Culture medium of diluted skimmed milk for the production of nisin in batch cultivations

Nisin is a promising alternative to chemical preservatives for use as a natural biopreservative in foods. This bacteriocin has also potential biomedical applications. Lactic acid bacteria are commonly cultivated in expensive standard complex media. We have evaluated the cell growth and nisin production of Lactococcus lactis in a low-cost natural medium consisting of diluted skimmed milk in a 2-L bioreactor. The assays were performed at 30 degrees C for 56 h, at varying agitation speeds and airflow rates: (1) 200 rpm (no airflow, and airflow at 0.5, 1.0 and 2.0 L/min); (2) 100 rpm (no airflow, and airflow at 0.5 L/min). Nisin activity was evaluated using agar diffusion assays. The highest nisin concentration, 49.88 mg/L (3.3 log AU/mL or 1,995.29 AU/mL), was obtained at 16 h of culture, 200 rpm and no airflow (k(L)a = 5.29 x 10(-3)). These results show that a cultivation medium composed of diluted skimmed milk supports cell growth to facilitate nisin biosynthesis.

Production and characterization of poly-(3-hydroxybutyrate) from recombinant Escherichia coli grown on cheap renewable carbon substrates

Although the biopolymer poly-(3-hydroxybutyrate), P[3HB], presents physicochemical properties that make it an alternative material to conventional plastics, its biotechnological production is quite expensive. As carbon substrates contribute greatly to P[3HB] production cost, the utilization of a cheaper carbon substrate and less demanding micro-organisms should decrease its cost. In the present study a 23 factorial experimental design was applied, aiming to evaluate the effects of using hydrolysed corn starch (HCS) and soybean oil (SBO) as carbon substrates, and cheese whey (CW) supplementation in the mineral medium (MM) on the responses, cell dried weigh (DCW), percentage P[3HB] and mass P[3HB] by recombinant Escherichia coli strains JM101 and DH10B, containing the P[3HB] synthase genes from Cupriavidus necator (ex-Ralstonia eutropha). The analysis of effects indicated that the substrates and the supplement and their interactions had positive effect on CDW. Statistically generated equations showed that, at the highest concentrations of HCS, SO and CW, theoretically it should be possible to produce about 2 g L(1) DCW, accumulating 50% P[3HB], in both strains. To complement this study, the strain that presented the best results was cultivated in MM added to HCS, SBO and CW ( in best composition observed) and complex medium (CM) to compare the obtained P[3HB] in terms of physicochemical parameters. The obtained results showed that the P[3HB] production in MM (1.29 g L(-1)) was approximately 20% lower than in CM (1.63 g L(-1)); however, this difference can be compensated by the lower cost of the MM achieved by the use of cheap renewable carbon sources. Moreover, using differential scanning calorimetry and thermogravimetry analyses, it was observed that the polymer produced in MM was the one which presented physicochemical properties (Tg and Tf) that were more similar to those found in the literature for P[3HB].

Enterococcus faecium isolated from Lombo, a Portuguese traditional meat product: characterisation of antibacterial compounds and factors affecting bacteriocin production

Strain ST211CH, identified as a strain of Enterococcus faecium, isolated from Lombo produced a bacteriocin that inhibited the growth of Enterococcus spp., Listeria spp., Klebsiella spp., Lactobacillus spp., Pseudomonas spp., Staphylococcus spp. and Streptococcus spp. The mode of action of the bacteriocin named as bacteriocin ST211Ch was bactericidal against Enterococcus faecalis ATCC19443. As determined by Tricine-SDS-PAGE, the approximate molecular mass of the bacteriocin was 8.0 kDa. Loss in antimicrobial activity was recorded after treatment with proteolytic enzymes. Maximum activity of bacteriocin ST211Ch was measured in broth cultures of E. faecium strain ST211Ch after 24 h; thereafter, the activity was reduced. Bacteriocin ST211Ch remained active after exposure to various temperatures and pHs, as well as to Triton X-100, Tween-80, Tween-20, sodium dodecyl sulfate, NaCl, urea and EDTA. Effect of media components on production of bacteriocin ST211Ch was also studied. On the basis of PCR reactions targeting different bacteriocin genes, i.e. enterocins, curvacins and sakacins, no evidences for the presence of these genes in the total DNA of E. faecium strain ST211Ch was obtained. The bacterium most probably produced a bacteriocin different from those mentioned above. Based on the antimicrobial spectrum, stability and mode of action of bacteriocin ST211CH, E. faecium strain ST211Ch might be considered as a potential candidate with beneficial properties for use in biopreservation to control food spoilage bacteria.

