Enhanced 2,3-butanediol production in fed-batch cultures of free and immobilized Bacillus licheniformis DSM 8785

2,3-Butanediol (2,3-BD) is a valuable bulk chemical with particular use in industry. 2,3-BD has a potential as solvent and fuel additive, as carrier for pharmaceuticals, or as feedstock for the production of synthetic rubber. Until now, the highest 2,3-BD concentrations were obtained with risk group 2 microorganisms (e.g., Klebsiella oxytoca). In this study, the nonpathogenic bacterium Bacillus licheniformis DSM 8785 was used for 2,3-BD production from glucose. In batch experiments, a maximum 2,3-BD concentration of 72.6 g/L was reached from 180 g/L glucose after 86 h. The yield was 0.42 g/g glucose and the productivity was 0.86 g/(L h). During fed-batch cultivation, 2,3-BD production could be increased up to 144.7 g/L, with a productivity of 1.14 g/(L h). Additionally, repeated batch/fed-batch experiments were conducted using immobilized B. licheniformis in the form of LentiKats®. Results showed a high activity and stability of the immobilizates even after multiple medium replacements, as well as 2,3-BD concentrations, yields, and productivities similar to those obtained with free cells. To our knowledge, these results show the highest 2,3-BD concentration reported so far using a risk group 1 microorganism in general and B. licheniformis in particular. Furthermore, productivity lies in the same range with data reported from risk group 2 strains, which makes B. licheniformis DSM 8785 a suitable candidate for large-scale fermentation processes.     

Integrated production of lactic acid and biomass on distillery stillage

The possibilities of parallel lactic acid and biomass production in batch and fed-batch fermentation on distillery stillage from bioethanol production were studied. The highest lactic acid yield and productivity of 92.3 % and 1.49 g L^?1 h^?1 were achieved in batch fermentation with initial sugar concentration of 55 g L^?1. A significant improvement of the process was achieved in fed-batch fermentation where the concentration of lactic acid was increased to 47.6 % and volumetric productivity for 21 % over the batch process. A high number of Lactobacillus rhamnosus ATCC 7469 viable cells of 10⁹ CFU ml^?1 was attained at the end of fed-batch fermentation. The survival of 92.9 % of L. rhamnosus cells after 3 h of incubation at pH 2.5 validated that the fermentation media remained after lactic acid removal could be used as a biomass-enriched animal feed thus making an additional value to the process.     

A novel fed-batch based strategy for enhancing cell-density and recombinant cyprosin B production in bioreactors

Nowadays, the dairy industry is continuously looking for new and more efficient clotting enzymes to create innovative products. Cyprosin B is a plant aspartic protease characterized by clotting activity that was previously cloned in Saccharomyces cerevisiae BJ1991 strain. The production of recombinant cyprosin B by a batch and fed-batch culture was compared using glucose and galactose as carbon sources. The strategy for fed-batch cultivation involved two steps: in the first batch phase, the culture medium presented glucose 1 % (w/v) and galactose 0.5 % (w/v), while in the feed step the culture medium was constituted by 5 % (w/v) galactose with the aim to minimize the GAL7 promoter repression. Based on fed-batch, in comparison to batch growth, an increase in biomass (6.6-fold), protein concentration (59 %) and cyprosin B activity (91 %) was achieved. The recombinant cyprosin B was purified by a single hydrophobic chromatography, presenting a specific activity of 6 × 10⁴ U·mg^?1, corresponding to a purification degree of 12.5-fold and a recovery yield of 16.4 %. The SDS-PAGE analysis showed that recovery procedure is suitable for achieving the purified recombinant cyprosin B. The results show that the recombinant cyprosin B production can be improved based on two distinct steps during the fed-batch, presenting that this strategy, associated with a simplified purification procedure, could be applied to large-scale production, constituting a new and efficient alternative for animal and fungal enzymes widely used in cheese making.     

