher cnorB(p) mRNA levels than soil without NO(3) but was not different from the 10 and 500 µg NO(3)-N g(-1) treatments. cnorB(p) abundance in soils without glucose addition was significantly higher in soils with 500 µg NO(3)-N g(-1) soil compared to lower N-treated soils. Conversely, addition of 500 µg NO(3)-N g(-1) soil resulted in lower cnorB(p) abundance compared with soil without N-addition. Over 48 h, cumulative denitrification in soils with 500 µg glucose-C g(-1) soil, and 50 or 500 µg NO(3)-N g(-1) was higher than all other treatments. There was a positive correlation between cnorB(p) abundance and cumulative denitrification, but only in soils without glucose addition. Glucose-treated soils generally had higher cnorB(p) abundance and mRNA levels than soils without glucose added, however response of cnorB(p) abundance and mRNA levels to NO(3) supply depended on carbon availability.

Proteomic insights into cold adaptation of psychrotrophic and mesophilic Acidithiobacillus ferrooxidans strains.

Cold tolerant strains of Acidithiobacillus ferrooxidans play a role in metal leaching and acid mine drainage (AMD) production in northern latitude/boreal mining environments. In this study we used a proteomics and bioinformatics approach to decipher the proteome changes related to sustained growth at low temperatures to increase our understanding of cold adaptation mechanisms in A. ferrooxidans strains. Changes in protein abundance in response to low temperatures (5 and 15°C) were monitored and protein analyses of a psychrotrophic strain (D6) versus a mesophilic strain (F1) showed that both strains increased levels of 11 stress-related and metabolic proteins including survival protein SurA, trigger factor Tig, and AhpC-Tsa antioxidant proteins. However, a unique set of changes in the proteome of psychrotrophic strain D6 were observed. In particular, the importance of protein fate, membrane transport and structure for psychrotrophic growth were evident with increases in numerous chaperone and transport proteins including GroEL, SecB, ABC transporters and a capsule polysaccharide export protein. We also observed that low temperature iron oxidation coincides with a relative increase in the key iron metabolism protein rusticyanin, which was more highly expressed in strain D6 than in strain F1 at colder growth temperatures. We demonstrate that the psychrotrophic strain uses a global stress response and cold-active metabolism which permit growth of A. ferrooxidans in the extreme AMD environment in colder climates.

The composition and organization of cytoplasm in prebiotic cells.

This article discusses the hypothesized composition and organization of cytoplasm in prebiotic cells from a theoretical perspective and also based upon what is currently known about bacterial cytoplasm. It is unknown if the first prebiotic, microscopic scale, cytoplasm was initially contained within a primitive, continuous, semipermeable membrane, or was an uncontained gel substance, that later became enclosed by a continuous membrane. Another possibility is that the first cytoplasm in prebiotic cells and a primitive membrane organized at the same time, permitting a rapid transition to the first cell(s) capable of growth and division, thus assisting with the emergence of life on Earth less than a billion years after the formation of the Earth. It is hypothesized that the organization and composition of cytoplasm progressed initially from an unstructured, microscopic hydrogel to a more complex cytoplasm, that may have been in the volume magnitude of about 0.1-0.2 µm(3) (possibly less if a nanocell) prior to the first cell division.

Anaerobic biotransformation of high concentrations of chloroform by an enrichment culture and two bacterial isolates.

A fermentative enrichment culture (designated DHM-1) was developed that is capable of cometabolically biotransforming high concentrations of chloroform (CF) to nontoxic end products. Two Pantoea spp. were isolated from DHM-1 that also possess this dechlorination capability. Following acclimation to increasing levels of CF, corn syrup-grown DHM-1 was able to transform over 500 mg/liter CF in the presence of vitamin B(12) (approximately 3% of CF on a molar basis) at a rate as high as 22 mg/liter/day in a mineral salts medium. CO, CO(2), and organic acids were the predominant biodegradation products, suggesting that hydrolytic reactions predominate during CF transformation. DHM-1 was capable of growing on corn syrup in the presence of high concentrations of CF (as may be present near contaminant source zones in groundwater), which makes it a promising culture for bioaugmentation. Strains DHM-1B and DHM-1T transform CF at rates similar to that of the DHM-1 enrichment culture. The ability of these strains to grow in the presence of high concentrations of CF appears to be related to alteration of membrane fluidity or homeoviscous and homeophasic adaptation.

