Metabolic analysis of poly(3-hydroxybutyrate) production by recombinant Escherichia coli

Poly(3-hydroxybutyrate) (PHB) production by fermentation was examined under both restricted- and ample-oxygen supply conditions in a single fed-batch fermentation. Recombinant Escherichia coli transformed with the PHB production plasmid pSYL107 was grown to reach high cell density (227 g/l dry cell weight) with a high PHB content (78% of dry cell weight), using a glucose-based minimal medium. A simple flux model containing 12 fluxes was developed and applied to the fermentation data. A superior closure (95%) of the carbon mass balance was achieved. When the data were put into use, the results demonstrated a surprisingly large excretion of formate and lactate. Even though periods of severe oxygen limitation coincided with rapid acetate and lactate excretion, PHB productivity and carbon utilization efficiency were not significantly impaired. These results are very positive in reducing oxygen demand in an industrial PHA fermentation without sacrificing its PHA productivity, thereby reducing overall production costs.

Effects of mutations in acetate metabolism on high-cell-density growth of Escherichia coli

To study the role played by acetate metabolism during high-cell-density growth of Escherichia coli cells, we constructed isogenic null mutants of strain W3100 deficient for several genes involved either in acetate metabolism or the transition to stationary phase. We grew these strains under identical fed-batch conditions to the highest cell densities achievable in 8 h using a predictive-plus-feedback-controlled computer algorithm that maintained glucose at a set-point of 0.5 g/l, as previously described. Wild-type strains, as well as mutants lacking the sigma(s) subunit of RNA polymerase (rpoS), grew reproducibly to high cell densities (44-50 g/l dry cell weights, DCWs). In contrast, a strain lacking acetate kinase (ackA) failed to reach densities greater than 8 g/l. Strains lacking other acetate metabolism genes (pta, acs, poxB, iciR, and fadR) achieved only medium cell densities (15-21 g/l DCWs). Complementation of either the acs or the ackA mutant restored wild-type high-cell-density growth, on a dry weight basis, poxB and fadR strains produced approximately threefold more acetate than did the wild-type strain. In contrast, the pta, acs, or rpoS strains produced significantly less acetate per cell dry weight than did the wild-type strain. Our results show that acetate metabolism plays a critical role during growth of E. coli cultures to high cell densities. They also demonstrate that cells do not require the sigma(s) regulon to grow to high cell densities, at least not under the conditions tested.

Exigências nutricionais e operacionais para a produção de etanol pela levedura IQ-Ar/45-1 a partir do melaço em batelada alimentada

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Produção e purificação de L (+) ácido lático por Lactobacillus rhammosus utilizando processo de batelada alimentada

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Biofuels and high value added products from the yeast Rhodosporidium toruloides NCYC 921: Strategies towards a true cost-effective and environmentally sustainable integrated multiproduct driven biorefinery [Poster]

Single-cell oils (SCO) have been considered a promising source of 3rd generation biofuels mainly in the final form of biodiesel. However, its high production costs have been a barrier towards the commercialization of this commodity. The fast growing yeast Rhodosporidium toruloides NCYC 921 has been widely reported as a potential SCO producing yeast. In addition to its well-known high lipid content (that can be converted into biodiesel), is rich in high value added products such as carotenoids with commercial interest. The process design and integration may contribute to reduce the overall cost of biofuels and carotenoid production and is a mandatory step towards their commercialization. The present work addresses the biomass disruption, extraction, fractionation and recovery of products with special emphasis on high added valued carotenoids (beta-carotene, torulene, torularhodin) and fatty acids directed to biodiesel. The chemical structure of torularhodin with a terminal carboxylic group imposes an additional extra challenge in what concern its separation from fatty acids. The proposed feedstock is fresh biomass pellet obtained directly by centrifugation from a 5L fed-batch fermentation culture broth. The use of a wet instead of lyophilised biomass feedstock is a way to decrease processing energy costs and reduce downstream processing time. These results will contribute for a detailed process design. Gathered data will be of crucial importance for a further study on Life-Cycle Assessment (LCA).

