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Endoglucanases, exoglucanases and β-glucosidases act synergistically to hydrolyse cellulose into glucose monomers. Thus, this study aimed to improve production of a β-glucosidase from a newly isolated Neofusicoccum parvum strain F7 by optimizing the culture conditions and medium components using Plackett-Burman Design (PBD) and Box Behnken Design (BBD). β-Glucosidase production was significantly enhanced (p-value≤0.05) by 1.5-fold to 2.5 U/ml by BBD as compared to the preliminary one variable at a time (OVAT) experiments of (1.6 U/ml). The optimal conditions for enzyme production by BBD were 12 days of fermentation at 20°C, 175 rpm, 0.5% glycerol and 1.5% casein in 50 mM sodium phosphate (pH 6.0) buffer. Three β-glucosidase isoforms referred to as Bgl1, Bgl2, Bgl3 were purified and characterized from the optimized crude extract displaying IC50 values of 2.6, 22.6 and 319.5 mM for glucose, respectively. Bgl3 with a molecular weight of approximately 65 kDa displayed the highest tolerance to glucose among the isoforms. The optimum activity and stability for Bgl3 was observed at pH 4.0 in 50 mM sodium acetate buffer with 80% β-glucosidase residual activity retained for three hours. This isoform also retained 60% residual activity at 65°C for one hour which was then reduced to 40 % which remained stable for another 90 minutes. The β-Glucosidase activity of Bgl3 was not enhanced after the addition of metal ions in assay buffers. The Km and vmax for 4-nitrophenyl-β-D-glucopyranoside were found to be 1.18 mM and 28.08 µmol/min, respectively indicating high affinity to the substrate. The ability to withstand the presence of glucose in conjunction with its thermophilic nature indicates promise for the enzyme in industrial application.

The South African pulp and paper industry generates an estimated 0.5 million tons of pulp and paper mill sludge (PPMS) annually. As PPMS is generated, it requires safe, efficient, and economical co...

The search for a novel microbial producer of cellulases including a glucose tolerant β-glucosidase is a challenge as most are inhibited by their product glucose. This study aims to screen for cellulolytic fungi using qualitative and quantitative screening methods. Primary screening revealed 34 of 46 fungal isolates with β-glucosidase activity. Eleven and 13 of these also displayed endoglucanase and exoglucanase activities, respectively. During secondary screening, this number was reduced to 26 β-glucosidase producers with 13 also having endoglucanase and exoglucanase activities. Isolate C1 displayed enhanced production of β-glucosidases in the presence of 0.05 M glucose (69% higher activity). Optimisation of growth conditions for β-glucosidase production by one variable at a time experiments improved production for (isolates) PS1 (64%), MB5 (84%), and C2 (69%). Isolate PS1 identified as Chaetomella sp. BBA70074 displayed the highest tolerance to glucose, retaining 10% of β-glucosidase activity in the presence of 0.8 M glucose. Tolerance to glucose increased to 14% when produced under optimal conditions. β-Glucosidase had a molecular weight of 170 kDa with a pH and temperature optima of 6 and 70°C, respectively. Future studies will include optimisation of the production of the glucose tolerant enzyme by Chaetomella sp. BBA70074.

Biochar amendment effectively controlled the Phytophthora blight of pepper and suppressed the pathogen abundance, with biochar applied just before transplanting (BC0) producing greater effects than that applied 20 days before transplanting (BC20). Biochar treatments stimulated the proliferation of total bacteria, Bacillus spp., Pseudomonas spp. , Streptomyces spp., and Sphingomonas spp. The proliferative effect of BC20 treatment gradually weakened compared to that of BC0 treatment with extended planting time. Moreover, biochar amendment strongly promoted the antagonist percentage and antagonistic ability of total bacteria, Bacillus spp., and Pseudomonas spp. and the promoting effect of BC0 treatment was stronger than that of BC20 treatment. Biochar-enriched Bacillus and Streptomyces strains, followed by Pseudomonas strains, were the best in terms of reducing the abundance of P. capsici and controlling Phytophthora blight of pepper. In addition, MiSeq sequencing data indicated that biochar treatments altered the soil bacterial community and enriched some beneficial bacteria, with BC0 treatment producing greater effects than BC20 treatment. Overall, the biochar-induced improvement of soil properties (particularly the abundance of biocontrol bacteria such as Bacillus spp., Pseudomonas spp. and Streptomyces spp. and bacterial antagonisms against P. capsici) may constitute one of the important mechansims of biochar-mediated control of Phytophora blight of pepper.

