Production of bacterial cellulose by<Emphasis Type="Italic">Gluconacetobacter hansenii</Emphasis> PJK isolated from rotten apple

A cellulose-producing strain isolated from rotten apples was identified asGluconace-tobacter hansenii based on its physiological properties and the 16S rDNA complete sequencing method, and specifically namedGluconacetobacter hansenii PJK. The amount of bacterial cellulose (BC) produced byG. hansenii PJK in a shaking incubator was 1.5 times higher than that produced in a static culture. The addition of ethanol to the medium during cultivation enhanced the productivity of bacterial cellulose, plus the supplementation of 1% ethanol into the culture medium made the produced BC aggregate into a big lump and thus protected the bacterial-cellulose-producingG. hansenii PJK cells in the shear stress field from being converted into noncellulose-producing (Cel) mutants. Cells subcultured three times in a medium containing ethanol retained their ability to produce BC without any loss in the production yield.     

Modeling of lutein production by heterotrophic <Emphasis Type="Italic">Chlorella</Emphasis> in batch and fed-batch cultures

Chlorella is a promising alternative resource of lutein (xanthophyll) production as it can be cultivated heterotrophically in fermentors. In this paper, a kinetic model for lutein production by heterotrophic Chlorella pyrenoidosa was developed based on batch cultivations in 250-ml flasks and a 19-l fermentor. The model was validated by experimental data from two fed-batch cultivations performed in the same fermentor. The dynamic behavior of lutein production by C. pyrenoidosa with various concentrations of glucose and nitrogen was analyzed based on the kinetic model. Model-based analyses suggested that glucose concentrations between 5 and 24 g/l and nitrogen concentrations between 0.7 and 12 g/l during the cultivation were favorable for lutein production by heterotrophic C. pyrenoidosa. It also showed that fed-batch cultivations are more suitable for efficient production of lutein than batch ones. The results obtained in this study may contribute to commercial lutein production by heterotrophic Chlorella.     

A cultivation technique for <Emphasis Type="Italic">E. coli</Emphasis> fed-batch cultivations operating close to the maximum oxygen transfer capacity of the reactor

A cultivation strategy combining the advantages of temperature-limited fed-batch and probing feeding control is presented. The technique was evaluated in fed-batch cultivations with E. coli BL21(DE3) producing xylanase in a 3 liter bioreactor. A 20% increase in cell mass was achieved and the usual decrease in specific enzyme activity normally observed during the late production phase was diminished with the new technique. The method was further tested by growing E. coli W3110 in a larger bioreactor (50 l). It is a suitable cultivation technique when the O₂ transfer capacity of the reactor is reached and it is desired to continue to produce the recombinant protein.Revisions requested 13 April 2005; Revisions received 6 May 2005     

Fed-batch cultivation of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> on lignocellulosic hydrolyzate

Saccharomyces cerevisiae grows very poorly in dilute acid lignocellulosic hydrolyzate during the anaerobic fermentation for fuel ethanol production. However, yeast cells grown aerobically on the hydrolyzate have increased tolerance for the hydrolyzate. Cultivation of yeast on part of the hydrolyzate has therefore the potential of enabling increased ethanol productivity in the fermentation of the hydrolyzate. To evaluate the ability of the yeast to grow in the hydrolyzate, fed-batch cultivations were run using the ethanol concentration as input variable to control the feed-rate. The yeast then grew in an undetoxified hydrolyzate with a specific growth rate of 0.19 h⁻¹ by controlling the ethanol concentration at a low level during the cultivation. However, the biomass yield was lower for the cultivation on hydrolyzate compared to synthetic media: with an ethanol set-point of 0.25 g/l the yield was 0.46 g/g on the hydrolyzate, compared to 0.52 g/g for synthetic media. The main reason for the difference was not the ethanol production per se, but a significant production of glycerol at a high specific growth rate. The glycerol production may be attributed to an insufficient respiratory capacity.     