Changes in denitrifier abundance, denitrification gene mRNA levels, nitrous oxide emissions, and denitrification in anoxic soil microcosms amended with glucose and plant residues.

In agricultural cropping systems, crop residues are sources of organic carbon (C), an important factor influencing denitrification. The effects of red clover, soybean, and barley plant residues and of glucose on denitrifier abundance, denitrification gene mRNA levels, nitrous oxide (N(2)O) emissions, and denitrification rates were quantified in anoxic soil microcosms for 72 h. nosZ gene abundances and mRNA levels significantly increased in response to all organic carbon treatments over time. In contrast, the abundance and mRNA levels of Pseudomonas mandelii and closely related species (nirS(P)) increased only in glucose-amended soil: the nirS(P) guild abundance increased 5-fold over the 72-h incubation period (P < 0.001), while the mRNA level significantly increased more than 15-fold at 12 h (P < 0.001) and then subsequently decreased. The nosZ gene abundance was greater in plant residue-amended soil than in glucose-amended soil. Although plant residue carbon-to-nitrogen (C:N) ratios varied from 15:1 to 30:1, nosZ gene and mRNA levels were not significantly different among plant residue treatments, with an average of 3.5 x 10(7) gene copies and 6.9 x 10(7) transcripts g(-1) dry soil. Cumulative N(2)O emissions and denitrification rates increased over 72 h in both glucose- and plant-tissue-C-treated soil. The nirS(P) and nosZ communities responded differently to glucose and plant residue amendments. However, the targeted denitrifier communities responded similarly to the different plant residues under the conditions tested despite changes in the quality of organic C and different C:N ratios.

Membrane fluidity and fatty acid comparisons in psychrotrophic and mesophilic strains of Acidithiobacillus ferrooxidans under cold growth temperatures.

Psychrotrophic strains of Acidithiobacillus ferrooxidans have an important role in metal leaching and acid mine drainage (AMD) production in colder mining environments. We investigated cytoplasmic membrane fluidity and fatty acid alterations in response to low temperatures (5 and 15°C). Significant differences in membrane fluidity, measured by polarization (P) of 1,6-diphenyl-1,3,5-hexatriene (DPH), were found where the psychrotrophic strains had a significantly more rigid membrane (P range = 0.41-0.45) and lower transition temperature midpoints (T (m) = 2.0°C) and broader transition range than the mesophilic strains (P range = 0.38-0.39; T (m) = 2.0-18°C) at cold temperatures. Membrane remodeling was evident in all strains with a common trend of increased unsaturated fatty acid component in response to lower growth temperatures. In psychrotrophic strains, decreases in 12:0 fatty acids distinguished the 5°C fatty acid profiles from those of the mesophilic strains that showed decreases in 16:0, 17:0, and cyclo-19:0 fatty acids. These changes were also correlated with the observed changes in membrane fluidity (R (2) = 63-97%). Psychrotrophic strains employ distinctive modulation of cytoplasmic membrane fluidity with uncommon membrane phase changes as part of their adaptation to the extreme AMD environment in colder climates.

Quantitative identification of fecal water pollution sources by TaqMan real-time PCR assays using Bacteroidales 16S rRNA genetic markers.

PCR-based analysis of Bacteroidales 16S rRNA genes has emerged as a promising tool to identify sources of fecal water pollution. In this study, three TaqMan real-time PCR assays (BacGeneral, BacHuman, and BacBovine) were developed and evaluated for their ability to quantitatively detect general (total), human-specific, and bovine-specific Bacteroidales 16S rRNA genetic markers. The detection sensitivity was determined to be 6.5 copies of 16S rRNA gene for the BacGeneral and BacHuman assays and 10 copies for the BacBovine assay. The assays were capable of detecting approximately one to two cells per PCR. When tested with 70 fecal samples from various sources (human, cattle, pig, deer, dog, cat, goose, gull, horse, and raccoon), the three assays positively identified the target markers in all samples without any false-negative results. The BacHuman and BacBovine assays exhibited false-positive reactions with non-target samples in a few cases. However, the level of the false-positive reactions was about 50 times smaller than that of the true-positive ones, and therefore, these cross-reactions were unlikely to cause misidentifications of the fecal pollution sources. Microbial source-tracking capability was tested at two freshwater streams of which water quality was influenced by human and cattle feces, respectively. The assays accurately detected the presence of the corresponding host-specific markers upon fecal pollution and the persistence of the markers in downstream areas. The assays are expected to reliably determine human and bovine fecal pollution sources in environmental water samples.