Fungal endophytes have the capacity to biosynthesize secondary metabolites that are produced by their host plants. In this study, a dilactone terpenoid of C16 architecture was isolated from the fungal endophytes of Kigelia africana, in our attempt to identify anti-Pseudomonas aeruginosa metabolites. Thirty-eight fungal isolates were cultured for biomolecule production over a period of thirty days. Extracts from three (ZF 34, ZF 52 and ZF 91) of the fungi showed good anti-P. aeruginosa activity, with ZF 52 presenting the best MIC of 19.53 µg/mL and was accordingly subjected to chromatographic separation. Based on nuclear magnetic resonance (NMR) spectroscopy, high resolution mass spectrometry and single crystal X-ray diffraction (XRD) analyses, the isolated compound was identified as a C16-terpene dilactone, with a structure consistent with that of the known diterpene, CJ-14445. The isolated dilactone showed anti-P. aeruginosa activity with MIC of 0.61 µg/mL, signifying the antibacterial potential of the biomolecule. The bioactive fungal isolate (ZF 52) was identified as Neofusicoccum luteum based on genomic DNA sequencing. This is the first report of the endophyte N. luteum from K. africana and the first reported occurrence of CJ-14445 in the fungus.

Additional file 1: Figure S1. Pyrogram of commercial PHB. Figure S2. Pyrogram of commercial PHBV. Figure S3. Pyrogram of PHA extracted after cycle 1 of cyclic fed-batch fermentation. Figure S4. Pyrogram of PHA extracted after cycle 2 of cyclic fed-batch fermentation. Figure S5. Pyrogram of PHA extracted after cycle 3 of cyclic fed-batch fermentation. Figure S6. Pyrogram of PHA extracted after cycle 4 of cyclic fed-batch fermentation.

For some environments such as planktonic/aqueous environments, the separation of bacteria cells from eukaryotic cells prior to DNA extraction using filtration is relatively straightforward. However, for woodchips, the bacteria are attached/embedded within the wood matrix, which prevents easy removal of bacterial cells. In this study, a method for the selective extraction of DNA from bacteria inhabiting Eucalyptus spp. woodchips has been developed. The objective was to compare milled and unmilled woodchips processed via three detachment methods, viz., sonication, vortexing and shaking followed by filtration using Teflon filters according to three relevant criteria: DNA yield, DNA purity and quality of DNA. Highest DNA yield was obtained by milling and vortexing for 10 min (77.50 ± 5.17 ng/μl), followed by milling and vortexing for 2 min (61.00 ± 6.56 ng/μl), unmilled and vortexing for 10 min (38.67 ± 5.17 ng/μl) and milled and shaking for 2 h (31.62 ± 5.17 ng/μl). The lowest DNA yield was obtained by using unmilled woodchips and 5 min of sonication treatment (7.00 ± 1.22 ng/μl). There was no significant difference in DNA purity for milled or unmilled woodchips processed via the three detachment methods. Duration of cell detachment treatment did not significantly influence DNA yield and purity. Following optimisation experiments, it was possible to extract bacterial DNA using milled woodchips and 10 minute vortexing devoid of DNA from the host background and other associated eukaryotes and of sufficient quality and quantity for metagenomic analysis.