Overproduction of glutathione by <Emphasis Type="SmallCaps">l</Emphasis>-cysteine addition and a temperature-shift strategy

The present study investigated the effects of three constituent amino acids on glutathione production in flask culture of Candida utilis. Although l-glutamic acid and glycine had little impact on cell growth and glutathione biosynthesis, l-cysteine positively influenced glutathione production, despite inhibiting cell growth when it was added prior to stationary phase. Adding 8 mmol/L of l-cysteine to the culture broth at 16 h boosted glutathione production by 91%, increasing the intracellular glutathione content by 106% compared to untreated controls. A temperature-shift strategy, in which we shifted batch and fed-batch cultures of C. utilis from 30 to 26°C, also significantly enhanced glutathione production. Applying both strategies (i.e. adding 20 mmol/L l-cysteine and shifting the temperature from 30 to 26°C) at 33 h enhanced the glutathione concentration and the intracellular glutathione content to 1,312 mg/L and 3.75%, respectively, during fed-batch cultivation (glucose feeding at a constant rate of 18.3 g/h). The average specific glutathione production rate under this condition was 129% higher than that of the control without strategy.     

Cellulose synthesis by Komagataeibacter rhaeticus strain P 1463 isolated from Kombucha

Isolate B17 from Kombucha was estimated to be an efficient producer of bacterial cellulose (BC). The isolate was deposited under the number P 1463 and identified as Komagataeibacter rhaeticus by comparing a generated amplified fragment length polymorphism (AFLP™) DNA fingerprint against a reference database. Static cultivation of the K. rhaeticus strain P 1463 in Hestrin and Schramm (HS) medium resulted in 4.40 ± 0.22 g/L BC being produced, corresponding to a BC yield from glucose of 25.30 ± 1.78 %, when the inoculum was made with a modified HS medium containing 10 g/L glucose. Fermentations for 5 days using media containing apple juice with analogous carbon source concentrations resulted in 4.77 ± 0.24 g/L BC being synthesised, corresponding to a yield from the consumed sugars (glucose, fructose and sucrose) of 37.00 ± 2.61 %. The capacity of K. rhaeticus strain P 1463 to synthesise BC was found to be much higher than that of two reference strains for cellulose production, Komagataeibacter xylinus DSM 46604 and Komagataeibacter hansenii DSM 5602^T, and was also considerably higher than that of K. hansenii strain B22, isolated from another Kombucha sample. The BC synthesised by K. rhaeticus strain P 1463 after 40 days of cultivation in HS medium with additional glucose supplemented to the cell culture during cultivation was shown to have a degree of polymerization of 3300.0 ± 122.1 glucose units, a tensile strength of 65.50 ± 3.27 MPa and a length at break of 16.50 ± 0.83 km. For the other strains, these properties did not exceed 25.60 ± 1.28 MPa and 15.20 ± 0.76 km.

     

Production of ethanol by filamentous and yeast-like forms of <Emphasis Type="Italic">Mucor indicus</Emphasis> from fructose,glucose, sucrose,and molasses

The fungus Mucor indicus is found in this study able to consume glucose and fructose, but not sucrose in fermentation of sugarcane and sugar beet molasses. This might be an advantage in industries which want to selectively remove glucose and fructose for crystallisation of sucrose present in the molasses. On the other hand, the fungus assimilated sucrose after hydrolysis by the enzyme invertase. The fungus efficiently grew on glucose and fructose and produced ethanol in synthetic media or from molasses. The cultivations were carried out aerobically and anaerobically, and manipulated toward filamentous or yeast-like morphology. Ethanol was the major metabolite in all the experiments. The ethanol yield in anaerobic cultivations was between 0.35 and 0.48 g/g sugars consumed, depending on the carbon source and the growth morphology, while a yield of as low as 0.16 g/g was obtained during aerobic cultivation. The yeast-like form of the fungus showed faster ethanol production with an average productivity of 0.90 g/l h from glucose, fructose and inverted sucrose, than the filamentous form with an average productivity of 0.33 g/l h. The biomass of the fungus was also analyzed with respect to alkali-insoluble material (AIM), chitin, and chitosan. The biomass of the fungus contained per g maximum 0.217 g AIM and 0.042 g chitosan in yeast-like cultivation under aerobic conditions.     