Improvement of mannitol production by Lactobacillus brevis mutant 3-A5 based on dual-stage pH control and fed-batch fermentations

Lactobacillus brevis 3-A5 was isolated and expected to produce mannitol efficiently by regulating pH in batch and fed-batch fermentations. In 48 h batch fermentations with free and constant pH, the optimal pH for cell growth and mannitol production in the first 24 h of incubation was 5.5, whereas that for mannitol production in the second 24 h of incubation was 4.5. To achieve high cell density and mannitol yield simultaneously, a dual-stage pH control strategy was proposed based on the kinetic analysis of mannitol production. The pH value was controlled at 5.5 for the first 12 h of fermentation and subsequently shifted to 4.5 until the fermentation was completed. Under dual-stage pH control fermentation, a 103 g/L yield of mannitol with a volumetric production rate of 3.7 g/L/h was achieved after 28 h. The dual-stage pH control fed-batch fermentation strategy was further developed to improve mannitol yield, wherein the yield increased by 109 % to 215 g/L after 98 h of fermentation. This value is the highest yield of mannitol ever reported using L. brevis.     

Upstream process optimization of polyhydroxybutyrate (PHB) by Alcaligenes latus using two-stage batch and fed-batch fermentation strategies

This research focused on optimizing the upstream process time for production of polyhydroxybutyrate (PHB) from sucrose by two-stage batch and fed-batch fermentation with Alcaligenes latus ATCC 29714. The study included selection of strain, two-stage batch fermentations with different time points for switching to nitrogen limited media (14, 16 or 18?h) and fed-batch fermentations with varied time points (similar to two stage) for introducing nitrogen limited media. The optimal strain to produce PHB using sucrose as carbon source was A. latus ATCC 29714 with maximum-specific growth rate of 0.38?±?0.01?h^?1 and doubling time of 1.80?±?0.05?h. Inducing nitrogen limitation at 16?h and ending second stage at 26?h gave optimal performance for PHB production, resulting in a PHB content of 46.7?±?12.2?% (g PHB per g dry cell weight) at the end of fermentation. This was significantly higher (P?≤?0.05) (approximately 7?%) than the corresponding fed batch run in which nitrogen limitation was initiated at 16?h.     

Poly(β-hydroxybutyric acid) thermoplastic production by Alcaligenes latus: Behavior of fed-batch cultures

Fed-batch culture of Alcaligenes latus, ATCC 29713, was investigated for producing the intracellular bioplastic poly(β–hydroxybutyric acid), PHB. Constant rate feeding, exponentially increasing feeding rate, and pH-stat fed batch methods were evaluated. pH-stat fed batch culture reduced or delayed accumulation of the substrate in the broth and led to significantly enhanced PHB productivity relative to the other modes of feeding. Presence of excessive substrate appeared to inhibit PHB synthesis, but not the production of cells. In fed-batch culture, the maximum specific growth rate (0.265?h^?1) greatly exceeded the value (0.075?h^?1) previously observed in batch culture of the same strain. Similarly, the maximum PHB production rate (up to 1.15?g?·?l^?1?·?h^?1) was nearly 8-fold greater than values observed in batch operations. Fed-batch operation was clearly superior to batch fermentation for producing PHB. A low growth rate was not a prerequisite for PHB accumulation, but a reduced or delayed accumulation of substrate appeared to enhance PHB accumulation. Under the best conditions, PHB constituted up to 63% of dry cell mass after 12?h of culture. The average biomass yield coefficient on sucrose was about 0.35, or a little less than in batch fermentations. The highest PHB concentrations attained were about 18?g?·?l^?1.     

Development of a novel solid-state fermentation strategy for the production of poly-3-hydroxybutyrate using polyurethane foams by Bacillus sphaericus NII 0838

The extensive use of synthetic plastics has caused serious waste disposal problems in our environment. Poly-3-hydroxybutyrates (PHB) are eco-friendly bacterial polyesters which are produced under unbalanced nutrient conditions. Few reports are available on PHB production by solid state fermentation (SSF). We have developed a novel SSF bioprocess in which polyurethane foam (PUF) is used as a physical inert support for the production of PHB by Bacillus sphaericus NII 0838. Media engineering for optimal PHB production was carried out using response surface methodology (RSM) adopting a Box–Behnken design. The factors optimized by RSM were inoculum size, pH and (NH₄)₂SO₄ concentration. Under optimized conditions—6.5 % inoculum size, 1.7 % (w/v) (NH₄)₂SO₄ and pH 9.0—PHB production and biomass were 0.169?±?0.03 and 0.4?±?0.002 g/g PUF, respectively. This is the first report on PHB production by SSF using PUF as an inert support. Our results demonstrate that SSF can be used as an alternative strategy for the production of PHB.     