Batch and continuous fermentation methods for the production of dextransucrase

The available literature concerning dextransucrase and dextran production and purification has been reviewed along with the reaction mechanisms of the enzyme. A discussion of basic fermentation theory is included, together with a brief description of bioreactor hydrodynamics and general biotechnology. The various fermenters used in this research work are described in detail, along with the various experimental techniques employed. The micro-organism Leuconostoc mesenteroides NRRL B512 (F) secretes dextransucrase in the presence of an inducer, sucrose, this being the only known inducer of the enzyme. Dextransucrase is a growth related product and a series of fed-batch fermentations have been carried out to extend the exponential growth phase of the organism. These experiments were carried out in a number of different sized vessels, ranging in size from 2.5 to 1,000 litres. Using a 16 litre vessel, dextransucrase activities in excess of 450 DSU/cm3 (21.67 U/cm3) have been obtained under non-aerated conditions. It has also been possible to achieve 442 DSU/cm3 (21.28 U/cm3) using the 1,000 litre vessel, although this has not been done consistently. A 1 litre and a 2.5 litre vessel were used for the continuous fermentations of dextransucrase. The 2.5 litre vessel was a very sophisticated MBR MiniBioreactor and was used for the majority of continuous fermentations carried out. An enzyme activity of approximately 108 DSU/cm3 (5.20 U/cm3) was achieved at a dilution rate of 0.50 h-1, which corresponds to the maximum growth rate of the cells under the process conditions. A number of continuous fermentations were operated for prolonged periods of time, with experimental run-times of up to 389 h being recorded without any incidence of contamination. The phenomenon of enzyme enhancement on hold-up of up to 100% was also noted during these fermentations, with dextransucrase of activity 89.7 DSU/cm3 (4.32 U/cm3) being boosted to 155.7 DSU/cm3 (7.50 U/cm3) following 24 hours of hold-up. These findings support the recommendation of a second reactor being placed in series with the existing vessel.

CO(2) from Alcoholic Fermentation for Continuous Cultivation of Arthrospira (Spirulina) platensis in Tubular Photobioreactor Using Urea as Nitrogen Source

Carbon dioxide released from alcoholic fermentation accounts for 33% of the whole CO(2) involved in the use of ethanol as fuel derived from glucose. As Arthrospira platensis can uptake this greenhouse gas, this study evaluates the use of the CO(2) released from alcoholic fermentation for the production of Arthrospira platensis. For this purpose, this cyanobacterium was cultivated in continuous process using urea as nitrogen source, either using CO(2) from alcoholic fermentation, without any treatment, or using pure CO(2) from cylinder. The experiments were carried out at 120 mu mol photons m(-2) s(-1) in tubular photobioreactor at different dilution rates (0.2 <= D <= 0.8 d(-1)). Using CO(2) from alcoholic fermentation, maximum steady-state cell concentration (2661 +/- 71 mg L(-1)) was achieved at D 0.2 d(-1), whereas higher dilution rate (0.6 d(-1)) was needed to maximize cell productivity (839 mg L(-1) d(-1)). This value was 10% lower than the one obtained with pure CO(2), and there was no significant difference in the biomass protein content. With D 0.8 d(-1), it was possible to obtain 56% +/- 1.5% and 50% +/- 1.2% of protein in the dry biomass, using pure CO(2) and CO(2) from alcoholic fermentation, respectively. These results demonstrate that the use of such cost free CO(2) from alcoholic fermentation as carbon source, associated with low cost nitrogen source, may be a promising way to reduce costs of continuous cultivation of photosynthetic microorganisms, contributing at the same time to mitigate the greenhouse effect. (C) 2011 American Institute of Chemical Engineers Biotechnol. Prog., 27: 650-656, 2011

Influence of carbon source and the fermentation process on levan production by Zymomonas mobilis analyzed by the surface response method

The aim of this study is to assess sugar cane juice and sucrose as substrates, the batch and fed batch processes and their interaction in the levan production using a complete factorial design. Zymomonas mobilis was cultivated in different sugar cane juice and sucrose concentrations in two fermentation processes at 25 °C for 20 h. A complete factorial design (2³) was used to analyze the effects of the type and concentration of the substrate, as well as the batch and fed batch processes. A complete second factorial design (2²) was used to observe the importance of sugar cane juice. The results indicated that the batch process improved the levan production reaching 40.14 g/L. The addition of sugar cane juice was not statistically significant for levan formation, however sugar cane juice stimulated biomass, sorbitol and ethanol production. The best medium for levan production was 150 g/L sucrose in batch.

In vitro fermentation of carbohydrates by porcine faecal inocula and their influence on Salmonella Typhimurium growth in batch culture systems

The aim of this study was to evaluate in vitro the influence of fermentable carbohydrates on the activity of porcine microbiota and survival of Salmonella Typhimurium in a batch culture system simulating the porcine hindgut. The carbohydrates tested were xylooligosaccharides, a mixture of fructooligosaccharides/inulin (FIN), fructooligosaccharides (FOS), gentiooligosaccharides (GEO) and lactulose (LAC). These ingredients stimulated the growth of selected Bifidobacterium and Lactobacillus species in pure cultures. In batch cultures, the carbohydrates influenced some fermentation parameters. For example, GEO and FIN significantly increased lactic acids compared with the control (no added carbohydrate). With the exception of LAC, the test carbohydrates increased the production of short-chain fatty acid (SCFA) and modified SCFA profiles. Quantitative analysis of bacterial populations by FISH revealed increased counts of the Bifidobacterium group compared with control and, with exception of FOS, increased Lactobacillus, Leuconostoc and Weissella spp. counts. Salmonella numbers were the lowest during the fermentation of LAC. This work has looked at carbohydrate metabolism by porcine microbiota in a pH-controlled batch fermentation system. It provides an initial model to analyse interactions with pathogens.