Immobilization of Zymomonas mobilis 2716, for the protection of cellular activity

Alginate-immobilized Zymomonas mobilis cells produced 17.8% (v/v) ethanol in less than 24 h, with an ethanol yield of 97%, compared with 88% for free cells, using a fed-batch cultivation technique. The substrate, glucose, was added intermittently in powder form to foster nucleation of the CO₂ formed. Repeated-batch cultivation led to complete utilization of approximately 200 g glucose/l in 7.5 h with a 98% conversion efficiency to ethanol. Free cells used the glucose less efficiently (conversion efficiency of 78%), and even after 100 h the glucose was not fully consumed. Freeze-substitution electron microscopy studies showed that immobilized cells generally displayed lesser blebbing and membrane disruption than free cells. These studies further suggest that membrane blebbing may be due to an effect of high initial glucose levels, and not due to the accumulation of end-products ethanol and CO₂.L.A. Kirk, H.W. Doelle and R.I. Webb are with the Department of Microbiology, University of Queensland, Brisbane, QId 4072, Australia. R.I. Webb is also with the Microscopy and Microanalysis Centre, University of Queensland, Brisbane, QId 4072, Australia;     

Isolation of a novel high erythritol-producing <Emphasis Type="Italic">Pseudozyma tsukubaensis</Emphasis> and scale-up of erythritol fermentation to industrial level

This study isolated a novel erythritol-producing yeast strain, which is capable of growth at high osmolarity. Characteristics of the strain include asexual reproduction by multilateral budding, absence of extracellular starch-like compounds, and a negative Diazonium blue B color reaction. Phylogenetic analysis based on the 26S rDNA sequence and physiological analysis indicated that the strain belongs to the species Pseudozyma tsukubaensis and has been named P. tsukubaensis KN75. When P. tsukubaensis KN75 was cultured aerobically in a fed-batch culture with glucose as a carbon source, it produced 245 g/L of erythritol, corresponding to 2.86 g/L/h productivity and 61% yield, the highest erythritol yield ever reported by an erythritol-producing microorganism. Erythritol production was scaled up from a laboratory scale (7 L fermenter) to pilot (300 L) and plant (50,000 L) scales using the dissolved oxygen as a scale-up parameter. Erythritol production at the pilot and plant scales was similar to that at the laboratory scale, indicating that the production of erythritol by P. tsukubaensis KN75 holds commercial potential.     

Enhanced cutinase production of Thermobifida fusca by a two-stage batch and fed-batch cultivation strategy

In this study, cutinase production by Thermobifida fusca WSH03-11 was investigated with mixed short-chain organic acids as co-carbon sources to demonstrate the possibility of producing high value-added products from organic wastes. T. fusca WSH03-11 was cultured with different combinations of butyrate, acetate, and lactate with a purpose of increasing cutinase activity. The optimum proportion of butyrate, acetate, and lactate was 4:1:3. In batch cultivation, acetate and lactate were consumed quickly, while the consumption of butyrate was depressed in the presence of acetate with a concentration higher than 0.5 g/L. Based on these results, a two-stage batch and fed-batch cultivation strategy was proposed: a batch culture with acetate and lactate as the co-carbon sources in the first 10 h, and then a fed-batch culture with a constant butyrate feeding rate of 12 mL/h during 11∼20 h. By this two-stage cultivation strategy, cutinase activity, dry cell weight, and consumption rate of butyrate were increased by 70%, 103.4%, and 4.3-fold, respectively, compared to those of the batch cultivation. These results provided a novel and efficient way to produce high value-added products from organic wastes.     