Highlights • Phenol red screening plates is the best method for detecting lipolytic activity. • Substrate specificity is affected by temperature and pH. • Essential to test substrates at various pH and temperature to determine optima. • Lipolytic enzymes indigenous to Eucalyptus sp. can assist in pitch control., This study highlights the importance of determining substrate specificity at variable experimental conditions. Lipases and esterases were isolated from microorganisms cultivated from Eucalyptus wood species and then concentrated (cellulases removed) and characterized. Phenol red agar plates supplemented with 1% olive oil or tributyrin was ascertained to be the most favourable method of screening for lipolytic activity. Lipolytic activity of the various enzymes were highest at 45–61 U/ml at the optimum temperature and pH of between at 30–35 °C and pH 4–5, respectively. Change in pH influenced the substrate specificity of the enzymes tested. The majority of enzymes tested displayed a propensity for longer aliphatic acyl chains such as dodecanoate (C12), myristate (C14), palmitate (C16) and stearate (C18) indicating that they could be characterised as potential lipases. Prospective esterases were also detected with specificity towards acetate (C2), butyrate (C4) and valerate (C5). Enzymes maintained up to 95% activity at the optimal pH and temperature for 2–3 h. It is essential to test substrates at various pH and temperature when determining optimum activity of lipolytic enzymes, a method rarely employed. The stability of the enzymes at acidic pH and moderate temperatures makes them excellent candidates for application in the treatment of pitch during acid bi-sulphite pulping, which would greatly benefit the pulp and paper industry.

Taxonomic and metabolic profiles of bacterial inhabitants of Eucalyptus spp. woodchips from storage piles were analysed using whole genome shotgun metagenomics sequencing. The majority of the bacterial species present in the metagenome from the Eucalyptus spp. woodchips are found within four bacterial phyla: Proteobacteria (77%), Bacteroidetes (15%), Acidobacteria (3%) and Actinobacteria (2%). The most abundant species in the metagenome were Burkholderia spp. (25%; Proteobacteria) and Chryseobacterium spp. (37%; Bacteroidetes). Other species prevalent in this bacterial metagenome are Rhodanobacter spp. (7%), Klebsiella spp. (6%) and Pseudomonas spp. (5%). Both PhyloSift and Metagenome Rapid Annotation Subsystem Technology tools to estimate taxonomic diversity showed that the phylum Proteobacteria was the predominant group among the bacteria in the bacterial metagenome from the Eucalyptus spp. woodchips, followed by Bacteroidetes, Acidobacteria and Actinobacteria. Functional analysis revealed that clustering-based subsystem and carbohydrate metabolism which are key to degradation of the lignocellulose were the most abundant SEED subsystems representing 14% and 11% of the bacterial metagenome, respectively. Approximately 8% of the sequences were grouped under “miscellaneous functions” and 3% under metabolism of aromatic compounds. One thousand two hundred forty-seven potential contigs encoding biomass-degrading enzymes including glycoside hydrolases (GH, 525 contigs), glycosyl transferases (GT, 452 contigs) carbohydrate binding modules (CBM, 149 contigs), carbohydrate esterases (CE, 70 contigs), polysaccharide lyases (PL, 16 contigs) and auxiliary activity (AA, 35 contigs) were identified. Both taxonomic and functional classifications suggest that the bacterial inhabitants of the Eucalyptus spp. woodchips present a broader metabolic potential than the degradation of plant biomass.

Pulp and paper mill sludge is a waste stream derived from the pulp and paper making industry, comprised of organic and inorganic material in the form of cellulose, hemicellulose, lignin and ash. In South Africa, approximately fivefour hundred thousand wet tonnes are produced per annum and is currently disposed via landfilling or incineration. However, these disposal methods raise environmental and financial concerns. This waste stream is an attractive feedstock for fermentable sugars, mainly glucose, recovery and can be redirected for valorisation as a feedstock for microbial fermentation to produce value-added products. Sugar recovery by enzymatic hydrolysis, as opposed to acidic hydrolysis, is a promising approach but is hampered by the lignin and inorganic material found in pulp and paper mill sludge. Several treatment steps to reduce or remove these components prior to enzymatic hydrolysis are assessed in this review. Pretreatment improves hydrolysis of cellulosic fibres and ensures a substantial yield of sugars.

