Production of green biodegradable plastics of poly(3-hydroxybutyrate) from renewable resources of agricultural residues

This work describes potential opportunities for utilization of agro-industrial residues to produce green biodegradable plastics of poly(3-hydroxybutyrate) (PHB). Wheat straws were examined with good efficacy of carbon substrates using Cupriavidus necator. Production was examined in separate hydrolysis and fermentation (SHF) in the presence and absence of WS hydrolysis enzymes, and in simultaneous saccharification and fermentation (SSF) with enzymes. Results showed that production of PHB in SSF was more efficient in terms of viable cell count, cell dry weight, and PHB production and yield compared to those of SHF and glucose-control cultures. While glucose control experiment produced 4.6 g/L PHB; SSF produced 10.0 g/L compared to 7.1 g/L in SHF when utilizing enzymes during WS hydrolysis. Results showed that most of sugars produced during the hydrolysis were consumed in SHF (~98 %) compared to 89.2 % in SSF. Results also demonstrated that a combination of glucose and xylose can compensate for the excess carbon required for enhancing PHB production by C. necator. However, higher concentration of sugars at the beginning of fermentation in SHF can lead to cell inhibition and consequently catabolite repressions. Accordingly, results demonstrated that the gradual release of sugars in SSF enhanced PHB production. Moreover, the presence of sugars other than glucose and xylose can eliminate PHB degradation in medium of low carbon substrate concentrations in SSF.     

Enhancing polyhydroxybutyrate production from high cell density fed-batch fermentation of Bacillus megaterium BA-019

This study demonstrated the improved polyhydroxybutyrate (PHB) production via high cell density cultivation of Bacillus megaterium BA-019 with balanced initial total sugar concentration and carbon to nitrogen (C/N) weight ratio. In the 10 L stirred fermentor operated at 30 °C, pH 7.0, 600 rpm, and 1.0 vvm air, with the initial total sugar concentration of 60 g/L and urea at the C/N weight ratio of 10:1, 32.48 g/L cell biomass with the corresponding PHB weight content of 26.94 % and volumetric productivity of 0.73 g/L h were obtained from batch cultivation. Continuing cultivation by intermittent feeding of the sugarcane molasses along with urea at the C/N weight ratio of 12.5:1 gave much improved biomass and PHB production (90.71 g/L biomass with 45.84 % PHB content and 1.73 g/L h PHB productivity). Similar biomass and PHB yields were obtained in the 90 L stirred fermentor when using the impeller tip speed as the scale-up criterion.     

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.     

Process optimization and production of polyhydroxybutyrate using palm jaggery as economical carbon source by marine sponge-associated Bacillus licheniformis MSBN12

The Polyhydroxybutyrate (PHB) producer, Bacillus licheniformis MSBN12 was isolated from the marine sponge Callyspongia diffusa. The PHB production of B. licheniformis MSBN12 was optimized using a four-factor Box-Behnken design to find the interactive effects of variables such as palm jaggery, wheat bran, seawater, and incubation temperature. The maximum yield of PHB (6.38 g/L) was achieved through response surface methodology-based optimization and the optimized conditions were further used for the batch and fed-batch fermentation. Maximum biomass was reached at 48 and 36 h of incubation with PHB accumulation of 62.91 and 67.16 % (w/w of dry cells) for batch and fed-batch process. The production of PHB under fed-batch process with B. licheniformis MSBN12 was increased threefold over shake flask culture when palm jaggery as sole carbon source. The ¹H NMR data was extrapolated with peaks of the PHB reference standard and confirmed as PHB analog.     

Comparative study on the production of poly(3-hydroxybutyrate) by thermophilic Chelatococcus daeguensis TAD1: a good candidate for large-scale production

In spite of numerous advantages on operating fermentation at elevated temperatures, very few thermophilic bacteria with polyhydroxyalkanoates (PHAs)-accumulating ability have yet been found in contrast to the tremendous mesophiles with the same ability. In this study, a thermophilic poly(3-hydroxybutyrate) (PHB)-accumulating bacteria (Chelatococcus daeguensis TAD1), isolated from the biofilm of a biotrickling filter used for NOx removal, was extensively investigated and compared to other PHB-accumulating bacteria. The results demonstrate that C. daeguensis TAD1 is a growth-associated PHB-accumulating bacterium without obvious nutrient limitation, which was capable of accumulating PHB up to 83.6 % of cell dry weight (CDW, w/w) within just 24 h at 45 °C from glucose. Surprisingly, the PHB production of C. daeguensis TAD1 exhibited strong tolerance to high heat stress as well as nitrogen loads compared to that of other PHB-accumulating bacterium, while the optimal PHB amount (3.44?±?0.3 g l^?1) occurred at 50 °C and C/N?=?30 (molar) with glucose as the sole carbon source. In addition, C. daeguensis TAD1 could effectively utilize various cheap substrates (starch or glycerol) for PHB production without pre-hydrolyzed, particularly the glycerol, exhibiting the highest product yield (Y _P/S, 0.26 g PHB per gram substrate used) as well as PHB content (80.4 % of CDW, w/w) compared to other carbon sources. Consequently, C. daeguensis TAD1 is a viable candidate for large-scale production of PHB via utilizing starch or glycerol as the raw materials.     