URA3基因影响青蒿酸酿酒酵母工程菌中试发酵产量

CRISPR/Cas9基因编辑技术已经被广泛应用于工程酿酒酵母的基因插入、基因替换和基因敲除,通过使用选择标记进行基因编辑具有简单高效的特点。前期利用CRISPR/Cas9系统敲除青蒿酸生产菌株酿酒酵母(Saccharomyces cerevisiae) 1211半乳糖代谢负调控基因GAL80,获得菌株S. cerevisiae 1211-2,在不添加半乳糖诱导的情况下,青蒿酸摇瓶发酵产量达到了740 mg/L。但在50 L中试发酵实验中,S. cerevisiae 1211-2很难利用对青蒿酸积累起到决定性作用的碳源-乙醇,青蒿酸的产量仅为亲本菌株S.cerevisiae 1211的20%–25%。我们推测因遗传操作所需的筛选标记URA3突变,影响了其生长及青蒿酸产量。随后我们使用重组质粒pML104-KanMx4-u连同90 bp供体DNA成功恢复了URA3基因,获得了工程菌株S. cerevisiae 1211-3。S. cerevisiae 1211-3能够在葡萄糖和乙醇分批补料的发酵罐中正常生长,其青蒿酸产量超过20g/L,与亲本菌株产量相当。研究不但获得了不加半乳糖诱导的青...     

Characterization of new isolated <Emphasis Type="Italic">Ralstonia</Emphasis><Emphasis Type="Italic">eutropha</Emphasis> strain A-04 and kinetic study of biodegradable copolyester poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-4-hydroxybutyrate) production

A new isolated bacterial strain A-04 capable of producing high content of polyhydroxyalkanoates (PHAs) was morphologically and taxonomically identified based on biochemical tests and 16S rRNA gene analysis. The isolate is a member of the genus Ralstonia and close to Ralstonia eutropha. Hence, this study has led to the finding of a new and unexplored R. eutropha strain A-04 capable of producing PHAs with reasonable yield. The kinetic study of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] production by the R. eutropha strain A-04 was examined using butyric acid and γ–hydroxybutyric acid as carbon sources. Effects of substrate ratio and mole ratio of carbon to nitrogen (C/N) on kinetic parameters were investigated in shake flask fed-batch cultivation. When C/N was 200, that is, nitrogen deficient condition, the specific production rate of 3-hydroxybutyrate (3HB) showed the highest value, whereas when C/N was in the range between 4 and 20, the maximum specific production rate of 4-hydroxybutyrate (4HB) was obtained. Thus, the synthesis of 3HB was growth-limited production under nitrogen-deficient condition, whereas the synthesis of 4HB was growth-associated production under nitrogen-sufficient condition. The mole fraction of 4HB units increased proportionally as the ratio of γ–hydroxybutyric acid in the feed medium increased at any value of C/N ratio. Based on these kinetic studies, a simple strategy to improve P(3HB-co-4HB) production in shake flask fed-batch cultivation was investigated using C/N and substrate feeding ratio as manipulating variable, and was successfully proved by the experiments. The nucleotide sequence 1,378 bp reported in this study will appear in the GenBank nucleotide sequence database under accession number EF988626.     

Effect of reactor surface on production of bacterial cellulose and water soluble oligosaccharides by <Emphasis Type="Italic">Gluconacetobacter hansenii</Emphasis> PJK

The effect of agar plates on the bacterial cellulose (BC) production in a static culture was investigated in order to find the role of agar component as a surface modifying agent. Two types of surface modified reactors (SMRs: SMRD and SMRB) were prepared by coating the bottom of the reactors with agar dissolved in distilled water and basal medium, respectively. The SMRs were used for BC and water soluble oligosaccharides (WSOS) production. Control was done by the same procedure using reactors without agar plate. In both types of SMRs, the maximum production rate was observed after the second day of cultivation compared to third day of cultivation in the case of the control. The maximum productions of BC 5.308 and 5.472 g/L were observed at the first batch using SMRs prepared with agar dissolved in distilled water (SMRDs) and SMRs prepared with agar dissolved in a basal medium (SMRBs), respectively. Similarly, in the daily-culture and successive batch strategy experiments the maximum amount of WSOS produced in the SMRs was almost double that of the control. The highest water holding capacity value 92.21 g/g was observed for BC formed in the SMRs prepared with 3.0% of agar. FTIR and XRD analyses were carried out to study the structural features of the prepared BC.     

