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The accumulation of petrochemical plastic waste is detrimental to the environment. Polyhydroxyalkanoates (PHAs) are bacterial-derived polymers utilized for the production of bioplastics. PHA-plastics exhibit mechanical and thermal properties similar to conventional plastics. However, high production cost and obtaining high PHA yield and productivity impedes the widespread use of bioplastics. This study demonstrates the concept of cyclic fed-batch fermentation (CFBF) for enhanced PHA productivity by Bacillus thuringiensis using a glucose-rich hydrolyzate as the sole carbon source. The statistically optimized fermentation conditions used to obtain high cell density biomass (OD600 of 2.4175) were: 8.77 g L−1 yeast extract; 66.63% hydrolyzate (v/v); a fermentation pH of 7.18; and an incubation time of 27.22 h. The CFBF comprised three cycles of 29 h, 52 h, and 65 h, respectively. After the third cyclic event, cell biomass of 20.99 g L−1, PHA concentration of 14.28 g L−1, PHA yield of 68.03%, and PHA productivity of 0.219 g L−1 h−1 was achieved. This cyclic strategy yielded an almost threefold increase in biomass concentration and a fourfold increase in PHA concentration compared with batch fermentation. FTIR spectra of the extracted PHAs display prominent peaks at the wavelengths unique to PHAs. A copolymer was elucidated after the first cyclic event, whereas, after cycles CFBF 2–4, a terpolymer was noted. The PHAs obtained after CFBF cycle 3 have a slightly higher thermal stability compared with commercial PHB. The cyclic events decreased the melting temperature and degree of crystallinity of the PHAs. The approach used in this study demonstrates the possibility of coupling fermentation strategies with hydrolyzate derived from lignocellulosic waste as an alternative feedstock to obtain high cell density biomass and enhanced PHA productivity.

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...

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.

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.

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.

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.

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.

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.