Lipophilic extractives naturally occurring in wood tend to coalesce during pulping to form pitch deposits, which have particularly undesirable effects on the pulping process and quality of pulp produced. A chemical characterization of different eucalypt species [Eucalyptus nitens, E. grandis, and E. dunnii (of different site qualities)] wood and generated pulp was performed. This study aimed at determining the effects of wood storage at -20 °C (for 6 months), by examining their chemical composition and indigenous microflora. Fatty acids were the main lipophilic compounds among E. dunnii (SQ3 and SQ4) and E. grandis wood extractives. The wood of E. nitens posed the least risk for pitch deposit formation, making it the most suitable Eucalyptus species for pulping. Storage of wood chips at -20 °C had a similar effect as the traditional method of seasoning (storage of wood outdoors prior to pulping) used for the reduction of lipophilic extractives. A 25 to 44% reduction of total extractives was observed in the raw material after storage. Variations in bacterial and fungal communities were observed after storage, and should be taken into consideration when conducting lab scale trials. If storage of wood chips is necessary for lab testing, it should be retained for a maximum of 3 months at -20 °C.

In the pulp and paper industry, during the manufacturing process, the agglomeration of pitch particles (composed of triglycerides, fatty acids, and esters) leads to the formation of black pitch deposits in the pulp and on machinery, which impacts on the process and pulp quality. Traditional methods of pitch prevention and treatment are no longer feasible due to environmental impact and cost. Consequently, there is a need for more efficient and environmentally friendly approaches. The application of lipolytic enzymes, such as lipases and esterases, could be the sustainable solution to this problem. Therefore, an understanding of their structure, mechanism, and sources are essential. In this report, we review the microbial sources for the different groups of lipolytic enzymes, the differences between lipases and esterases, and their potential applications in the pulping industry.

Eucalyptus is the predominant exotic wood species used in South African pulp and paper industry. Once chipped and stored in piles, the wood becomes vulnerable to microbial degradation and spontaneous combustion. The denaturing gradient gel electrophoresis (DGGE) technique was optimized for the detection of microbial diversity in the wood. Wood chips were collected and milled to different specifications. The 16S and 18S rRNA genes were amplified using 338F-GC/518R and 933F-GC/1387R for bacteria and NS26/518R-GC and EF4F/518R for fungi. Several gel gradients were examined to determine optimal separation. A comparison of DGGE profiles revealed greater diversity in the milled wood chips amplified using primer sets of 338F-GC/518R (16S) and NS26/518R-GC (18S) with gradients of 30/60% (16S) and 25/50% (18S), respectively. Once optimized, this protocol was tested against five samples to assess its applicability to wood chip samples. Profiles were generated and amplicons excised from gels, re-amplified and sequenced to determine origin of DNA. Using this technique, 18 bacterial and 12 fungal species were identified, compared to ten bacterial and nine fungal isolates which were identified using the culturing technique and standard rRNA gene sequence analysis. The optimised DGGE is an appropriate tool for microbial community studies of Eucalyptus wood chips. Key words: Wood chips, Eucalyptus, community analysis using denaturing gradient gel electrophoresis (DGGE), microfloral variations.