Ethanol production from wheat straw by recombinant Escherichia coli strain FBR5 at high solid loading

Ethanol production by a recombinant bacterium from wheat straw (WS) at high solid loading by separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) was studied. The yield of total sugars from dilute acid pretreated WS (150 g/L) after enzymatic saccharification was 86.3 ± 1.5 g/L. The pretreated WS was bio-abated by growing a fungal strain aerobically in the liquid portion for 16 h. The recombinant Escherichia coli strain FBR5 produced 41.1 ± 1.1 g ethanol/L from non-abated WS hydrolyzate (total sugars, 86.6 ± 0.3 g/L) in 168 h at pH 7.0 and 35 °C. The bacterium produced 41.8 ± 0.0 g ethanol/L in 120 h from the bioabated WS by SHF. It produced 41.6 ± 0.7 g ethanol/L in 120 h from bioabated WS by fed-batch SSF. This is the first report of the production of above 4% ethanol from a lignocellulosic hydrolyzate by the recombinant bacterium.     

Development of a serial bioreactor system for direct ethanol production from starch using<Emphasis Type="Italic">Aspergillus niger</Emphasis> and<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Aspergillus niger hyphae were found to grow with unliquefied potato starch under aerobic conditions, but did not grow under anaerobic conditions. The raw culture ofA. niger catalyzed saccharification of potato starch to glucose, producing approximately 12 g glucose/L/day/ The extracellular enzyme activity was decreased in proportion to incubation time, and approximately 64% of initial activity was maintained after 3 days. At 50°C,A. niger hyphae growth stopped, while the extracellular enzyme activity peaked. On the basis of theA. niger growth property and enzyme activity, we designed a serial bioreactor system composed of four different reactors. Fungal hyphae were cultivated in reactor I at 30°C, uniquefied starch was saccharified to glycose by a fungal hyphae culture in reactors II and III at 50°C, and glucose was fermented to ethanol bySaccharomyces cerevisiae in reactor IV. The total glucose produced by fungal hyphae in reactor I and saccharification in reactor II was about 42 g/L/day. Ethanol production in reactor IV was approximately 22 g/L/day, which corresponds to about 79% of the theoretical maximum produced from 55 g starch/L/day.     

<Emphasis Type="Italic">Pichia pastoris</Emphasis> is a valuable host for the expression of genes encoding membrane proteins from the hyperthermophilic Archeon <Emphasis Type="Italic">Pyrococcus abyssi</Emphasis>

We present here the experimental strategies, first results and identified bottlenecks of a structural genomics initiative on membrane proteins of the hyperthermophilic archaea Pyrococcus abyssi. Five ORFs coding for putative membrane proteins have been cloned and expressed in the methylotrophic Pichia pastoris expression system, using two different constructs, with or without the signal sequence α-mating factor of Saccharomyces cerevisiae. A c-myc epitope and 6 His codons were added at the 3′-end of the targeted genes to allow immunodetection of the recombinant proteins and to facilitate their further purification. We have selected at least one producer clone for each protein of interest and for almost every construction. All the membrane proteins were produced in Erlenmeyer flasks culture and in fed-batch cultivation for large-scale preparation. The proteins were detected in the membrane fractions of P. pastoris. Production efficiencies were relatively low in both production conditions but the quantities of biomass obtained during fed-batch cultivation have allowed us to collect sufficient amount of material for further purification. The proteins were extracted, solubilized and partially purified. Large-scale purification will be necessary for further structural work.     

Production of recombinant β-galactosidase in bioreactors by fed-batch culture using DO-stat and linear control

β-Galactosidase enzymes continue to play an important role in food and pharmaceutical industries. These enzymes hydrolyze lactose in its constituent monosaccharides, glucose and galactose. The industrial use of enzymes presents an increase in process costs reflecting in higher final product value. An alternative to enhance processes’ productivity and yield would be the use of recombinant enzymes and their large-scale fed-batch production. The overexpression of recombinant β-galactosidase from Kluyveromyces sp. was carried out in 2-L bioreactors using Escherichia coli strain BL21 (DE3) as host. Effect of induction time on recombinant enzyme expression was studied by adding 1?mM isopropyl thiogalactoside (IPTG) at 12?h, 18?h and 24?h of cultivation. Glucose feeding strategies were compared employing feedback-controlled DO-stat and ascendant linear pump feeding in bioreactor fed-batch cultivations. Linear feeding strategy with IPTG addition at 18?h of cultivation resulted in approximately 20?g/L and 17,745?U/L of biomass and β-galactosidase activity, respectively. On the other hand, although the feedback-controlled DO-stat feeding strategy induced at 12?h of cultivation led to lower final biomass of 18?g/L, it presented an approximately 2.5 increase in enzymatic activity, resulting in 42,367?U/L, and most importantly it led to the most prominent specific enzymatic activity of approximately 40?U/mg_protein. Comparing to previous results, these results suggest that the DO-stat feeding is a promising strategy for recombinant β-galactosidase enzyme production.     