Conversion of acid hydrolysate of oil palm empty fruit bunch to L-lactic acid by newly isolated Bacillus coagulans JI12

Cost-effective conversion of lignocellulose hydrolysate to optically pure lactic acid is commercially attractive but very challenging. Bacillus coagulans JI12 was isolated from natural environment and used to produce L-lactic acid (optical purity?>?99.5 %) from lignocellulose sugars and acid hydrolysate of oil palm empty fruit bunch (EFB) at 50 °C and pH 6.0 without sterilization of the medium. In fed-batch fermentation with 85 g/L initial xylose and 55 g/L xylose added after 7.5 h, 137.5 g/L lactic acid was produced with a yield of 98 % and a productivity of 4.4 g/L?h. In batch fermentation of a sugar mixture containing 8.5 % xylose, 1 % glucose, and 1 % L-arabinose, the lactic acid yield and productivity reached 98 % and 4.8 g/L?h, respectively. When EFB hydrolysate was used, 59.2 g/L of lactic acid was produced within 9.5 h at a yield of 97 % and a productivity of 6.2 g/L?h, which are the highest among those ever reported from lignocellulose hydrolysates. These results indicate that B. coagulans JI12 is a promising strain for industrial production of L-lactic acid from lignocellulose hydrolysate.     

Production of Polyhydroxyalkanoate Co-polymer by Bacillus thuringiensis

Integrative processes for the production of bioenergy and biopolymers are gaining importance in recent years as alternatives to fossil fuels and synthetic plastics. In the present study, Bacillus thuringiensis strain EGU45 has been used to generate hydrogen (H₂), polyhydroxybutyrate (PHB) and new co-polymers (NP). Under batch culture conditions with 250 ml synthetic media, B. thuringiensis EGU45 produced up to 0.58 mol H₂/mol of glucose. Effluent from the H₂ production stage was incubated under shaking conditions leading to the production of PHB up to 95 mg/l along with NP of levulinic acid up to 190 mg/l. A twofold to fourfold enhancement in PHB and up to 1.5 fold increase in NP yields was observed on synthetic medium (mixture of M-9+GM-2 medium in 1:1 ratio) containing at 1–2 % glucose concentration. The novelty of this work lies in developing modified physiological conditions, which induce bacterial culture to produce NP.     

Batch, fed-batch and repeated fed-batch fermentation processes of the marine thraustochytrid Schizochytrium sp. for producing docosahexaenoic acid

Different fermentation processes, including batch, fed-batch and repeated fed-batch processes by Schizochytrium sp., were studied and compared for the effective DHA-rich microbial lipids production. The comparison between different fermentation processes showed that fed-batch process was a more efficient cultivation strategy than the batch process. Among the four different feeding strategies, the glucose concentration feed-back feeding strategy had achieved the highest fermentation results of final cell dry weight, total lipids content, DHA content and DHA productivity of 72.37, 48.86, 18.38 g l^?1 and 138.8 mg l^?1 h^?1, respectively. The repeated fed-batch process had the advantages of reducing the time and cost for seed culture and inoculation between each fermentation cycles. The results of fermentation characteristics and lipid characterization of the repeated fed-batch process indicated that this repeated fed-batch process had promising industrialization prospect for the production of DHA-rich microbial lipids.     

Effect of nitrogen and/or oxygen concentration on poly(3-hydroxybutyrate) accumulation by <Emphasis Type="Italic">Halomonas boliviensis</Emphasis>

The behaviour of Halomonas boliviensis during growth in fed-batch culture under different kind of nutrient restrictions was examined. The metabolic switch between growth and accumulation phase is determined by the limitation in one or more essential nutrient for bacterial growth. The aim of this study was to test the effect of applying limitations of a essential nutrient, such as nitrogen, and the influence of different O₂ concentrations on poly(3-hydroxybutyrate) (PHB) production during the accumulation phase. Single limitations of nitrogen and oxygen provoke PHB accumulations of 45 and 37 % (g g^?1), respectively, while N limitation with low O₂ supply causes the highest PHB accumulation of 73 %. The characterization of the PHB production with the strain H. boliviensis would allow a better optimization of the process and enrich the knowledge about the PHB production from strains different than Cupriavidus necator.     