Effect of Feed Strategy on Methane Production and Performance of an AnSBBR Treating Effluent from Biodiesel Production

The aim of this work was to investigate the effect of different feeding times (2, 4 and 6 h) and applied volumetric organic loads (4.5, 6.0 and 7.5 gCOD L-1 day(-1)) on the performance of an anaerobic sequencing batch biofilm reactor (AnSBBR) treating effluent from biodiesel production. Polyurethane foam cubes were used as inert support in the reactor, and mixing was accomplished by recirculating the liquid phase. The effect of feeding time on reactor performance showed to be more pronounced at higher values of applied volumetric organic loads (AVOLs). Highest organic material removal efficiencies achieved at AVOL of 4.5 gCOD L-1 day(-1) were 87 % at 4-h feeding against 84 % at 2-h and 6-h feeding. At AVOL of 6.0 gCOD L-1 day(-1), highest organic material removal efficiencies achieved with 4-h and 6-h feeding were 84 %, against 71 % at 2-h feeding. At AVOL of 7.5 gCOD L-1 day(-1), organic material removal efficiency achieved with 4-h feeding was 77 %. Hence, longer feeding times favored minimization of total volatile acids concentration during the cycle as well as in the effluent, guaranteeing process stability and safety.

Hydrogen production and consumption of organic acids by a phototrophic microbial consortium

Alternative fuel sources have been extensively studied. Hydrogen gas has gained attention because its combustion releases only water, and it can be produced by microorganisms using organic acids as substrates. The aim of this study was to enrich a microbial consortium of photosynthetic purple non-sulfur bacteria from an Upflow Anaerobic Sludge Blanket reactor (UASB) using malate as carbon source. After the enrichment phase, other carbon sources were tested, such as acetate (30 mmol l(-1)), butyrate (17 mmol l(-1)), citrate (11 mmol l(-1)), lactate (23 mmol l(-1)) and malate (14.5 mmol l(-1)). The reactors were incubated at 30 degrees C under constant illumination by 3 fluorescent lamps (81 mu mol m(-2) s(-1)). The cumulative hydrogen production was 7.8, 9.0, 7.9, 5.6 and 13.9 mmol H-2 l(-1) culture for acetate, butyrate, citrate, lactate and malate, respectively. The maximum hydrogen yield was 0.6, 1.4, 0.7, 0.5 and 0.9 mmol H-2 mmol(-1) substrate for acetate, butyrate, citrate, lactate and malate, respectively. The consumption of substrates was 43% for acetate, 37% for butyrate, 100% for citrate, 49% for lactate and 100% for malate. Approximately 26% of the clones obtained from the Phototrophic Hydrogen-Producing Bacterial Consortium (PHPBC) were similar to Rhodobacter, Rhodospirillum and Rhodopseudomonas, which have been widely cited in studies of photobiological hydrogen production. Clones similar to the genus Sulfurospirillum (29% of the total) were also found in the microbial consortium. Copyright (C) 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.