Background Women make up more than 50% of adults living with human immunodeficiency virus (HIV) infection or the acquired immunodeficiency syndrome (AIDS) in sub-Saharan Africa. Thus, female-initiated HIV prevention methods are urgently needed. Methods We performed a randomized, double-blind, placebo-controlled trial of cellulose sulfate, an HIV-entry inhibitor formulated as a vaginal gel, involving women at high risk for HIV infection at three African and two Indian sites. The primary end point was newly acquired infection with HIV type 1 or 2. The secondary end point was newly acquired gonococcal or chlamydial infection. The primary analysis was based on a log-rank test of no difference in the distribution of time to HIV infection, stratified according to site. Results A total of 1398 women were enrolled and randomly assigned to receive cellulose sulfate gel (706 participants) or placebo (692 participants) and had follow-up HIV test data. There were 41 newly acquired HIV infections, 25 in the cellulose sulfate group and 16 in the placebo group, with an estimated hazard ratio of infection for the cellulose sulfate group of 1.61 (P=0.13). This result, which is not significant, is in contrast to the interim finding that led to the trial being stopped prematurely (hazard ratio, 2.23; P=0.02) and the suggestive result of a preplanned secondary (adherence-based) analysis (hazard ratio, 2.02; P=0.05). No significant effect of cellulose sulfate as compared with placebo was found on the risk of gonorrheal infection (hazard ratio, 1.10; 95% confidence interval [CI], 0.74 to 1.62) or chlamydial infection (hazard ratio, 0.71; 95% CI, 0.47 to 1.08). Conclusions Cellulose sulfate did not prevent HIV infection and may have increased the risk of HIV acquisition. (ClinicalTrials.gov number, NCT00153777 ; and Current Controlled Trials number, ISRCTN95638385.)

For indirect DNA extraction for metagenomics studies, bacterial cells can be effectively separated from sample debris by using a simple size exclusion technique, such as filtration, and thereafter lysed. The requirement for the optimal recovery of cells in filtrates is critical to achieve sufficient DNA yield and a representative population. Particles smaller than the filter pore size are expected to be found in the filtrate, whereas particles larger than the filter pore sizes are excluded. However, this is not always the case. It is established that the membrane pore size influences filtration efficiency to some degree. In addition the physicochemical characteristics of the filter suspension and characteristics of the microbial cells being filtered influence the exclusion property of a membrane. This review provides an overview of membrane filtration techniques and the factors that affect filterability of bacteria cells through a filter membrane.

In this study we report on the green synthesis of silver nanoparticles using extracts from selected morphological parts of Zanthoxylum capense. UV–vis spectra of the biosynthesised silver nanoparticles (AgNPs) revealed absorption peaks at around 450 nm, indicative of the nanoparticles' surface plasmon resonance, whilst infrared vibrational frequencies indicated the presence of flavonoids, alkaloids, and free and bonded sugars which could be responsible for the reduction and stabilisation of the AgNPs. 1H-NMR fingerprinting of the aqueous knob extract confirmed the active bio-reducing phytochemical of the knobs to be 6-O-p-coumaroyl-β-D-glucopyranoside. The nature, shape and morphology of the biosynthesised AgNPs were examined using transmission electron microscopy (TEM), selected area electron diffraction (SAED), scanning electron microscopy (SEM) and energy dispersive x-ray (EDX) analysis. Z. capense AgNPs were mostly spherical in shape with particle sizes in the range of 4–28 nm, 7–20 nm and 4–32 nm for leaves, knobs and roots, respectively. Leaf extracts were the most efficient in the synthesis of AgNPs with an average yield of 0.027 g AgNPs per g of plant (dry mass). The AgNPs were more effective than sodium hypochlorite (NaOCl) and sodium dichloroisocyanurate (NaDCC) in the control of in vitro fungal contamination in nodal explants of Z. capense up to two weeks. Shoots induced from the surface sterilised explants were further used for shoot multiplication on benzyl aminopurine (BAP) and kinetin (KIN). BAP at 0.5 mg l−1 gave the highest percentage (88.6%) of explants bearing shoots with an average of 4.78 shoots per explant. A total of 15 fungal endophyte strains associated with Z. capense were identified using molecular methods.