A Novel EPS-Producing Strain of Bacillus licheniformis Isolated from a Shallow Vent Off Panarea Island (Italy)

A haloalkaliphilic, thermophilic Bacillus strain (T14), isolated from a shallow hydrothermal vent of Panarea Island (Italy), produced a new exopolysaccharide (EPS). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain T14 was highly related (99 % similarity) to Bacillus licheniformis DSM 13^T and Bacillus sonorensis DSM 13779^T. Further DNA–DNA hybridization analysis revealed 79.40 % similarity with B. licheniformis DSM 13^T and 39.12 % with B. sonorensis DSM 13779^T. Sucrose (5 %) was the most efficient carbon source for growth and EPS production. The highest EPS production (366 mg l^?1) was yielded in fermenter culture at 300 rpm after 48 h of incubation. The purified fraction EPS1 contained fructose/fucose/glucose/galactosamine/mannose in a relative proportion of 1.0:0.75:0.28:tr:tr and possessed a molecular weight of 1,000 kDa displaying a trisaccharide unit constituted by sugars with a β-manno-pyranosidic configuration. Screening for biological activity showed anti-cytotoxic effect of EPS1 against Avarol in brine shrimp test, indicating a potential use in the development of novel drugs.     

Effect of phosphate supply and aeration on poly-β-hydroxybutyrate production in Azotobacter chroococcum

The effects of phosphate supply and aeration on cell growth and PHB accumulation were investigated in Azotobacter chroococcum 23 with the aim of increasing PHB production. Phosphate limitation favoured PHB formation in Azotobacter chroococcum 23, but inhibited growth. Azotobacter chroococcum 23 cells demonstrated intensive uptake of orthophosphate during exponential growth. At the highest phosphate concentration (1·5 g/litre) and low aeration the amount of intracellular orthophosphate/g residual biomass was highest. Under conditions of fed-batch fermentation the possibility of controlling the PHB production process by the phosphate level in the cultivation medium was demonstrated. A 36 h fed-batch fermentation resulted in a biomass yield of 110 g/litre with a PHB cellular concentration of 75% dry weight, PHB content 82·5 g/litre, PHB yield Y_P/S = 0·24 g/g and process productivity 2·29 g/litre·h.     

Application of enzymatic apple pomace hydrolysate to production of 2,3-butanediol by alkaliphilic <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> NCIMB 8059

2,3-Butanediol (2,3-BD) synthesis by a nonpathogenic bacterium Bacillus licheniformis NCIMB 8059 from enzymatic hydrolysate of depectinized apple pomace and its blend with glucose was studied. In shake flasks, the maximum diol concentration in fed-batch fermentations was 113 g/L (in 163 h, from the hydrolysate, feedings with glucose) while in batch processes it was around 27 g/L (in 32 h, from the hydrolysate and glucose blend). Fed-batch fermentations in the 0.75 and 30 L fermenters yielded 87.71 g/L 2,3-BD in 160 h, and 72.39 g/L 2,3-BD in 94 h, respectively (from the hydrolysate and glucose blend, feedings with glucose). The hydrolysate of apple pomace, which was for the first time used for microbial 2,3-BD production is not only a source of sugars but also essential minerals.     

Utilization of oil extracted from spent coffee grounds for sustainable production of polyhydroxyalkanoates

Spent coffee grounds (SCG), an important waste product of the coffee industry, contain approximately 15 wt% of coffee oil. The aim of this work was to investigate the utilization of oil extracted from SCG as a substrate for the production of poly(3-hydroxybutyrate) (PHB) by Cupriavidus necator H16. When compared to other waste/inexpensive oils, the utilization of coffee oil resulted in the highest biomass as well as PHB yields. Since the correlation of PHB yields and the acid value of oil indicated a positive effect of the presence of free fatty acids in oil on PHB production (correlation coefficient R ²?=?0.9058), superior properties of coffee oil can be probably attributed to the high content of free fatty acids which can be simply utilized by the bacteria culture. Employing the fed-batch mode of cultivation, the PHB yields, the PHB content in biomass, the volumetric productivity, and the Y _P/S yield coefficient reached 49.4 g/l, 89.1 wt%, 1.33 g/(l h), and 0.82 g per g of oil, respectively. SCG are annually produced worldwide in extensive amounts and are disposed as solid waste. Hence, the utilization of coffee oil extracted from SCG is likely to improve significantly the economic aspects of PHB production. Moreover, since oil extraction decreased the calorific value of SCG by only about 9 % (from 19.61 to 17.86 MJ/kg), residual SCG after oil extraction can be used as fuel to at least partially cover heat and energy demands of fermentation, which should even improve the economic feasibility of the process.     

