Hernandulcin Production in Elicited Hairy Roots of Phyla scaberrima: Toward Sustainable Production of a Non-Caloric Sweetener with Nutraceutical Properties.

Herein we report on the generation of hairy root lines of P. scaberrima able to produce hernandulcin (HE), a non-caloric sweetener with nutraceutical properties. From ten different lines analyzed, three synthesized up to 100 mg ⋅ L-1 HE under the batch culture conditions standardized in this investigation. Adding elicitors (salicylic acid, chitin, Glucanex, polyethylene glycol) and biosynthetic precursors (farnesol and (+)-epi-alpha-bisabolol) significantly altered HE accumulation. Chitin and Glucanex enhanced HE production from 130 to 160 mg ⋅ L-1 , whereas farnesol and (+)-epi-alpha-bisabolol from 165 to 200 mg ⋅ L-1 without dependence on biomass accumulation. Improved batch cultures containing liquid Murashige & Skoog medium (MS; pH 7), added with 4 % sucrose, 0.5 mg ⋅ L-1 naphthaleneacetic acid, 100 mg ⋅ L-1 Glucanex, 150 mg ⋅ L-1 chitin, 250 mg ⋅ L-1 farnesol, and 150 mg ⋅ L-1 (+)-epi-alpha-bisabolol at 25 °C (12 h light/12 h darkness), triggered HE accumulation to 250 mg ⋅ L-1 in 25 days. The efficiency of each recombinant line is discussed.

Yarrowia lipolytica produces lipid-rich biomass in medium mimicking lignocellulosic biomass hydrolysate.

In recent years, lignocellulosic biomass has become an attractive low-cost raw material for microbial bioprocesses aiming the production of biofuels and other valuable chemicals. However, these feedstocks require preliminary pretreatments to increase their utilization by microorganisms, which may lead to the formation of various compounds (acetic acid, formic acid, furfural, 5-hydroxymethylfurfural, p-coumaric acid, vanillin, or benzoic acid) with antimicrobial activity. Batch cultures in microplate wells demonstrated the ability of Yarrowia strains (three of Y. lipolytica and one of Y. divulgata) to grow in media containing each one of these compounds. Cellular growth of Yarrowia lipolytica W29 and NCYC 2904 (chosen strains) was proven in Erlenmeyer flasks and bioreactor experiments where an accumulation of intracellular lipids was also observed in culture medium mimicking lignocellulosic biomass hydrolysate containing glucose, xylose, acetic acid, formic acid, furfural, and 5-HMF. Lipid contents of 35% (w/w) and 42% (w/w) were obtained in bioreactor batch cultures with Y. lipolytica W29 and NCYC 2904, respectively, showing the potential of this oleaginous yeast to use lignocellulosic biomass hydrolysates as feedstock for obtaining valuable compounds, such as microbial lipids that have many industrial applications. KEY POINTS: • Yarrowia strains tolerate compounds found in lignocellulosic biomass hydrolysate • Y. lipolytica consumed compounds found in lignocellulosic biomass hydrolysate • 42% (w/w) of microbial lipids was attained in bioreactor batch cultures.

Impact of inorganic iron and haem on the human gut microbiota; An in vitro batch-culture approach.

Although iron is an essential nutrient for humans, as well as for almost all other organisms, it is poorly absorbed (~15%) from the diet such that most passes through the upper gut into the large intestine. The colonic microbiota is thus exposed to, and potentially influenced by, such residual iron which could have an impact on human health. The aim of the research described here is to determine how the major forms of dietary iron (inorganic iron and haem) influence metabolic activity and composition of the human gut microbiota by utilizing an in vitro parallel, pH-controlled anaerobic batch culture approach. Controlled iron provision was enabled by the design of a 'modified' low-iron gut-model medium whereby background iron content was reduced from 28 to 5 µM. Thus, the impact of both low and high levels of inorganic and haem iron (18-180 µM and 7.7-77 µM, respectively) could be explored. Gut-microbiota composition was determined using next generation sequencing (NGS) based community profiling (16S rRNA gene sequencing) and flow-fluorescent in situ hybridization (FISH). Metabolic-end products (organic acids) were quantified using gas chromatography (GC) and iron incorporation was estimated by inductively coupled plasma optical emission spectroscopy (ICP-OES). Results showed that differences in iron regime induced significant changes in microbiota composition when low (0.1% w/v) fecal inoculation levels were employed. An increase in haem levels from 7.7 to 77 µM (standard levels employed in gut culture studies) resulted in reduced microbial diversity, a significant increase in Enterobacteriaceae and lower short chain fatty acid (SCFA) production. These effects were countered when 18 µM inorganic iron was also included into the growth medium. The results therefore suggest that high-dietary haem may have a detrimental effect on health since the resulting changes in microbiota composition and SCFA production are indicators of an unhealthy gut. The results also demonstrate that employing a low inoculum together with a low-iron gut-model medium facilitated in vitro investigation of the relationship between iron and the gut microbiota.