Formulation of fermentation media from flour-rich waste streams for microbial lipid production by Lipomyces starkeyi

Flour-rich waste (FRW) and by-product streams generated by bakery, confectionery and wheat milling plants could be employed as the sole raw materials for generic fermentation media production, suitable for microbial oil synthesis. Wheat milling by-products were used in solid state fermentations (SSF) of Aspergillus awamori for the production of crude enzymes, mainly glucoamylase and protease. Enzyme-rich SSF solids were subsequently employed for hydrolysis of FRW streams into nutrient-rich fermentation media. Batch hydrolytic experiments using FRW concentrations up to 205 g/L resulted in higher than 90%(w/w) starch to glucose conversion yields and 40% (w/w) total Kjeldahl nitrogen to free amino nitro-gen conversion yields. Starch to glucose conversion yields of 98.2, 86.1 and 73.4% (w/w) were achieved when initial FRW concentrations of 235, 300 and 350 g/L were employed in fed-batch hydrolytic experiments, respectively. Crude hydrolysates were used as fermentation media in shake flask cultures with the oleaginous yeast Lipomyces starkeyi DSM 70296 reaching a total dry weight of 30.5 g/L with a microbial oil content of 40.4% (w/w), higher than that achieved in synthetic media. Fed-batch bioreactor cultures led to a total dry weight of 109.8 g/L with a microbial oil content of 57.8% (w/w) and productivity of 0.4 g/L/h.

Biorefinery development through utilization of biodiesel industry by-products as sole fermentation feedstock for 1,3-propanediol production

Rapeseed meal (RSM) hydrolysate was evaluated as substitute for commercial nutrient supplements in 1,3-propanediol (PDO) fermentation using the strain Clostridium butyricum VPI 1718. RSM was enzymatically converted into a generic fermentation feedstock, enriched in amino acids, peptides and various micro-nutrients, using crude enzyme consortia produced via solid state fermentation by a fungal strain of Aspergillus oryzae. Initial free amino nitrogen concentration influenced PDO production in batch cultures. RSM hydrolysates were compared with commercial nutrient supplements regarding PDO production in fed-batch cultures carried out in a bench-scale bioreactor. The utilization of RSM hydrolysates in repeated batch cultivation resulted in a PDO concentration of 65.5 g/L with an overall productivity of 1.15 g/L/h that was almost 2 times higher than the productivity achieved when yeast extract was used as nutrient supplement.

Effect of 3,4,4 '-trichlorocarbanilide on growth of lactic acid bacteria contaminants in alcoholic fermentation

3,4,4'-trichlorocarbanilide (TCC) was rested as a new method of bacterial growth control for S. cerevisiae alcoholic fermentations of diluted high test molasses (HTM). Minimal inhibitory concentration (MIC) was tested to determine the necessary concentration of TCC to control bacterial growth. The fed-batch alcoholic fermentation process was used with cell recycle similar to industrial conditions and Lactobacillus fermentum CCT 1407 was mixed in the first inoculum to grow with the yeast. Yeast extract was added into the must to stimulate bacterial growth. The best results of TCC's MIC to bacterial growth of Lactobacillus fermentum and Leuconostoc mesenteroides (< 0.125-1.0 mu g/ml) and Saccharomyces cerevisiae (16 mu g/ml) occurred when it was combined with sodium dodecylsulphate (SDS) in a 1: 4 TCC/SDS ratio (wt/wt) in distilled water solution. 1.8 g/l TCC entrapped in calcium alginate added to the must with yeast extract inhibited the growth of Lactobacillus fermentum CCT 1407 maintaining a controlled acidity, higher yeast viability and up to 20.8% of improvement in the average of alcoholic efficiency. Addition of 0.075 g/l TCC entrapped in calcium alginate and 1.67 mg/l SDS in the wort with yeast extract (0-5.0 g/l), inhibited and controlled the extensive bacterial contamination for 19 cycles of fermentation. (C) 1998 Published by Elsevier B.V. Ltd.

