Predictive simulation of the water-energy-food nexus for the City of Cape Town.

The City of Cape Town (CoCT), South Africa faced a critical situation between 2015 and 2018 in which the municipal water supply was almost completely exhausted. This situation, commonly referred to as Day Zero in South Africa emanated from a decline in rainfall, resulting in one of the most severe droughts in history. The crisis was also aggravated by rapid population growth and urbanization. CoCT was on the verge of becoming the first city in the past decade to experience a complete cessation of water supply for urban and agricultural purposes. In addition to the effects of low rainfall and population surge, urban energy consumption and increased food demand impacted directly the available water resources. To evaluate the interlinkages between water utilization, water production, energy supply and demand, and food production and demand, this study employed a system dynamics modeling (SDM) approach. The model was developed as a stock and flow diagram utilizing Stella Architect and encompassed five interconnected nodes: water, energy, food, land, and population. The findings revealed that by the end of the 20-year modeling period, the volume of accessible and stored water in all the major dams will be approximately 459 million cubic meters, with residential use accounting for about 85 % of urban water use and agriculture accounting for approximately30.37 % of total water demand. The model illustrates the impacts of precipitation rate, runoff, and evaporation on variables such as land-use change and population dynamics. It is anticipated that the outcomes of this study will serve as valuable inputs for decision-making processes, not only within the CoCT as it aims to mitigate or prevent the recurrence of Day Zero, but also for other cities facing similar challenges.

Stress modulation as a means to improve yeasts for lignocellulose bioconversion.

The second-generation (2G) fermentation environment for lignocellulose conversion presents unique challenges to the fermentative organism that do not necessarily exist in other industrial fermentations. While extreme osmotic, heat, and nutrient starvation stresses are observed in sugar- and starch-based fermentation environments, additional pre-treatment-derived inhibitor stress, potentially exacerbated by stresses such as pH and product tolerance, exist in the 2G environment. Furthermore, in a consolidated bioprocessing (CBP) context, the organism is also challenged to secrete enzymes that may themselves lead to unfolded protein response and other stresses. This review will discuss responses of the yeast Saccharomyces cerevisiae to 2G-specific stresses and stress modulation strategies that can be followed to improve yeasts for this application. We also explore published -omics data and discuss relevant rational engineering, reverse engineering, and adaptation strategies, with the view of identifying genes or alleles that will make positive contributions to the overall robustness of 2G industrial strains. KEYPOINTS: • Stress tolerance is a key driver to successful application of yeast strains in biorefineries. • A wealth of data regarding stress responses has been gained through omics studies. • Integration of this knowledge could inform engineering of fit for purpose strains.

The synergistic application of quinone reductase and lignin peroxidase for the deconstruction of industrial (technical) lignins and analysis of the degraded lignin products.

The effect of combined quinone reductase (QR) and lignin peroxidase (LiP) on the depolymerization of technical lignins isolated from soda-anthraquinone (SAQ), steam explosion (S-E), and two sulfite processes (NaE and NaPE) was investigated. While LiP is best known for its ability to degrade lignins, it may also cause lignin re-polymerization due to the random coupling of phenoxy radicals and quinoid intermediates. This study evidenced that the addition of the bioreactor produced QR can to some extent limit the lignin re-polymerization by LiP. The synergistic application of QR and LiP lowered the molecular weights (Mw) of SAQ, NaE, S-E, and NaPE lignins by 31%, 34%, 41%, and 52%, respectively. The thermogravimetric analysis also showed that the thermal stability of the four lignins was reduced, whereas gas chromatography-mass spectrometry analysis showed that the degradation products included monomeric phenols. Therefore, the combined QR and LiP system is a promising approach for lignin valorization.

Synergistic codon optimization and bioreactor cultivation toward enhanced secretion of fungal lignin peroxidase in Pichia pastoris: Enzymatic valorization of technical (industrial) lignins.