Gamma aminobutyric acid production by commercially available probiotic strains.

AIMS: Certain bacteria can produce gamma aminobutyric acid (GABA) from glutamate in the human intestinal tract, leading to the possibility of altering GABA levels through diet. To this end, we assessed the ability of seven commercially available probiotic supplements to produce GABA. METHOD AND RESULTS: Probiotic strains were compared for GABA production in pure culture. The bacteria were inoculated at a concentration of 107 CFU ml-1 in 10 ml MRS supplemented with monosodium glutamate (1% w/v), both with and without oligofructose-enriched inulin (OFI) (1% w/v). Two strains with the highest production of GABA were further assessed for 48 h in pH-controlled anaerobic batch cultures inoculated with faecal bacteria. Liquid chromatography-mass spectrometry (LC-MS) was used for quantification of GABA and microbiota composition was determined through 16S rRNA gene sequencing. Levilactobacillus brevis LB01 (CGMCC 16921) and Lactiplantibacillus plantarum 299v (DSM 9843) were the most efficient producers of GABA. High GABA levels (28.32 mmol l-1 ± 0.29) were produced by the probiotic strain L. brevis LB01 at pH 5.4-5.6. This was significantly higher than the levels of GABA produced by L. plantarum (4.8 mmol l-1 ± 6.8) and a negative control (2.9 mM ± 3.1). The addition of OFI did not further stimulate GABA production under the conditions tested. The ability of these strains to produce GABA in-vitro was further evaluated in a faecal microbiota environment. Once again, L.brevis LB01 produced the highest levels of GABA (40.24 mmol l-1 ± 20.98). CONCLUSIONS: L. brevis LB01 was found to be the most efficient probiotic strain, of those tested, for GABA production.

Process intensification at the expression system level for the production of 1-phosphate aldolase in antibiotic-free E. coli fed-batch cultures.

To successfully design expression systems for industrial biotechnology and biopharmaceutical applications; plasmid stability, efficient synthesis of the desired product and the use of selection markers acceptable to regulatory bodies are of utmost importance. In this work we demonstrate the application of a set of IPTG-inducible protein expression systems -- harboring different features namely, antibiotic vs auxotrophy marker; two-plasmids vs single plasmid expression system; expression levels of the repressor protein (LacI) and the auxotrophic marker (glyA) -- in high-cell density cultures to evaluate their suitability in bioprocess conditions that resemble industrial settings. Results revealed that the first generation of engineered strain showed a 50% reduction in the production of the model recombinant protein fuculose-1-phosphate aldolase (FucA) compared to the reference system from QIAGEN. The over-transcription of glyA was found to be a major factor responsible for the metabolic burden. The second- and third-generation of expression systems presented an increase in FucA production and advantageous features. In particular, the third-generation expression system is antibiotic-free, autotrophy-selection based and single-plasmid and, is capable to produce FucA at similar levels compared to the original commercial expression system. These new tools open new avenues for high-yield and robust expression of recombinant proteins in E. coli.

Assessment of stool collection and storage conditions for in vitro human gut model studies.