Production of poly(3-hydroxybutyric acid) by fed-batch culture of Alcaligenes eutrophus with glucose concentration control

Alcaligenes eutrophus NCIMB 11599 was cultivated to produce poly(3-hydroxybutyric acid) (PHB) from glucose by the automatic fed-batch culture technique. The glucose concentration of the culture broth was controlled at 10 to 20 g/L by two methods: using exit gas data obtained from a mass spectrometer and using an on-line glucose analyzer. The effect of ammonium limitation on PHB synthesis at different culture phases was studied. The final cell concentration, PHB concentration, and PHB productivity increased as ammonia feeding was stopped at a higher cell concentration. High concentrations of PHB (121 g/L) and total cells (164 g/L) were obtained in 50 h when ammonia feeding was stopped at the cell concentration of 70 g/L. The maximum PHB content reached 76% of dry cell weight and the productivity was 2.42 g/L h with the yield of 0.3 g PHB/g glucose.     

Enhanced isopropanol and <Emphasis Type="Italic">n</Emphasis>-butanol production by supplying exogenous acetic acid via co-culturing two clostridium strains from cassava bagasse hydrolysate

The focus of this study was to produce isopropanol and butanol (IB) from dilute sulfuric acid treated cassava bagasse hydrolysate (SACBH), and improve IB production by co-culturing Clostridium beijerinckii (C. beijerinckii) with Clostridium tyrobutyricum (C. tyrobutyricum) in an immobilized-cell fermentation system. Concentrated SACBH could be converted to solvents efficiently by immobilized pure culture of C. beijerinckii. Considerable solvent concentrations of 6.19 g/L isopropanol and 12.32 g/L butanol were obtained from batch fermentation, and the total solvent yield and volumetric productivity were 0.42 g/g and 0.30 g/L/h, respectively. Furthermore, the concentrations of isopropanol and butanol increased to 7.63 and 13.26 g/L, respectively, under the immobilized co-culture conditions when concentrated SACBH was used as the carbon source. The concentrations of isopropanol and butanol from the immobilized co-culture fermentation were, respectively, 42.62 and 25.45 % higher than the production resulting from pure culture fermentation. The total solvent yield and volumetric productivity increased to 0.51 g/g and 0.44 g/L/h when co-culture conditions were utilized. Our results indicated that SACBH could be used as an economically favorable carbon source or substrate for IB production using immobilized fermentation. Additionally, IB production could be significantly improved by co-culture immobilization, which provides extracellular acetic acid to C. beijerinckii from C. tyrobutyricum. This study provided a technically feasible and cost-efficient way for IB production using cassava bagasse, which may be suitable for industrial solvent production.     

Utilization of by-products derived from bioethanol production process for cost-effective production of lactic acid

The by-products of bioethanol production such as thin stillage (TS) and condensed distillers solubles (CDS) were used as a potential nitrogen source for economical production of lactic acid. The effect of those by-products and their concentrations on lactic acid fermentation were investigated using Lactobacillus paracasei CHB2121. Approximately, 6.7 g/L of yeast extract at a carbon source to nitrogen source ratio of 15 was required to produce 90 g/L of lactic acid in the medium containing 100 g/L of glucose. Batch fermentation of TS medium resulted in 90 g/L of lactic acid after 48 h, and the medium containing 10 % CDS resulted in 95 g/L of lactic acid after 44 h. Therefore, TS and CDS could be considered as potential alternative fermentation medium for the economical production of lactic acid. Furthermore, lactic acid fermentation was performed using only cassava and CDS for commercial production of lactic acid. The volumetric productivity of lactic acid [2.94 g/(L·h)] was 37 % higher than the productivity obtained from the medium with glucose and CDS.     