Shifting trends in pathogen dynamics on a changing planet.

In this perspective we offer views on how microbial activities may change in response to the impact of human activities and an increasing human population. New knowledge is forthcoming in microbiology and immunology on the biology of emerging pathogens. With the exposure of human populations to changing environments, through climate change and human encroachment into previously unpopulated areas, there is likely to be increased incidences of diseases caused by existing, emerging and previously unrecognised pathogens.

Single chain variable fragment antibodies selected by phage display against the sporozoite surface antigen S16 of Cryptosporidium parvum.

Two human single chain variable fragment (scFv) libraries were used to select clones that bound to the surface glycoprotein S16 of Cryptosporidium parvum. Panning of the Tomlinson libraries I and J resulted in the isolation of nine distinct clones. Of the four clones which had full-length scFv, three contained stop codons. The remaining five clones were truncated, with four missing the heavy chain, and one missing most of the light chain. The full-length clones exhibited better binding to native C. parvum proteins and recombinant S16 than the truncated clones, with the exception of one truncated clone. None of the selected clones cross-reacted with Giardia lamblia, Escherichia coli, Streptococcus pyogenes, Listeria monocytogenes, Bacillus cereus or another immunogenic target of C. parvum, P23. Clones expressed as the soluble scFv-gIIIp construct were able to detect C. parvum native proteins and sporozoites. Panning from naïve libraries was an useful method for isolation and identification of recombinant antibodies that have the potential for use in pathogen detection and immunotherapy.

Diversity of nirK denitrifying genes and transcripts in an agricultural soil.

Environmental conditions can change dramatically over a crop season and among locations in an agricultural field and can increase denitrification and emissions of the potent greenhouse gas nitrous oxide. In a previous study, changes in the overall size of the denitrifier community in a potato crop field were relatively small and did not correlate with variations in environmental conditions or denitrification rates. However, denitrifying bacteria are taxonomically diverse, and different members of the community may respond differently to environmental changes. The objective of this research was to understand which portion of the nirK denitrifying community is active and contributes to denitrification under conditions in a potato crop field. Denaturing gradient gel electrophoresis (DGGE) of nirK genes in soil-extracted DNA showed changes in the composition of the nirK denitrifier community over the growing season and among spatial locations in the field. By contrast, the composition of the active nirK denitrifier community, as determined by DGGE analysis of nirK transcripts derived from soil-extracted mRNA, changed very little over time, although differences in the relative abundance of some specific transcripts were observed between locations. Our results indicate that the soil denitrifier populations bearing nirK genes are not all contributing to denitrification and that the denitrifying populations that are active are among the most abundant and ubiquitous nirK-bearing denitrifiers. Changes in the community composition of the total and active nirK denitrifiers were not strongly correlated with changes in environmental factors and denitrification activity.

Effect of pH and temperature on denitrification gene expression and activity in Pseudomonas mandelii.

Pseudomonas mandelii liquid cultures were studied to determine the effect of pH and temperature on denitrification gene expression, which was quantified by quantitative reverse transcription-PCR. Denitrification was measured by the accumulation of nitrous oxide (N(2)O) in the headspace in the presence of acetylene. Levels of gene expression of nirS and cnorB at pH 5 were 539-fold and 6,190-fold lower, respectively, than the levels of gene expression for cells grown at pH 6, 7, and 8 between 4 h and 8 h. Cumulative denitrification levels were 28 micromol, 63 micromol, and 22 micromol at pH 6, 7, and 8, respectively, at 8 h, whereas negligible denitrification was measured at pH 5. P. mandelii cells grown at 20 degrees C and 30 degrees C exhibited 9-fold and 94-fold increases in levels of cnorB expression between 0 h and 2 h, respectively, and an average 17-fold increase in levels of nirS gene expression. In contrast, induction of cnorB and nirS gene expression for P. mandelii cells grown at 10 degrees C did not occur in the first 4 h. Levels of cumulative denitrification at 10 h were 6.6 micromol for P. mandelii cells grown at 10 degrees C and 20 degrees C and 30 micromol for cells grown at 30 degrees C. Overall, levels of cnorB and nirS expression were relatively insensitive to pH values over the range of pH 6 to 8 but were substantially reduced at pH 5, whereas gene expression was sensitive to temperature, with induction and time to achieve maximum gene expression delayed as the temperature decreased from 30 degrees C. Low pH and temperature negatively affected denitrification activity.