Background South Africa, with its scientific capacity, good infrastructure and high HIV incidence rates, is ideally positioned to conduct large-scale HIV prevention trials. The HIV Prevention Research Unit of the South African Medical Research Council conducted four phase III and one phase IIb trials of women-initiated HIV prevention options in KwaZulu-Natal between 2003 and 2009. A total of 7046 women participated, with HIV prevalence between 25% and 45% and HIV incidence ranging from 4.5-9.1% per year. Unfortunately none of the interventions tested had any impact on reducing the risk of HIV acquisition; however, extremely valuable experience was gained, lessons learned and capacity built, while the communities gained associated benefits. Experience Our experience in conducting these trials ranged from setting up community partnerships to developing clinical research sites and dissemination of trial results. Community engagement included setting up community-based research sites with approval from both political and traditional leaders, and developing community advisory groups to assist with the research process. Community-wide education on HIV/sexually transmitted infection prevention, treatment and care was provided to over 90 000 individuals. Myths and misconceptions were addressed through methods such as anonymous suggestion boxes in clinic waiting areas and intensive education and counselling. Attempts were made to involve male partners to foster support and facilitate recruitment of women. Peer educator programmes were initiated to provide ongoing education and also to facilitate recruitment of women to the trials. Recruitment strategies such as door-to-door recruitment and community group meetings were initiated. Over 90% of women enrolled were retained. Community benefits from the trial included education on HIV prevention, treatment and care and provision of ancillary care (such as Pap smears, reproductive health care and referral for chronic illnesses). Social benefits included training of home-based caregivers and sustainable ongoing HIV prevention education through peer educator programmes. Challenges Several challenges were encountered, including manipulation by participants of their eligibility criteria in order to enroll in the trial. Women attempted to co-enroll in multiple trials to benefit from financial reimbursements and individualised care. The trials became ethically challenging when participants refused to take up referrals for care due to stigma, denial of their HIV status and inadequate health infrastructure. Lack of disclosure of HIV status to partners and family members was particularly challenging. Some of the ethical dilemmas put to the test our responsibility as researchers and our obligation to provide health care to research participants. Conclusion Conducting these five trials in a period of six years provided us with invaluable insights into trial implementation, community participation, recruitment and retention, provision of care and dissemination of trial results. The critical mass of scientists trained as clinical trialists will continue to address the relentless HIV epidemic in our setting and ensure our commitment to finding a biomedical HIV prevention option for women in the future.

Phenolic compounds from plants offer a means for both the prevention and detoxification of mycotoxins that affect human health. This research investigates the control of fungal growth and toxin production by Fusarium verticillioides with plant phenolic compounds, namely chlorophorin, iroko and maakianin, benzoic acid, caffeic acid, ferulic acid, and vanillic acid. Inhibition by these compounds of fungal growth was determined by the agar overlay method and their effect on fumonisin B(1) (FB(1)) production was determined by high-performance liquid chromatography. Chlorophorin was the most effective compound in inhibiting fungal growth, followed by iroko, maakianin, vanillic acid and caffeic acid. Chlorophorin also was the most effective compound in reducing toxin production (94% reduction), followed by caffeic acid, ferulic acid, vanillic acid and iroko, which reduced FB(1) levels by 90-91%. The widespread occurrence of fumonisins world-wide and the lack of adequate prevention of fumonisins require 'biologically safe' alternatives to prevent the transfer of fungi and their health hazardous toxins into our daily foods and environment.

This study was undertaken to determine the effectiveness of modified atmospheres and packaging materials on the growth of Penicillium expansum and patulin production in apples. Granny Smith apples were surface sterilized with 76% ethanol and inoculated with 0.1 ml of a 1.1 x 10(7) spore/ml P. expansum spore suspension. The apples were packaged either in polyethylene (PE) or polypropylene (PP) and treated with three different gas combinations, viz., 58% CO2/42% N2, 48% CO2/52% N2, and 88% CO2/12% N2, and were then incubated for 14 days at 25 degrees C. Fungal growth was monitored every 2 to 4 days by measuring radial growth from the point of inoculation. After the 14th day, apples were pulped, and patulin was extracted, purified, and quantified by high-performance liquid chromatography. PP did not inhibit fungal growth in any of the atmospheres tested, and it only inhibited patulin production in atmospheric gas and 58% CO2/42% N2. PE was very effective and inhibited fungal growth by four- or fivefold, depending on the modified atmosphere. Patulin production in PE-packaged apples was almost completely inhibited by all three gas combinations. Gas chromatographic analysis of the PE-packaged samples before and after the incubation period showed that CO2 levels dropped and N2 levels increased for all of the atmospheres tested. Our studies showed conclusively that PE is an excellent packaging material for the storage of apples since it inhibited the growth of P. expansum, thereby allowing3.2 microg/ml of patulin to be produced, regardless of gaseous environment.