Lignin peroxidase (LiP) is a well-recognized enzyme for its ability to oxidize lignins, but its commercial availability is limited, which hinders the biotechnological application of LiP-based bioprocesses in lignocellulose biorefineries. This study evaluated a combination strategy to improve the expression of LiP to promote its practical use. The strategy included optimization of the lipH8 gene of Phanerochaete chrysosporium according to the codon usage of Pichia pastoris, followed by fed-batch fermentation using a 14 L bioreactor (10 L working volume). The combination strategy achieved a maximum volumetric LiPH8 activity of 4480 U L-1, protein concentration of 417 mg L-1 and a specific activity of 10.7 U mg-1, which was higher than previous reports. Biochemical characterization showed that the recombinant LiPH8 (rLiPH8) was optimum at pH 3.0, 25 ℃ and 0.4 mM H2O2. Using the optimized conditions, rLiPH8 was used to treat isolated technical lignins namely soda-anthraquinone (SAQ) lignin and steam explosion (S-E) lignin. High-performance gel permeation chromatography (HP-GPC) analysis showed that the molecular weight (Mw) of SAQ and S-E lignins were increased by 1.43-and 1.14-fold, respectively, after the enzymatic treatment. Thermogravimetric analysis (TGA) also showed that the thermal stability of the lignins was improved, indicating that the enzyme treatment of lignins with rLiPH8 resulted in lignin re-polymerization. As the first report on rLiPH8 production using P. pastoris, this study has shed light on the possible route for the enhancement of rLiPH8 production and its potential application for upgrading technical lignins.

A review on the demineralisation of pre- and post-pyrolysis biomass and tyre wastes.

Pyrolysis is an attractive technology to convert low-cost carbonaceous waste materials into fuels, energy and other value added products goods. During pyrolysis, the inorganic minerals present in the feedstock can cause problems to the equipment and give side reactions. Besides, the minerals present in the chars can hinder their possible applications. Therefore, it seems necessary to eliminate said contaminants in order to valorise the aforementioned goods. Demineralisation is a process widely used for purifying materials that are contaminated with inorganic matter. Although this technique is commonly used with waste materials that will undergo pyrolysis, or the products obtained from it, the studies analysing this practise are rather scattered. The aim of this paper was to compile and review the current literature concerning the demineralisation of carbonaceous waste (tyres and lignocellulosic biomass) materials. The chemistry involved, feedstock type and the effect of performing the purifying step before or after pyrolysis were addressed in this work. The review revealed that biomass samples should be demineralised before pyrolysis in order to affect not only the char but also the bio-oil quality. Depending on the form in which the minerals are linked to the structure, the solvent chosen will vary (from water to strong acids). However, water is the most popular option due to its price and easy disposal. In tyres, demineralisation should be performed after pyrolysis using strong acid and subsequently base. Due to the crosslinked chemical structure, rubber is highly resistant to chemicals thus the pre-treatment has to be avoided.

In situ enzyme aided adsorption of soluble xylan biopolymers onto cellulosic material.

The functional properties of cellulose fibers can be modified by adsorption of xylan biopolymers. The adsorption is improved when the degree of biopolymers substitution with arabinose and 4-O-methyl-glucuronic acid (MeGlcA) side groups, is reduced. α-l-Arabinofuranosidase (AbfB) and α-d-glucuronidase (AguA) enzymes were applied for side group removal, to increase adsorption of xylan from sugarcane (Saccharum officinarum L) bagasse (BH), bamboo (Bambusa balcooa) (BM), Pinus patula (PP) and Eucalyptus grandis (EH) onto cotton lint. The AguA treatment increased the adsorption of all xylans by up to 334%, whereas, the AbfB increased the adsorption of the BM and PP by 31% and 44%, respectively. A combination of AguA and AbfB treatment increased the adsorption, but to a lesser extent than achieved with AguA treatment. This indicated that the removal of the glucuronic acid side groups provided the most significant increase in xylan adsorption to cellulose, in particular through enzymatic treatment.

Semirational Directed Evolution of Loop Regions in Aspergillus japonicus ß-Fructofuranosidase for Improved Fructooligosaccharide Production.