Effect of mixed organic substrate on α-tocopherol production by <Emphasis Type="Italic">Euglena gracilis</Emphasis> in photoheterotrophic culture

Effects of organic carbon sources on cell growth and alpha-tocopherol productivity in wild and chloroplast-deficient W14ZUL strains of Euglena gracilis under photoheterotrophic culture were investigated. In both strains, the increase in cell growth was particularly high when glucose was added as the sole organic carbon source. On the other hand, alpha-tocopherol production per dry cell weight was enhanced by adding ethanol. Ethanol addition also increased the chlorophyll concentration in wild strain and mitochondria activity in W14ZUL strain. For effective alpha-tocopherol production, the effects of mixture of glucose and ethanol were investigated. The results showed that, when a mixture of glucose (6 g/l) and ethanol (4 g/l) was used, alpha-tocopherol productivity per culture broth was 3.89 x 10(-2) mg l(-1) h(-1), which was higher than the value obtained without addition of organic carbon source (0.92 x 10(-2) mg l(-1) h(-1)). In addition, under fed-batch cultivation using an internally illuminated photobioreactor, the alpha-tocopherol production per culture broth was 23.43 mg/l, giving a productivity of 16.27 x 10(-2) mg l(-1) h(-1).     

Citric Acid Production from Hydrolysate of Pretreated Straw Cellulose by Yarrowia lipolytica SWJ-1b Using Batch and Fed-Batch Cultivation

In this study, crude cellulase produced by Trichoderma reesei Rut-30 was used to hydrolyze pretreated straw. After the compositions of the hydrolysate of pretreated straw were optimized, the study showed that natural components of pretreated straw without addition of any other components such as (NH₄)₂SO₄, KH₂PO₄, or Mg²⁺ were suitable for citric acid production by Yarrowia lipolytica SWJ-1b, and the optimal ventilatory capacity was 10.0 L/min/L medium. Batch and fed-batch production of citric acid from the hydrolysate of pretreated straw by Yarrowia lipolytica SWJ-1b has been investigated. In the batch cultivation, 25.4 g/L and 26.7 g/L citric acid were yields from glucose and hydrolysate of straw cellulose, respectively, while the cultivation time was 120 hr. In the three-cycle fed-batch cultivation, citric acid (CA) production was increased to 42.4 g/L and the cultivation time was extended to 240 hr. However, iso-citric acid (ICA) yield in fed-batch cultivation (4.0 g/L) was similar to that during the batch cultivation (3.9 g/L), and only 1.6 g/L of reducing sugar was left in the medium at the end of fed-batch cultivation, suggesting that most of the added carbon was used in the cultivation.     

DO-stat fed-batch production of 2-keto-d-gluconic acid from cassava using immobilized Pseudomonas aeruginosa

Bioconversion of cassava-derived glucose to 2-keto-d-gluconic acid (2-KDG) using resting cells of immobilized Pseudomonas aeruginosa IFO 3448 was investigated. The tuberous roots of cassava were selected as the feedstock as they are inexpensive and widely available, and possess high amounts of starch (approximately 70% (w/w) of dry mass). Immobilized bacteria was used in a fed-batch fermenter and recycled over a period of 2 weeks. Given that the formation of 2-KDG from glucose requires oxygen as a reagent, and that high glucose concentrations are detrimental to the production yield of 2-KDG by resting cells, a DO-stat control strategy was used, whereby the feed rate of cassava hydrolysate was regulated by coupling it with the control variable, dissolved oxygen. For 319 h of operation including three cycles of repeated fed batch, 72 g of 2-KDG was produced from hydrolysate derived from 110 g of dried cassava at a maximum production rate of 0.55 g/L/h and an average concentration of 35 g/L.