BACKGROUND: The role of the gut microbiota in health and disease is becoming increasingly apparent. Faeces is the most accessible sample to collect from human volunteers for studying the gut microbiota. However, the impact of stool collection and storage conditions on microbial and metabolic profiles have not been fully evaluated. By understanding the effect of different stool collection and storage conditions on microbial and metabolic composition, we can consider these parameters in the design of in vitro fermentation studies. METHODS: Stool samples from 3 volunteers were stored under 5 different conditions to mimic methods that researchers may use to collect and store stool samples for study of the gut microbiota, including: fresh sample used within 10 min; stored on wet ice (4 °C) for 60 min; stored in an anaerobic chamber in a temperature-controlled bag (4 °C) for 60 min; freezing at -20 °C for 60 min and freezing at -20 °C for 60 min and then at -80 °C for 2 weeks. The stored samples were added to basal medium in batch culture fermenters alone (negative control) or with 5 g 2'-Fucosyllactose (2'FL) Human Milk Oligosaccharide (HMO) (as a positive fermentation control). Samples were collected at 3 timepoints (0, 12 and 24 h) for analysis by Flow Cytometry-Fluorescent In Situ Hybridisation (FC-FISH) and 1H-Nuclear Magnetic Resonance (NMR) spectroscopy to assess the impact on microbial and metabolic profiles, respectively. RESULTS: Freezing stool significantly impacted microbial numbers and activity during in vitro fermentations, whereas storing the stool on wet ice (4 °C) or in an anaerobic chamber at 4 °C for 60 min had minimal effects on microbial and metabolic profiles throughout the 24 h batch culture fermentation experiments. DISCUSSION: For in vitro batch culture fermentation studies where it may not be practical or possible to use fresh stool, either storing the stool on wet ice (4 °C) or in an anaerobic chamber at 4 °C for 60 min could be plausible alternatives to maintain microbial and metabolic profiles for analysis.

Monoculture-based consumer-resource models predict species dominance in mixed batch cultures of dinoflagellates.

Global change will disturb the frequency, scale and distribution of harmful algal blooms (HABs), but we are unable to predict future HABs due to our limited understanding of how physicochemical changes in the environment affect interspecific competition between dinoflagellates. Trait-based mechanistic modelling is an important tool to unravel and quantify various direct and indirect interactions between species. The present study explores whether MacArthur's consumer-resource model can be used as a viable base model to predict dinoflagellate growth in closed multispecies systems. To this end, two batch culture experiments (294 cultures in total) with monocultures and multispecies cultures of Alexandrium minutum, Prorocentrum lima, P. micans, Protoceratium reticulatum and Scrippsiella trochoidea were performed. Despite changes to the relative (different nitrate concentrations) and absolute nutrient availability (dilutions of L1 medium), P. micans outcompeted all other species in mixed cultures. Consumer-resource modelling parameterized using monoculture growth correctly predicted this species dominance (R² between 0.80 and 0.95). Parameter estimates revealed that P. micans had a faster uptake of nitrogen when compared to its competitors, but did not differ in resource efficiency and natural mortality rate. Yet, while the model accurately predicted community dynamics during the growth phase, it was not able to predict their dynamics beyond the point of quiescence. Consumer-resource modelling was shown to differentiate the roles of resource assimilation, resource efficiency, and natural mortality rates in batch culture experiments with minimal data requirements beyond common measurements. The results suggest that consumer-resource models provide a promising basis for trait-based modelling of interspecific competition between (harmful) algae.

Investigation into the impact of tyrosine on the product formation and quality attributes of mAbs in rCHO cell cultures.

Tyrosine (Tyr) is crucial to the maintenance of the monoclonal antibody (mAb) titers and quality attributes in fed-batch cultures of recombinant Chinese hamster ovary (rCHO) cells. However, the relation between tyrosine and these aspects is not yet fully defined. In order to further elucidate such a relation, two groups of fed-batch experiments with high tyrosine (H-T) or low tyrosine (L-T) additions producing an IgG1 monoclonal antibody against CD20 were implemented to investigate the intracellular and extracellular effects of tyrosine on the culture performance. It was found that the scarcity of tyrosine led to the distinctive reduction in both viable cell density and antibody specific production rate, hence the sharply reduced titer, possibly related to the impaired translation efficiency caused by the substrate limitation of tyrosine. In addition, alterations to the critical quality attributes were detected in the L-T group, compared to those in the H-T condition. Notable decrease in the contents of intact antibody was found under the L-T condition because of the elevated reductive level in the supernatant. Moreover, the aggregate content in the L-T condition was also reduced, probably resulting from the accumulation of extracellular cystine. In particular, the lysine variant content noticeably increased with tyrosine limitation owing to the downregulation of two carboxypeptidases, i.e., CpB and CpH. Overall, understanding the role of tyrosine in these aspects is fundamental to the increase of product titers and control of critical quality attributes in the monoclonal antibody production of rCHO cell fed-batch cultures. KEY POINTS: • Tyrosine is essential in the maintenance of product titers and the control of product qualities in high cell density cultivations in rCHO cell. • This study revealed the bottleneck of decreased qmAbupon the deficiency of tyrosine. • The impact of tyrosine on the critical product qualities and the underlying mechanisms were also thoroughly assessed.