Chlorine dioxide against bacteria and yeasts from the alcoholic fermentation

The ethanol production in Brazil is carried out by fed-batch or continuous process with cell recycle, in such way that bacterial contaminants are also recycled and may be troublesome due to the substrate competition. Addition of sulphuric acid when inoculum cells are washed can control the bacterial growth or alternatively biocides are used. This work aimed to verify the effect of chlorine dioxide, a well-known biocide for bacterial decontamination of water and equipments, against contaminant bacteria ( Bacillus subtilis, Lactobacillus plantarum, Lactobacillus fermentum and Leuconostoc mesenteroides) from alcoholic fermentation, through the method of minimum inhibitory concentration ( MIC), as well as its effect on the industrial yeast inoculum. Lower MIC was found for B. subtilis ( 10 ppm) and Leuconostoc mesenteroides ( 50 ppm) than for Lactobacillus fermentum ( 75 ppm) and Lactobacillus plantarum ( 125 ppm). Additionally, these concentrations of chlorine dioxide had similar effects on bacteria as 3 ppm of Kamoran (R) ( recommended dosage for fermentation tanks), exception for B. subtilis, which could not be controlled at this Kamoran (R) dosage. The growth of industrial yeasts was affected when the concentration of chlorine dioxide was higher than 50 ppm, but the effect was slightly dependent on the type of yeast strain. Smooth yeast colonies ( dispersed cells) seemed to be more sensitive than wrinkled yeast colonies ( clustered cells/pseudohyphal growth), both isolated from an alcohol-producing unit during the 2006/2007 sugar cane harvest. The main advantage in the usage of chlorine dioxide that it can replace antibiotics, avoiding the selection of resistant populations of microorganisms.

Arthrospira (Spirulina) platensis cultivation in tubular photobioreactor: Use of no-cost CO2 from ethanol fermentation

The present study aimed at evaluating the production of Arthrospira platensis in tubular photobioreactor using CO2 from ethanol fermentation. The results of these cultivations were compared to those obtained using CO2 from cylinder at different protocols of simultaneous ammonium sulfate and sodium nitrate feeding. Maximum cell concentration (X-m), cell productivity (P-x), nitrogen-to-cell conversion factor (Y-X/N), and biomass composition (total lipids and proteins) were selected as responses and evaluated by analysis of variance. The source of CO2 did not exert any significant statistical influence on these responses, which means that the flue gas from ethanol fermentation could successfully be used as a carbon source as well as to control the medium pH, thus contributing to reduce the greenhouse effect. The results taken as a whole demonstrated that the best combination of responses mean values (X-m = 4.543 g L-1; P-x = 0.460 g L-1 d(-1); Y-X/N = 15.6 g g(-1); total lipids = 8.39%; total proteins = 18.7%) was obtained using as nitrogen source a mixture of 25% NaNO3 and 75% (NH4)(2)SO4, both expressed as nitrogen. (C) 2011 Elsevier Ltd. All rights reserved.

Fermentation methods for dextransucrase production

Several fermentation methods for the production of the enzyme dextransucrase have been employed. The theoretical aspects of these fermentation techniques have been given in the early chapters of this thesis together with a brief overview of enzyme biotechnology. A literature survey on cell recycle fermentation has been carried out followed by a survey report on dextransucrase production, purification and the reaction mechanism of dextran biosynthesis. The various experimental apparatus as employed in this research are described in detail. In particular, emphasis has been given to the development of continuous cell recycle fermenters. On the laboratory scale, fed-batch fermentations under anaerobic low agitation conditions resulted in dextransucrase activities of about 450 DSU/cm3 which are much higher than the yields reported in the literature and obtained under aerobic conditions. In conventional continuous culture the dilution rate was varied in the range between 0.375 h-1 to 0.55 h-1. The general pattern observed from the data obtained was that the enzyme activity decreased with increase in dilution rate. In these experiments the maximum value of enzyme activity was ∼74 DSU/cm3. Sparging the fermentation broth with CO2 in continuous culture appears to result in a decrease in enzyme activity. In continuous total cell recycle fermentations high steady state biomass levels were achieved but the enzyme activity was low, in the range 4 - 27 DSU/cm3. This fermentation environment affected the physiology of the microorganism. The behaviour of the cell recycle system employed in this work together with its performance and the factors that affected it are discussed in the relevant chapters. By retaining the whole broth leaving a continuous fermenter for between 1.5 - 4 h under controlled conditions, the enzyme activity was enhanced with a certain treatment from 86 DSU/cm3 to 180 DSU/cm3 which represents a 106% increase over the enzyme activity achieved by a steady-state conventional chemostat. A novel process for dextran production has been proposed based on the findings of this latter part of the experimental work.