The effect of nine different oils was evaluated on the growth of Aspergillus parasiticus and Fusarium moniliforme. The experimental design to examine the inhibition of mycotoxins involved the incorporation of each of seven oils into broth and patty cultures. The fungal mycotoxin was identified by high-pressure liquid chromatography. Clove oil (eugenol) was the most inhibitory to the growth of A. parasiticus and F. moniliforme, followed by cinnamon (cinnamic aldehyde), oregano (thymol and carvacol) and mace oils (myristin). Neem and eucalyptus oil (cineole) did not affect fungal growth. The feasibility of implementing the results of this study to control mycotoxin toxicity was examined by costoring whole and ground cloves with mycotoxin-infected grain. Addition of both whole and ground cloves markedly reduced the aflatoxin contamination of the grain. These results clearly suggest that commonly occurring mycotoxigenic fungi can be controlled with clove oil (eugenol), thus spice oil successfully inhibited the growth of A. parasiticus and F. moniliforme, regulated the production of fumonisins, and prevented the formation of aflatoxins. The social implication of this finding is that rural communities can prevent the formation of fungal toxins in contaminated grain by simple measures. Dans cette etude, l'effet de 9 huiles d'epices sur la multiplication de moisissures produisant de l'aflatoxine et de la fumonisine (Aspergillus parasiticus et Fusarium monoliforme) est evalue par la methode de recouvrement en gelose. L'huile de clou de girofle (eugenol) est la plus efficace pour l'inhibition de A. parasiticus et F. monoliforme, suivie de la cannelle (aldehyde cinnamique), l'origan (thymol et carcvacrol) et muscade (myristine).

The xylose reductase gene (XYL1) was isolated from Pichia stipitis and Candida shehatae, cloned into YEp-based vectors under the control of ADH2 and PGK1 promoter/terminator cassettes and introduced into Saccharomyces cerevisiae Y294 by electroporation. Shake-flask fermentations were carried out with 5% xylose and 1% galactose, glucose or maltose as co-substrates. Xylose uptake was similar in both the recombinant strains when different co-substrates were used and slowed once the co-substrate was depleted. The recombinant strains converted xylose to xylitol with yields approaching the theoretical maxima. Xylitol production was most rapid when the co-substrate was still present. Approximately 50% of the xylose was not metabolized due to the depletion of the co-substrate.

DNA-DNA binding experiments on selected fusants of Candida shehatae and Pichia stipitis showed that the nucleus of these strains was composed predominantly of Pichia DNA. Electrophoretic karyotyping revealed that the fusants contained four chromosomes, similar to those found in the Pichia parental strain. In addition, the fusants showed only marginal increases in cell DNA content when compared with the parents. Karyogamy was confirmed, however, by the isolation of recombinant phenotypic segregants, induced by meiotic and mitotic segregation. The results suggest that the fusion led to integration of Candida genes, rather than whole chromosomes, with the entire genome of P. stipitis.

In sub-Saharan Africa, almost 60% of HIV infections are among women [1], and the number of new HIV infections in women worldwide continues to escalate. The high incidence of HIV in many African countries provides the optimum environment for research on technologies that could prevent women from becoming infected, including microbicides. In this article, we discuss the recent highly publicised closure of a trial of cellulose sulphate (CS), which we conducted. We discuss the impact of the closure on the participants, the community at the trial site and the public at large, the public health sector, national regulatory bodies, the media, and on other ongoing microbicide trials. The local lessons that we learnt from the closure may provide guiding principles for researchers and advocates in the HIV prevention field as a whole, who may face similar situations in the future.