In Vitro Gas Production from Batch Cultures of Stomach and Hindgut Digesta of Horses Adapted to a Prebiotic Dose of Fructooligosaccharides and Inulin.

Fructooligosaccharides (FOS) and inulin may modulate hindgut fermentation. It was tested if digesta batch cultures taken from horses adapted to FOS and inulin show different fermentation compared with such taken from nonsupplemented horses. Six horses received 0.15 g FOS and inulin/kg body weight/d via Jerusalem artichoke meal (JAM) upon a hay-based diet; six horses received corncob meal without grains (CMG) as placebo. The horses were euthanized after 20 days. Digesta samples were taken from stomach, cecum, ventral colon ascendens (VCA), and colon transversum (CT). Digesta batch cultures were incubated 48 hours to measure in vitro gas production as well as pre- and post-incubation pH and oxidation-reduction potential (ORP). A distinct fermentation of the surplus of fructans present in the inoculum was found with JAM-adapted batch cultures. Gas production was accelerated in inoculated gastric contents of horses adapted to JAM compared with CMG adapted ones (7.8 vs. 16.4 hours to achieve half of the 48 hours gas quantity, respectively; P > .05). Although buffered, pH decreased during fermentation. Postincubation pH was lower with JAM than CMG-adapted batch cultures (P > .05). Preinoculation ORP was lower with stomach batch cultures adapted to CMG than with such adapted to JAM. The ORP increased twofold from pre- to post-incubation with the latter. Asymptotic maximal gas production decreased gradually using cecum, VCA, or CT digesta. Parts of FOS and inulin of digesta are fermented in the stomach, which reduce possible effects on hindgut fermentation. Elevated fermentation may considerably impact stomach health.

Direct Effects of Temperature on Growth of Different Tropical Phytoplankton Species.

Temperature increase may influence competition among phytoplankton species, potentially intensifying cyanobacteria blooms that can be favored by direct and indirect effects of temperature. In this study, we aimed to clarify how cyanobacteria can be favored by the direct effects of increased temperature compared to diatoms and chlorophytes. Strains of the most representative species of a eutrophic coastal lagoon (Microcystis aeruginosa, Planktothrix agardhii, Desmodesmus communis, and Cyclotella meneghiniana) were used to test the hypothesis that cyanobacteria would be favored by the direct effect of temperature increase. First, we evaluated the effect of temperature increase on growth in monocultures (batch and chemostats) at 25 and 30 °C and after in mixed cultures (chemostats). In batch monocultures, the cyanobacteria showed higher growth rates in 30 °C than in 25 °C. However, in continuous culture experiments (chemostats), growth rates of M. aeruginosa and P. agardhii were not affected by temperature, but the strains showed higher biovolume in steady-state with the temperature increase. In continuous mixed cultures, M. aeruginosa was always dominant and C. meneghiniana was excluded, regardless of temperature tested. D. communis was able to coexist with lower biomass. This study shows that rising temperatures can be detrimental to diatoms, even for a tropical strain. Although some studies indicate that the dominance of cyanobacteria in warmer climates may be due to the indirect effect of warming that will promote physical conditions in the environment more favorable to cyanobacteria, the outcomes of mixed cultures demonstrate that the direct effect of temperature can also favor the dominance of cyanobacteria.

High-Throughput Screening of Chlorella Vulgaris Growth Kinetics inside a Droplet-Based Microfluidic Device under Irradiance and Nitrate Stress Conditions.