The Aspergillus japonicus ß-fructofuranosidase catalyzes the industrially important biotransformation of sucrose to fructooligosaccharides. Operating at high substrate loading and temperatures between 50 and 60°C, the enzyme activity is negatively influenced by glucose product inhibition and thermal instability. To address these limitations, the solvent-exposed loop regions of the ß-fructofuranosidase were engineered using a combined crystal structure- and evolutionary-guided approach. This semirational approach yielded a functionally enriched first-round library of 36 single-amino-acid-substitution variants with 58% retaining activity, and of these, 71% displayed improved activities compared to the parent. The substitutions yielding the five most improved variants subsequently were exhaustively combined and evaluated. A four-substitution combination variant was identified as the most improved and reduced the time to completion of an efficient industrial-like reaction by 22%. Characterization of the top five combination variants by isothermal denaturation assays indicated that these variants displayed improved thermostability, with the most thermostable variant displaying a 5.7°C increased melting temperature. The variants displayed uniquely altered, concentration-dependent substrate and product binding as determined by differential scanning fluorimetry. The altered catalytic activity was evidenced by increased specific activities of all five variants, with the most improved variant doubling that of the parent. Variant homology modeling and computational analyses were used to rationalize the effects of amino acid changes lacking direct interaction with substrates. Data indicated that targeting substitutions to loop regions resulted in improved enzyme thermostability, specific activity, and relief from product inhibition.

Application of endo-ß-1,4,D-mannanase and cellulase for the release of mannooligosaccharides from steam-pretreated spent coffee ground.

Spent coffee ground (SCG), a present waste stream from instant coffee production, represents a potential feedstock for mannooligosaccharides (MOS) production. MOS can be used in nutraceutical products for humans/animals or added to instant coffee, increasing process yield and improving product health properties. The SCG was evaluated for MOS production by steam pretreatment and enzymatic hydrolysis with a recombinant mannanase and a commercial cellulase cocktail (Acremonium, Bioshigen Co. Ltd, Japan). The mannanase was produced using a recombinant strain of Yarrowia lipolytica, used to produce and secrete endo-1,4-ß,D-mannanase from Aspergillus aculeatus in bioreactor cultures. Endo-1,4-ß,D-mannanase was produced with an activity of 183.5 U/mL and 0.23 mg protein/mL. The enzyme had an optimum temperature of 80 °C, and the activity in the supernatant was improved by 150 % by supplementation with 0.2 % sodium benzoate and 35 % sorbitol as a preservative and stabiliser, respectively. The steam pretreatment of SCG improved the enzymatic digestibility of SCG, thus reducing the required enzyme dosage for MOS release. Combined enzymatic hydrolysis of untreated or steam-pretreated (150, 190 and 200 °C for 10 min) SCG with mannanase and cellulase cocktail resulted in 36-57 % (based on mannan content) of MOS production with a degree of polymerization of up to 6. The untreated material required at least 1 % of both mannanase and cellulase loading. The optimum mannanase and cellulase loadings for pretreated SCG hydrolysis were between 0.3 and 1 and 0.4 and 0.8 %, respectively. Statistical analysis suggested additive effect between cellulase cocktail and mannanase on MOS release, with no indication of synergism observed.

Optimization of carbon and nitrogen medium components for biomass production using non-Saccharomyces wine yeasts.

UNLABELLED: The impact of different nitrogen and carbon sources on biomass production of the non-Saccharomyces wine yeast species Lachancea thermotolerans, Metschnikowia pulcherrima and Issatchenkia orientalis was assessed. Using a molasses-based medium, yeast extract and corn steep liquor as well as ammonium sulphate and di-ammonium phosphate (DAP) as nitrogen sources were compared in shake-flask cultures. A medium with 20 g l⁻¹ sugar (diluted molasses) and 500 mg l⁻¹ total yeast assimilable nitrogen, from yeast extract, gave the highest biomass concentrations and yields. Invertase pretreatment was required for cultures of M. pulcherrima and I. orientalis, and respective biomass yields of 0.7 and 0.8 g g⁻¹ were achieved in aerobic bioreactor cultures. The absence of ethanol production suggested Crabtree-negative behaviour by these yeasts, whereas Crabtree-positive behaviour by L. thermotolerans resulted in ethanol and biomass concentrations of 5.5 and 11.1 g l⁻¹, respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: Recent studies demonstrate that non-Saccharomyces yeasts confer positive attributes to the final composition of wine. However, optimal process conditions for their biomass production have not been described, thereby limiting commercial application. In this study, industrial media and methods of yeast cultivation were investigated to develop protocols for biomass production of non-Saccharomyces yeast starter cultures for the wine industry.