Effect of nitrate and acetylene on nirS, cnorB, and nosZ expression and denitrification activity in Pseudomonas mandelii.

Nitrate acts as an electron acceptor in the denitrification process. The effect of nitrate in the range of 0 to 1,000 mg/liter on Pseudomonas mandelii nirS, cnorB, and nosZ gene expression was studied, using quantitative reverse transcription-quantitative PCR. Denitrification activity was measured by using the acetylene blockage method and gas chromatography. The effect of acetylene on gene expression was assessed by comparing denitrification gene expression in P. mandelii culture grown in the presence or absence of acetylene. The higher the amount of NO(3)(-) present, the greater the induction and the longer the denitrification genes remained expressed. nirS gene expression reached a maximum at 2, 4, 4, and 6 h in cultures grown in the presence of 0, 10, 100, and 1,000 mg of KNO(3)/liter, respectively, while induction of nirS gene ranged from 12- to 225-fold compared to time zero. cnorB gene expression also followed a similar trend. nosZ gene expression did not respond to NO(3)(-) treatment under the conditions tested. Acetylene decreased nosZ gene expression but did not affect nirS or cnorB gene expression. These results showed that nirS and cnorB responded to nitrate concentrations; however, significant denitrification activity was only observed in culture with 1,000 mg of KNO(3)/liter, indicating that there was no relationship between gene expression and denitrification activity under the conditions tested.

Mutants of the pentose-fermenting yeast Pichia stipitis with improved tolerance to inhibitors in hardwood spent sulfite liquor.

Mutants of Pichia stipitis NRRL Y-7124 able to tolerate and produce ethanol from hardwood spent sulfite liquor (HW SSL) were obtained by UV mutagenesis. P. stipitis cells were subjected to three successive rounds of UV mutagenesis, each followed by screening first on HW SSL gradient plates and then in diluted liquid HW SSL. Six third generation mutants with greater tolerance to HW SSL as compared to the wild type (WT) were isolated. The WT strain could not grow in HW SSL unless it was diluted to 65% (v/v). In contrast, the third generation mutants were able to grow in HW SSL diluted to 75% (v/v). Mutants PS301 and PS302 survived even in 80% (v/v) HW SSL, although there was no increase in cell number. All the third generation mutants exhibited higher growth rates but significantly lower growth yields on xylose or glucose compared to the WT. The mutants fermented 4% (w/v) glucose as efficiently as the WT and fermented 4% (w/v) xylose more efficiently with a higher ethanol yield than the WT. In a medium containing 4% (w/v) each of xylose and glucose, all the third generation mutants utilized glucose as efficiently and xylose more efficiently than the WT. This resulted in higher ethanol yield by the mutants. The mutants retained the ability to utilize galactose and mannose and ferment them to ethanol. Arabinose was consumed slowly by both the mutants and WT with no ethanol production. In 60% (v/v) HW SSL, the mutants utilized and fermented glucose, mannose, galactose and xylose while the WT could not ferment any of these sugars.

Single-chain variable fragment antibodies selected by phage display against the sporozoite surface antigen P23 Of Cryptosporidium parvum.

Cryptosporidium parvum, an apicomplexan parasite transmitted via animal fecal wastes, is the causative agent of cryptosporidiosis. Clones were selected from 2 synthetic naïve human single-chain variable fragment (scFv) phagemid libraries that bound to the recombinant P23 protein of C. parvum. Panning the Tomlinson I and J phagemid libraries resulted in 6 distinct clones. Two clones had full-length scFv sequences, while the remaining clones were either truncated or missing a section of the heavy chain. Despite these differences, all clones were able to detect both native C. parvum proteins and recombinant P23. None of the selected clones cross-reacted with Escherichia coli, Streptococcus pyogenes, Listeria monocytogenes, Bacillus cereus, Giardia lamblia (cysts or trophozoites), or with S16, another dominant surface antigen on C. parvum sporozoites. Clones expressed as the scFv-gIIIp fusion construct in soluble form detected C. parvum. Panning from naïve libraries is a useful method for isolation and identification of recombinant antibodies that have the potential for use in pathogen detection and immunotherapy.