Biodiesel is an eco-friendly renewable fuel that can be derived from microalgae. Maximization of biomass and lipid productivities are considered the main challenges for algal biodiesel production. Since conventional batch cultures are time-, space-, and reagent-consuming with many restrictions to apply many replicates, microfluidic technology has recently emerged as an alternative low-cost and efficient technology with high throughput repeatability and reproducibility. Different applications of microfluidic devices in algal biotechnology have been reported, including cell identification, sorting, trapping, and metabolic screening. In this work, Chlorella vulgaris was investigated by encapsulating in a simple droplet-based micro-array device at different light intensities of 20, 80, and 200 µmol/m2/s combined with different nitrate concentrations of 17.6, 8.8, and 4.4 mM. The growth results for C. vulgaris within microfluidic device were compared to the conventional batch culture method. In addition, the effect of combined stress of deficiencies in irradiance and nitrogen availability were studied to illustrate their impact on the metabolic profiling of microalgae. The results showed that the most optimum favorable culturing conditions for Chlorella vulgaris growth within the microfluidic channels were 17.6 mM and 80 µmol/m2/s.

Evaluation of the prebiotic potential of arabinoxylans extracted from wheat distillers' dried grains with solubles (DDGS) and in-process samples.

Distillers' dried grains with solubles (DDGS) is a low-value agro-industrial by-product, rich in arabinoxylans (AX), which is produced by commercial distillery and bioethanol plants. In a first approach, we investigated the prebiotic potential of four fractions comprising arabinoxylan oligosaccharides (AXOS) and xylooligosaccharides (XOS) obtained by enzymatic hydrolysis of AX fractions derived from DDGS and wet solids (in-process sample of DDGS production process). Anaerobic batch cultures in controlled pH conditions were used to test the prebiotic activity of the samples. Results did not show significant differences between the enzymatic treatments used, and all AXOS/XOS were extensively fermented after 24 h. In addition, significant increases (P < 0.05) in Bifidobacterium and total short-chain fatty acids (SCFAs) were observed after 24 h of fermentation. Finally, DDGS-derived hydrolysates were separated on an anionic semi-preparative column to prepare AXOS/XOS fractions with degree of polymerisation (DP) greater than 3. Bifidogenic activity and an increase of SCFAs were again observed after 24 h of fermentation, although this time, the selectivity was higher and the fermentation slower, suggesting that the fermentation of this substrate could take place (at least partially) in the distal part of the colon with highly desirable beneficial effects.

In vitro evaluation of prebiotic properties derived from rice bran obtained by debranning technology.

The prebiotic ability of several rice bran fractions obtained by debranning (RBD) using human microbiota was studied in anaerobic batch cultures with agitation and pH-controlled. Fraction C (3.8-5% w/w pearling) from RBD increased the number of bifidobacteria and lactobacteria compared with the positive control, raftilose P95. RBD fermentation induced changes in the short-chain fatty acid (SCFA) profile. In addition, Fraction C revealed the highest growth of positive lactobacteria than commercial control. The present work illustrates the prebiotic capacity of RBD to modulate human microbiota and highlights that fraction C could be an economical source for use in human food as well as an interesting alternative to valorise a by-product of cereal industry.

Growth modelling of Nitrosomonas europaea ATCC® 19718 and Nitrobacter winogradskyi ATCC® 25391: A new online indicator of the partial nitrification.

The aim of the present work was to study the growth of two nitrifying bacteria. For modelling the nitrifying subsystem of the MELiSSA loop, Nitrosomonas europaea ATCC® 19718 and Nitrobacter winogradskyi ATCC® 25931 were grown separately and in cocultures. The kinetic parameters of a stoichiometric mass balanced Pirt model were identified: µmax=0.054h(-1), decay rate b=0.003h(-1) and maintenance rate m=0.135gN-NH4(+)·gX(-1)·h(-1) for Nitrosomonas europaea; µmax=0.024h(-1), b=0.001h(-1) and m=0.467gN-NO2(-)·gX(-1)·h(-1) for Nitrobacter winogradskyi. A predictive structured model of nitrification in co-culture was developed. The online evolution of the addition of KOH is correlated to the nitritation; the dissolved oxygen concentration is correlated to both nitritation and nitratation. The model suitably represents these two variables so that transient partial nitrification is assessed. This is a clue for avoiding partial nitrification by predictive functional control.

Paralytic shellfish toxin production by the dinoflagellate Alexandrium pacificum (Chinhae Bay, Korea) in axenic, nutrient-limited chemostat cultures and nutrient-enriched batch cultures.