Optimization of dilute sulfuric acid pretreatment to maximize combined sugar yield from sugarcane bagasse for ethanol production.

Increasing fermentable sugar yields per gram of biomass depends strongly on optimal selection of varieties and optimization of pretreatment conditions. In this study, dilute acid pretreatment of bagasse from six varieties of sugarcane was investigated in connection with enzymatic hydrolysis for maximum combined sugar yield (CSY). The CSY from the varieties were also compared with the results from industrial bagasse. The results revealed considerable differences in CSY between the varieties. Up to 22.7 % differences in CSY at the optimal conditions was observed. The combined sugar yield difference between the best performing variety and the industrial bagasse was 34.1 %. High ratio of carbohydrates to lignin and low ash content favored the release of sugar from the substrates. At mild pretreatment conditions, the differences in bioconversion efficiency between varieties were greater than at severe condition. This observation suggests that under less severe conditions the glucose recovery was largely determined by chemical composition of biomass. The results from this study support the possibility of increasing sugar yields or improving the conversion efficiency when pretreatment optimization is performed on varieties with improved properties.

Screening a random mutagenesis library of a fungal ß-fructofuranosidase using FT-MIR ATR spectroscopy and multivariate analysis.

Short-chain fructooligosaccharides (scFOS) are valuable health-promoting food additives. During the batch production of scFOS from sucrose the ß-fructofuranosidase catalyst is subject to product inhibition by glucose. Engineering the enzyme for reduced sensitivity to glucose could improve product yields or process productivity while preserving the simple industrial batch design. Random mutagenesis is a useful technique for engineering proteins but should be coupled to a relevant high-throughput screen. Such a screen for sucrose and scFOS quantification remains elusive. This work presents the development of a screening method displaying potential high-throughput capacity for the evaluation of ß-fructofuranosidase libraries using Fourier transform mid-infrared attenuated total reflectance (FT-MIR ATR) spectroscopy and multivariate analysis. A calibration model for the quantification of sucrose in enzyme assay samples ranged from 5 to 200 g/l and the standard error of prediction was below 13 g/l. A library of the Aspergillus japonicus fopA gene was generated by error prone PCR and screened in Saccharomyces cerevisiae. Using FT-MIR ATR spectroscopy, potential hits were identified as those variants that converted more sucrose in the presence of the glucose inhibitor than the parent. Subsequent analysis of reaction products generated by top performers using high-performance liquid chromatography identified a variant producing higher scFOS levels than the parent. At the peak difference in performance the variant produced 28 % more scFOS from the same amount of sucrose. This study highlights the application of FT-MIR ATR spectroscopy to a variant discovery pipeline in the directed evolution of a ß-fructofuranosidase for enhanced scFOS production.

Enzymatic hydrolysis of spent coffee ground.