Effects of carbon sources on growth and extracellular polysaccharide production of Nostoc flagelliforme under heterotrophic high-cell-density fed-batch cultures

Dissociated cells separated from a natural colony of Nostoc flagelliforme were cultivated heterotrophically in the darkness on glucose under fed-batch culture conditions. The effects of carbon sources (glucose, fructose, xylose, and sucrose) and concentrations on cell growth and extracellular polysaccharide (EPS) production were investigated. At harvest, the culture contained 1.325 g L^?1 of biomass and 117.2 mg L^?1 of EPS, respectively. The gravimetric EPS production rate was 16.7 mg g^?1 cell dry weight day^?1, which was 2.1 times higher than previously reported. Using sigmoid model, batch fermentation of N. flagelliforme was kinetically simulated to obtain equations including substrate consumption, biomass growth, and EPS accumulation. Results from a simulation correlated well with the experimental ones, indicating that this method could be useful in studying EPS production from batch and fed-batch cultures.     

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.     

Production of flavonoids and polysaccharide by adding elicitor in different cellular cultivation processes of Glycyrrhiza uralensis Fisch

In this study, callus and cell suspension were induced from seedlings of licorice (G. uralensis). In addition, it was revealed that the appropriate concentration of sucrose could promote the callus growth and increase the content of polysaccharide. The methyl jasmonate (MJ) and phenylalanine (PHE) could enhance the callus growth and content of flavonoids for G. uralensis. For producing more flavonoids and polysaccharide, two-stage cultivation was performed. In the first step, 30 g L^?1 sucrose was fed into a 5-L balloon-type bubble bioreactor on 8th day of culture to enhance cell production and metabolite production. In a two-stage cultivation process, PHE (2 mM) and MJ (5 mg L^?1) were added into a 5-L balloon-type bubble bioreactor after 10 days of culture. Using a fed-batch cultivation strategy (30 g L^?1 sucrose was fed into a 5-L balloon-type bubble bioreactor on 8th day), polysaccharide production was enhanced to 1.19 g L^?1, which was 2.12-fold greater than that in batch cultivation. The flavonoids yield (55.42 mg L^?1) which was about 22 % higher than that in batch cultivation was obtained on 21st day. In a two-stage cultivation process, the polysaccharide content was increased by 1.14- and 2.12-fold compared with fed-batch cultivation and batch cultivation on 15th day. Meanwhile, total flavonoids yield (132.36 mg L^?1) on 15th day, was increased by 2.26- and 2.67-fold compared with fed-batch cultivation and batch cultivation. In conclusion, two-stage cultivation process combined with the sucrose and elicitor treatment could promote both the callus growth and the secondary metabolites accumulation.     

Feeding strategies for the enhanced production of α-arbutin in the fed-batch fermentation of Xanthomonas maltophilia BT-112

To develop a cost-effective method for the enhanced production of α-arbutin using Xanthomonas maltophilia BT-112 as a biocatalyst, different fed-batch strategies such as constant feed rate fed-batch, constant hydroquinone (HQ) concentration fed-batch, exponential fed-batch and DO-control pulse fed-batch (DPFB) on α-arbutin production were investigated. The research results indicated that DPFB was an effective method for α-arbutin production. When fermentation with DO-control pulse feeding strategy to feed HQ and yeast extract was applied, the maximum concentrations of α-arbutin and cell dry weight were 61.7 and 4.21 g/L, respectively. The α-arbutin production was 394 % higher than that of the control (batch culture) and the molar conversion yield of α-arbutin reached 94.5 % based on the amount of HQ supplied (240 mM). Therefore, the results in this work provide an efficient and easily controlled method for industrial-scale production of α-arbutin.