Blooms of Alexandrium pacificum (formerly Alexandrium tamarense) are common in Chinhae Bay (Korea), presumably linked to anthropogenic eutrophication. Here we examine PSP toxin content and composition in axenic chemostat and batch cultures of A. pacificum using growth conditions that differed according to dilution rate, nutrient limitations, and enrichments. Phosphate (P)-limited cells in chemostat cultures had higher toxin content and a toxin composition that differed from that of nitrogen (N)-limited cells at the highest growth rates. Therefore, toxin composition changes do occur in axenic cultures of A. pacificum following extended growth under steady state conditions. In nutrient-limited batch cultures that received N and P enrichment, the N-enriched cultures showed a more diverse toxin profile than the P-enriched cells; the toxin content of N-enriched cells was lower than in the P-enriched cultures. We infer the following order for the biosynthesis of individual toxins: C1, C2>GTX3>GTX1>neoSTX.

Verhulst and stochastic models for comparing mechanisms of MAb productivity in six CHO cell lines.

The present study validates previously published methodologies-stochastic and Verhulst-for modelling the growth and MAb productivity of six CHO cell lines grown in batch cultures. Cytometric and biochemical data were used to model growth and productivity. The stochastic explanatory models were developed to improve our understanding of the underlying mechanisms of growth and productivity, whereas the Verhulst mechanistic models were developed for their predictability. The parameters of the two sets of models were compared for their biological significance. The stochastic models, based on the cytometric data, indicated that the productivity mechanism is cell specific. However, as shown before, the modelling results indicated that G2 + ER indicate high productivity, while G1 + ER indicate low productivity, where G1 and G2 are the cell cycle phases and ER is Endoplasmic Reticulum. In all cell lines, growth proved to be inversely proportional to the cumulative G1 time (CG1T) for the G1 phase, whereas productivity was directly proportional to ER. Verhulst's rule, "the lower the intrinsic growth factor (r), the higher the growth (K)," did not hold for growth across all cell lines but held good for the cell lines with the same growth mechanism-i.e., r is cell specific. However, the Verhulst productivity rule, that productivity is inversely proportional to the intrinsic productivity factor (r x ), held well across all cell lines in spite of differences in their mechanisms for productivity-that is, r x is not cell specific. The productivity profile, as described by Verhulst's logistic model, is very similar to the Michaelis-Menten enzyme kinetic equation, suggesting that productivity is more likely enzymatic in nature. Comparison of the stochastic and Verhulst models indicated that CG1T in the cytometric data has the same significance as r, the intrinsic growth factor in the Verhulst models. The stochastic explanatory and the Verhulst logistic models can explain the differences in the productivity of the six clones.

Application of byproducts from food processing for production of 2,3-butanediol using Bacillus amyloliquefaciens TUL 308.

A nonpathogenic bacterial strain Bacillus amyloliquefaciens TUL 308 synthesized minor 2,3-butanediol (2,3-BD) amounts from glucose, fructose, sucrose, and glycerol, and efficiently produced the diol from molasses and hydrolysates of food processing residues. Batch fermentations yielded 16.53, 10.72, and 5 g/L 2,3-BD from enzymatic hydrolysates of apple pomace, dried sugar beet pulp, and potato pulp (at initial concentrations equivalent to 45, 20, and 30 g/L glucose, respectively), and 25.3 g/L 2,3-BD from molasses (at its initial concentration equivalent to 60 g/L saccharose). Fed-batch fermentations in the molasses-based medium with four feedings with either glucose or sucrose (in doses increasing their concentration by 25 g/L) resulted in around twice higher maximum 2,3-BD concentration (of about 60 and 50 g/L, respectively). The GRAS Bacillus strain is an efficient 2,3-BD producer from food industry byproducts.

Optimization of bioprocess conditions improves production of a CHO cell-derived, bioengineered heparin.