Spent coffee ground (SCG) is the main residue generated during the production of instant coffee by thermal water extraction from roasted coffee beans. This waste is composed mainly of polysaccharides such as cellulose and galactomannans that are not solubilised during the extraction process, thus remaining as unextractable, insoluble solids. In this context, the application of an enzyme cocktail (mannanase, endoglucanase, exoglucanase, xylanase and pectinase) with more than one component that acts synergistically with each other is regarded as a promising strategy to solubilise/hydrolyse remaining solids, either to increase the soluble solids yield of instant coffee or for use as raw material in the production of bioethanol and food additives (mannitol). Wild fungi were isolated from both SCG and coffee beans and screened for enzyme production. The enzymes produced from the selected wild fungi and recombinant fungi were then evaluated for enzymatic hydrolysis of SCG, in comparison to commercial enzyme preparations. Out of the enzymes evaluated on SCG, the application of mannanase enzymes gave better yields than when only cellulase or xylanase was utilised for hydrolysis. The recombinant mannanase (Man1) provided the highest increments in soluble solids yield (17 %), even when compared with commercial preparations at the same protein concentration (0.5 mg/g SCG). The combination of Man1 with other enzyme activities revealed an additive effect on the hydrolysis yield, but not synergistic interaction, suggesting that the highest soluble solid yields was mainly due to the hydrolysis action of mannanase.

Comparing cytosolic expression to peroxisomal targeting of the chimeric L1/L2 (ChiΔH-L2) gene from human papillomavirus type 16 in the methylotrophic yeasts Pichia pastoris and Hansenula polymorpha.

The chimeric ChiΔH-L2 gene from human papillomavirus type 16, consisting of structural proteins L1 and L2, was successfully expressed in the cytosol of both Pichia pastoris and Hansenula polymorpha during methanol induction. In addition, a novel approach was employed whereby ChiΔH-L2 was targeted to the peroxisome using peroxisomal targeting sequence 1 (PTS1) to compare ChiΔH-L2 yields in the peroxisome vs the cytosol. The ChiΔH-L2 gene was yeast-optimized and cloned into plasmids aimed at genomic integration. Levels of intracellular ChiΔH-L2 accumulation in the cytosol were highest in P. pastoris KM71 strain KMChiΔH-L2 (1.43 mg/l), compared to the maximum production level of 0.72 mg/l obtained with H. polymorpha. ChiΔH-L2 targeting to the peroxisome was successful; however, it appeared to negatively affect ChiΔH-L2 production in both P. pastoris and H. polymorpha.

Production and characterisation of recombinant α-L-arabinofuranosidase for production of xylan hydrogels.

A recombinant strain of the protease-deficient, non-acidifying pH mutant Aspergillus niger D15 (A. niger D15 [abfB]) strain was developed to secrete α-L-arabinofuranosidase (AbfB) free of endo-1,4-ß-xylanases for selective hydrolysis of xylan into hydrogels. The A. niger D15 [abfB] strain expressed the α-L-arabinofuranosidase abfB gene under the transcriptional control of the glyceraldehyde-3-phosphate dehydrogenase promoter (gpd(P)) and glucoamylase terminator (glaA(T)) in fermentation cultures containing 10 % glucose. The yield, activity, purity, kinetics and ability of the recombinant AbfB to selectively hydrolyse xylans into hydrogels were assessed. The recombinant AbfB secreted in 125-mL shake flasks and 10-L bioreactor fermentation cultures had specific activities against ρ-nitrophenyl-α-arabinofuranoside of up to 4.4 and 2.7 U g⁻¹ (dry weight), respectively. In addition, the recombinant AbfB was present as a single protein species on silver-stained 10 % sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The recombinant AbfB had optimal activity at 40-55 °C and pH 3.0 to pH 5.0 and was stable at temperature and pH of up to 60 °C and pH 6.0, respectively. About 20 % of the available arabinose in the xylan was released by the recombinant AbfB from the hydrolysis of low viscosity wheat and oat spelt arabinoxylans and about 9 and 5 % from bagasse and bamboo arabinoglucuronoxylans, respectively, that led to the formation of the hydrogels. Therefore, the constructed A. niger D15 [abfB] strain presented a microbial system for the production of recombinant AbfB with the required purity for the modification of xylans into hydrogels.

Next-generation cellulosic ethanol technologies and their contribution to a sustainable Africa.