Effects of photobioreactor configuration, nitrogen source and light intensity on the fed-batch cultivation of Arthrospira (Spirulina) platensis. Bioenergetic aspects

Bioenergetic analysis may be applied in order to predict microbial growth yields, based on the Gibbs energy dissipation and mass conservation principles of the overall growth reaction. The bioenergetics of the photoautotrophic growth of the cyanobacterium Arthrospira (Spirulina) platensis was investigated in different bioreactor configurations (tubular photobioreactor and open ponds) using different nitrogen sources (nitrate and urea) and under different light intensity conditions to determine the best growing conditions in terms of Gibbs energy dissipation, number of photons to sustain cell growth and phototrophic energy yields distribution in relation to the ATP and NADPH formation, and release of heat. Although an increase in the light intensity increased the Gibbs energy dissipated for cell growth and maintenance with both nitrogen sources, it did not exert any appreciable influence on the moles of photons absorbed by the system to produce one C-mol biomass. On the other hand, both bioenergetic parameters were higher in cultures with nitrate than with urea, likely because of the higher energy requirements needed to reduce the former nitrogen source to ammonia. They appreciably increased also when open ponds were substituted by the tubular photobioreactor, where a more efficient light distribution ensured a remarkably higher cell mass concentration. The estimated percentages of the energy absorbed by the cell showed that, compared with nitrate, the use of urea as nitrogen source allowed the system to address higher energy fractions to ATP production and light fixation by the photosynthetic apparatus, as well as a lower fraction released as heat. The best energy yields values on Gibbs energy necessary for cell growth and maintenance were achieved in up to 4-5 days of cultivation, indicating that it would be the optimum range to maintain cell growth. Thanks to this better bioenergetic situation, urea appears to be a quite promising low-cost, alternative nitrogen source for Arthrospira platensis cultures in photobioreactors. (C) 2011 Elsevier Ltd. All rights reserved.

Oxygen supply in Bacillus thuringiensis fermentations: bringing new insights on their impact on sporulation and delta-endotoxin production

The growth kinetics, sporulation, and toxicity of Bacillus thuringiensis var. israelensis were evaluated through the analysis of batch cultures with different dissolved oxygen (DO) profiles. Firstly, DO was maintained constant at 5%, 20%, or 50% throughout fermentation in order to identify the most suitable one to improve the main process parameters. Higher biomass concentration, cell productivity, and cell yield based on glucose were obtained with 50% DO. The higher aeration level also resulted in higher spore counts and markedly improved the toxic activity of the fermentation broth, which was 9-fold greater than that obtained with 5% DO (LC50 of 39 and 329 mg/L, respectively). Subsequently, using a two-stage oxygen supply strategy, DO was kept at 50% during the vegetative and transition phases until the maximum cell concentration was achieved. Then, DO was changed to 0%, 5%, 20%, or 100% throughout sporulation and cell lysis phases. The interruption of oxygen supply strongly reduced the spore production and thoroughly repressed the toxin synthesis. On the contrary, when DO was raised to 100% of saturation, toxic activity increased approximately four times (LC50 of 8.2 mg/L) in comparison with the mean values reached with lower DO levels, even though spore counts were lower than that from the 50% DO assay. When pure oxygen was used instead of normal air, it was possible to obtain 70% of the total biomass concentration achieved in the air assays; however, cultures did not sporulate and the toxin synthesis was consequently suppressed.

Hydrogen and Methane Production, Energy Recovery, and Organic Matter Removal from Effluents in a Two-Stage Fermentative Process

This study evaluates the potential for using different effluents for simultaneous H-2 and CH4 production in a two-stage batch fermentation process with mixed microflora. An appreciable amount of H-2 was produced from parboiled rice wastewater (23.9 mL g(-1) chemical oxygen demand [COD]) and vinasse (20.8 mL g(-1) COD), while other effluents supported CH4 generation. The amount of CH4 produced was minimum for sewage (46.3 mL g(-1) COD), followed by parboiled rice wastewater (115.5 mL g(-1) COD) and glycerol (180.1 mL g(-1) COD). The maximum amount of CH4 was observed for vinasse (255.4 mL g(-1) COD). The total energy recovery from vinasse (10.4 kJ g(-1) COD) corresponded to the maximum COD reduction (74.7 %), followed by glycerol (70.38 %, 7.20 kJ g(-1) COD), parboiled rice wastewater (63.91 %, 4.92 kJ g(-1) COD), and sewage (51.11 %, 1.85 kJ g(-1) COD). The relatively high performance of vinasse in such comparisons could be attributed to the elevated concentrations of macronutrients contained in raw vinasse. The observations are based on kinetic parameters of H-2 and CH4 production and global energy recovery of the process. These observations collectively suggest that organic-rich effluents can be deployed for energy recovery with sequential generation of H-2 and CH4.