Heparin is the most widely used anticoagulant drug in the world today. Heparin is currently produced from animal tissues, primarily porcine intestines. A recent contamination crisis motivated development of a non-animal-derived source of this critical drug. We hypothesized that Chinese hamster ovary (CHO) cells could be metabolically engineered to produce a bioengineered heparin, equivalent to current pharmaceutical heparin. We previously engineered CHO-S cells to overexpress two exogenous enzymes from the heparin/heparan sulfate biosynthetic pathway, increasing the anticoagulant activity ∼100-fold and the heparin/heparan sulfate yield ∼10-fold. Here, we explored the effects of bioprocess parameters on the yield and anticoagulant activity of the bioengineered GAGs. Fed-batch shaker-flask studies using a proprietary, chemically-defined feed, resulted in ∼two-fold increase in integrated viable cell density and a 70% increase in specific productivity, resulting in nearly three-fold increase in product titer. Transferring the process to a stirred-tank bioreactor increased the productivity further, yielding a final product concentration of ∼90 µg/mL. Unfortunately, the product composition still differs from pharmaceutical heparin, suggesting that additional metabolic engineering will be required. However, these studies clearly demonstrate bioprocess optimization, in parallel with metabolic engineering refinements, will play a substantial role in developing a bioengineered heparin to replace the current animal-derived drug.

Treatment of tropical forages with exogenous fibrolytic enzymes: effects on chemical composition and in vitro rumen fermentation.

The effects of three treatments of fibrolytic enzymes (cellulase from Trichoderma longibrachiatum (CEL), xylanase from rumen micro-organisms (XYL) and a 1:1 mixture of CEL and XYL (MIX) on the in vitro fermentation of two samples of Pennisetum clandestinum (P1 and P2), two samples of Dichanthium aristatum (D1 and D2) and one sample of each Acacia decurrens and Acacia mangium (A1 and A2) were investigated. The first experiment compared the effects of two methods of applying the enzymes to forages, either at the time of incubation or 24 h before, on the in vitro gas production. In general, the 24 h pre-treatment resulted in higher values of gas production rate, and this application method was chosen for a second study investigating the effects of enzymes on chemical composition and in vitro fermentation of forages. The pre-treatment with CEL for 24 h reduced (p < 0.05) the content of neutral detergent fibre (NDF) of P1, P2, D1 and D2, and that of MIX reduced the NDF content of P1 and D1, but XYL had no effect on any forage. The CEL treatment increased (p < 0.05) total volatile fatty acid (VFA) production for all forages (ranging from 8.6% to 22.7%), but in general, no effects of MIX and XYL were observed. For both P. clandestinum samples, CEL treatment reduced (p < 0.05) the molar proportion of acetate and increased (p < 0.05) that of butyrate, but only subtle changes in VFA profile were observed for the rest of forages. Under the conditions of the present experiment, the treatment of tropical forages with CEL stimulated their in vitro ruminal fermentation, but XYL did not produce any positive effect. These results showed clearly that effectiveness of enzymes varied with the incubated forage and further study is warranted to investigate specific, optimal enzyme-substrate combinations.

Antipathogenic activity of probiotics against Salmonella Typhimurium and Clostridium difficile in anaerobic batch culture systems: is it due to synergies in probiotic mixtures or the specificity of single strains?

Probiotics are currently being investigated for prevention of infections caused by enteric pathogens. The aim of this in vitro study was to evaluate the influence of three single probiotics: Lactobacillus casei NCIMB 30185 (PXN 37), Lactobacillus acidophilus NCIMB 30184 (PXN 35), Bifidobacterium breve NCIMB 30180 (PXN 25) and a probiotic mixture containing the above strains plus twelve other strains belonging to the Lactobacillus, Bifidobacterium, Lactococcus, Streptococcus and Bacillus genera on the survival of Salmonella Typhimurium and Clostridium difficile using pH-controlled anaerobic batch cultures containing mixed faecal bacteria. Changes in relevant bacterial groups and effects of probiotic addition on survival of the two pathogens were assessed over 24 h. Quantitative analysis of bacterial populations revealed that there was a significant increase in lactobacilli and/or bifidobacteria numbers, depending on probiotic addition, compared with the control (no added probiotic). There was also a significant reduction in S. Typhimurium and C. difficile numbers in the presence of certain probiotics compared with controls. Of the probiotic treatments, two single strains namely L. casei NCIMB 30185 (PXN 37), and B. breve NCIMB 30180 (PXN 25) were the most potent in reducing the numbers of S. Typhimurium and C. difficile. In addition, the supplementation with probiotics into the systems influenced some fermentations parameters. Acetate was found in the largest concentrations in all vessels and lactate and formate were generally detected in higher amounts in vessels with probiotic addition compared to controls.