The world is currently heavily dependent on oil, especially in the transport sector. However, rising oil prices, concern about environmental impact and supply instability are among the factors that have led to greater interest in renewable fuel and green chemistry alternatives. Lignocellulose is the only foreseeable renewable feedstock for sustainable production of transport fuels. The main technological impediment to more widespread utilization of lignocellulose for production of fuels and chemicals in the past has been the lack of low-cost technologies to overcome the recalcitrance of its structure. Both biological and thermochemical second-generation conversion technologies are currently coming online for the commercial production of cellulosic ethanol concomitantly with heat and electricity production. The latest advances in biological conversion of lignocellulosics to ethanol with a focus on consolidated bioprocessing are highlighted. Furthermore, integration of cellulosic ethanol production into existing bio-based industries also using thermochemical processes to optimize energy balances is discussed. Biofuels have played a pivotal yet suboptimal role in supplementing Africa's energy requirements in the past. Capitalizing on sub-Saharan Africa's total biomass potential and using second-generation technologies merit a fresh look at the potential role of bioethanol production towards developing a sustainable Africa while addressing food security, human needs and local wealth creation.

Competitive exclusion as a mode of action of a novel Bacillus cereus aquaculture biological agent.

AIMS: To determine the contribution of potential modes of action of a Bacillus cereus aquaculture biological control agent in inhibition of the fish pathogen, Aeromonas hydrophila. METHODS AND RESULTS: When B. cereus was tested in plate well inhibition studies, no production of antimicrobial compounds was detected. Bacillus cereus had a high growth rate (0.96 h(-1)), whereas Aer. hydrophila concentration decreased by c. 70% in co-culture experiments. In nutrient limitation studies, B. cereus had a significantly higher growth rate when cultured under glucose (P < 0.05) and iron (P < 0.01) limitation in comparison with Aer. hydrophila. Bacillus cereus glucose (0.30 g l(-1) h(-1)) and iron (0.60 mg l(-1) h(-1)) uptake rates were also significantly higher (P < 0.01) than the Aer. hydrophila glucose (0.14 g l(-1) h(-1)) and iron (0.43 mg l(-1) h(-1)) uptake rates. Iron uptake was facilitated by siderophore production shown in time profile studies where relative siderophore production was c. 60% through the late exponential and sporulation phases. CONCLUSIONS: Competitive exclusion by higher growth rate, competition for organic carbon and iron, facilitated by siderophore production, could be identified as mechanisms of pathogen growth inhibition by B. cereus. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first elucidation of the mechanism of action of our novel B. cereus biological agent in growth attenuation of pathogenic Aer. hydrophila. This study enhances the application knowledge and attractiveness for adoption of B. cereus NRRL 100132 for exploitation in aquaculture.

High-density spore production of a B. cereus aquaculture biological agent by nutrient supplementation.

Previous studies have demonstrated the efficacy of our Bacillus cereus isolate (NRRL 100132) in reducing concentrations of nitrogenous wastes and inhibiting growth of fish pathogens. In vivo efficacy and tolerance to a range of physiological conditions in systems used to rear Cyprinus carpio make this isolate an excellent candidate for aquaculture applications. Production cost is an important consideration in development of commercially relevant biological products, and this study examines the optimization of nutrient supplementation, which has an impact on high-density production of spores by fermentation. Corn steep liquor (CSL) was identified as a lower cost and more effective nutrient source in comparison to conventional nutrient substrates, in particular yeast extract and nutrient broth. The improved sporulation performance of B. cereus could be related to the increased availability of free amino acids, carbohydrates, and minerals in CSL, which had a positive effect on sporulation efficiency. The impact of nutrient concentration on spore yield and productivity was modeled to develop a tool for optimization of nutrient concentration in fermentation. An excellent fit of the model was confirmed in laboratory fermentation studies. A cost comparison revealed that production using liquid phytase and ultrafiltered-treated CSL was less expensive than spray-dried CSL and supported cultivation of B. cereus spores at densities higher than 1 x 10(10) CFU ml(-1).

Effect of an exponential feeding regime on the production of Rhodotorula araucariae epoxide hydrolase in Yarrowia lipolytica.