Biotechnological Utilization of Biodiesel-Derived Glycerol for the Production of Ribonucleotides and Microbial Biomass

Ten yeast strains were evaluated concerning their capabilities to assimilate biodiesel-derived glycerol in batch cultivation. The influence of glycerol concentration, temperature, pH and yeast extract concentration on biomass production was studied for the yeast selected. Further, the effect of agitation on glycerol utilization by the yeast Hansenula anomala was also studied. The yeast H. anomala CCT 2648 showed the highest biomass yield (0.30 g g(-1)) and productivity (0.19 g L-1 h(-1)). Citric acid, succinic acid, acetic acid and ethanol were found as the main metabolites produced. The increase of yeast extract concentration from 1 to 3 g L-1 resulted in high biomass production. The highest biomass concentration (21 g L-1), yield (0.45 g g(-1)) and productivity (0.31 g L-1 h(-1)), as well as ribonucleotide production (13.13 mg g(-1)), were observed at 700 rpm and 0.5 vvm. These results demonstrated that glycerol from biodiesel production process showed to be a feasible substrate for producing biomass and ribonucleotides by yeast species.

Influence of the use of rice bran extract as a source of nutrients on xylitol production

Xylose-to-xylitol bioconversion using 2.5 or 10% (v/v) rice bran extract was performed to verify the influence of this source of nutrients on Candida guilliermondii metabolism. Semisynthetic medium (SM) and sugarcane bagasse hemicellulosic hydrolysate detoxified with ion-exchange resins (HIE) or with alteration in pH combined with adsorption onto activated charcoal (HAC) were fermented in 125 mL Erlenmeyer flasks at 30 ºC and 200 rpm for 72 hours. Activated charcoal supplemented with 2.5% (v/v) rice bran extract was fermented by C. guilliermondii in a MULTIGEN stirred tank reactor using pH 5.0 and 22.9/hour oxygen transfer volumetric coefficient. Higher values of xylitol productivity (0.70, 0.71, and 0.62 g.Lh-1) and xylose-to-xylitol conversion yield (0.71, 0.69, and 0.63 g.g-1) were obtained with 2.5% (v/v) rice bran in semisynthetic medium, ion-exchange resins, and activated charcoal, respectively. Moreover, during batch fermentation, the xylitol volumetric productivity and fermentation efficiency values obtained were 0.53 g.Lh-1 and 61.1%, respectively.

Fermentative production of ribonucleotides from whey by Kluyveromyces marxianus: effect of temperature and pH

Ribonucleotides have shown many promising applications in food and pharmaceutical industries. The aim of the present study was to produce ribonucleotides (RNA) by Kluyveromyces marxianus ATCC 8,554 utilizing cheese whey, a dairy industry waste, as a main substrate under batch fermentation conditions. The effects of temperature, pH, aeration rate, agitation and initial cellular concentration were studied simultaneously through factorial design for RNA, biomass production and lactose consumption. The maximum RNA production (28.66 mg/g of dry biomass) was observed at temperature 30°C, pH 5.0 and 1 g/l of initial cellular concentration after 2 h of fermentation. Agitation and aeration rate did not influence on RNA concentration (p >0.05). Maximum lactose consumption (98.7%) and biomass production (6.0 g/l) was observed after 12 h of incubation. This study proves that cheese whey can be used as an adequate medium for RNA production by K. marxianus under the optimized conditions at industrial scale.