AIMS: To evaluate the effect of and exponential feeding regime on the production of epoxide hydrolase (EH) enzyme in recombinant Yarrowia lipolytica in comparison to a constant feed strategy. METHODS AND RESULTS: An exponential feed model was developed and fermentations were fed at six different exponential rates. A twofold increase in EH productivity and a 15% increase in volumetric EH activity was obtained by applying exponential glucose feed rates in fed-batch cultivation. These responses were modelled to obtain a theoretical optimum feed rate that was validated in duplicate fermentations. The model optimum of 0.06 h(-1) resulted in a volumetric EH activity of c. 5500 U l(-1) h(-1) and a maximum activity of 206,000 U l(-1). This correlated well with model predictions, with a variance of <10%. CONCLUSIONS: The use of an exponential feed strategy at a rate of 0.06 h(-1) yielded best results for all key responses which show a clear improvement over a constant feed strategy. SIGNIFICANCE AND IMPACT OF THE STUDY: The study was the first evaluation of an exponential feed strategy on recombinant Y. lipolytica for the production of EH enzyme. The results suggest a strategy for the commercial production of a valuable pharmaceutical enzyme.

Isolation and selection of Bacillus spp. as potential biological agents for enhancement of water quality in culture of ornamental fish.

AIMS: To isolate, select and evaluate Bacillus spp. as potential biological agents for enhancement of water quality in culture of ornamental fish. METHODS AND RESULTS: Natural isolates obtained from mud sediment and Cyprinus carpio were purified and assessed in vitro for efficacy based on the inhibition of growth of pathogenic Aeromonas hydrophila and the decrease in concentrations of ammonium, nitrite, nitrate and phosphate ions. Based on suitability to predefined characteristics, the isolates B001, B002 and B003 were selected and evaluated in vitro in the presence of Aer. hydrophila and in a preliminary in vivo trial with C. carpio. The inhibitory effect on pathogen growth and the decrease in concentrations of waste ions was demonstrated. Based on 16S RNA sequence homology, the isolates were identified as Bacillus subtilis, Bacillus cereus and Bacillus licheniformis, respectively. Isolate B002 did not contain the anthrax virulence plasmids pOX1, pOX2 or the B. cereus enterotoxin. CONCLUSIONS: Selected isolates effected synergistic reduction in pathogen load and the concentrations of waste ions in vitro and in vivo and are safe for use in ornamental aquaculture. SIGNIFICANCE AND IMPACT OF THE STUDY: A new approach for assessment of biological agents was demonstrated and has yielded putative isolates for development into aquaculture products.

The metabolic burden of the PGK1 and ADH2 promoter systems for heterologous xylanase production by Saccharomyces cerevisiae in defined medium.

Five recombinant S. cerevisiae strains were cultivated under identical conditions to quantify the molecular basis of the metabolic burden of heterologous gene expression, and to evaluate mechanisms for the metabolic burden. Two recombinant S. cerevisiae strains, producing Trichoderma reesei xylanase II under control of either the PGK1 or ADH2 promoters, were compared quantitatively with three references strains, where either the heterologous xylanase II (XYN2) gene, or the heterologous gene and the promoter and terminator were omitted from the recombinant plasmid. Neither the replication of multiple copies of the 2-microm-based YEp352 plasmid nor the replication the foreign XYN2 gene represented a metabolic burden to the cell, as the growth of the host organism was not affected. The inclusion of a glycolytic promoter on the recombinant plasmid, however, reduced the maximum specific growth rate (12% to 15%), biomass yield on glucose (8% to 11%), and specific glucose consumption rate (6% to 10%) of the recombinant strains. The presence of the heterologous XYN2 gene on the recombinant plasmid caused a further reduction in the maximum specific growth rate (11% to 14%), biomass yield (4%), and specific glucose consumption rate (12%) of the host strain during active gene expression, which was dictated by the regulatory characteristics of the promoter utilized. The metabolic effect of foreign gene expression was disproportionally large, with respect to on the amount of heterologous protein produced. This was most likely due to an increased energetic demand for the expression of a foreign gene and/or a competition for limiting amounts of transcription or translation factors, biosynthetic precursors or metabolic energy.