Production of poly(3-hydroxybutyrate) by high cell density fed-batch culture of Alcaligenes eutrophus with phospate limitation

High cell density fed-batch fermentation of Alcaligenes eutrophus was carried out for the production of poly(3-hydroxybutyrate) (PHB) in a 60-L fermentor. During the fermentation, pH was controlled with NH(4)OH solution and PHB accumulation was induced by phosphate limitation instead of nitrogen limitation. The glucose feeding was controlled by monitoring dissolved oxygen (DO) concentration and glucose concentration in the culture broth. The glucose concentration fluctuated within the range of 0-20 g/L. We have investigated the effect of initial phosphate concentration on the PHB production when the initial volume was fixed. Using an initial phosphate concentration of 5.5 g/L, the fed-batch fermentation resulted in a final cell concentration of 281 g/L, a PHB concentration of 232 g/L, and a PHB productivity of 3.14 g/L . h, which are the highest values ever reported to date. In this case, PHB content, cell yield from glucose, and PHB yield from glucose were 80, 0.46, and 0.38% (w/w), respectively. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 28-32, 1997.     

Ethanol production from galactose by a newly isolated Saccharomyces cerevisiae KL17

A wild-type yeast strain with a good galactose-utilization efficiency was newly isolated from the soil, and identified and named Saccharomyces cerevisiae KL17 by 18s RNA sequencing. Its performance of producing ethanol from galactose was investigated in flask cultures with media containing various combination and concentrations of galactose and glucose. When the initial galactose concentration was 20 g/L, it showed 2.2 g/L/h of substrate consumption rate and 0.63 g/L/h of ethanol productivity. Although they were about 70 % of those with glucose, such performance of S. cerevisiae KL17 with galactose was considered to be quite high compared with other strains reported to date. Its additional merit was that its galactose metabolism was not repressed by the existence of glucose. Its capability of ethanol production under a high ethanol concentration was demonstrated by fed-batch fermentation in a bioreactor. A high ethanol productivity of 3.03 g/L/h was obtained with an ethanol concentration and yield of 95 and 0.39 g/L, respectively, when the cells were pre-cultured on glucose. When the cells were pre-cultured on galactose instead of glucose, fermentation time could be reduced significantly, resulting in an improved ethanol productivity of 3.46 g/L/h. The inhibitory effects of two major impurities in a crude galactose solution obtained from acid hydrolysis of galactan were assessed. Only 5-Hydroxymethylfurfural (5-HMF) significantly inhibited ethanol fermentation, while levulinic acid (LA) was benign in the range up to 10 g/L.     

Effect of caffeine concentration on biomass production, caffeine degradation, and morphology of Aspergillus tamarii

The aim of the present study was to evaluate the effect of the initial caffeine concentration (1–8 g/L) on growth and caffeine consumption by Aspergillus tamarii as well as pellet morphology, in submerged fermentation. Caffeine was used as sole nitrogen source. At 1 g/L of initial caffeine concentration, caffeine degradation was not affected, resulting in a production of 8.7 g/L of biomass. The highest biomass production (12.4–14.8 g/L) was observed within a range of 2 to 4 g/L of initial caffeine concentration. At these initial caffeine concentrations, after 96 h of fermentation, 41–51 % of the initial caffeine was degraded. Using an initial caffeine concentration of 2–3 g/L, the highest specific growth rate was observed (μ?=?0.069 1/h). Biomass production decreased at 8 g/L of initial caffeine concentration. A. tamarii formed mainly pellets at all concentrations tested. The size of the pellet decreased at a caffeine concentration of 8 g/L.     

Novel extremely acidic lipases produced from Bacillus species using oil substrates

The extremely acidophilic microorganisms Bacillus pumilus and Bacillus subtilis were isolated from soil collected from the commercial edible oil and fish oil extraction industry. Optimization of conditions for acidic lipase production from B. pumilus and B. subtilis using palm oil and fish oil, respectively, was carried out using response surface methodology. The extremely acidic lipases, thermo-tolerant acidic lipase (TAL) and acidic lipase (AL), were produced by B. pumilus and B. subtilis, respectively. The optimum conditions for B. pumilus obtaining the maximum activity (1,100 U/mL) of TAL were fermentation time, 96 h; pH, 1; temperature, 50 °C; concentration of palm oil, 50 g/L. After purification, a 7.1-fold purity of lipase with specific activity of 5,173 U/mg protein was obtained. The molecular weight of the TAL was 55 kDa. The AL from B. subtilis activity was 214 U/mL at a fermentation time of 72 h; pH, 1; temperature, 35 °C; concentration of fish oil, 30 g/L; maltose concentration, 10 g/L. After purification, an 11.4-fold purity of lipase with specific activity of 2,189 U/mg protein was obtained. The molecular weight of the extremely acidic lipase was 22 kDa. The functional groups of lipases were determined by Fourier transform-infrared (FT-IR) spectroscopy.     

Cloning of <Emphasis Type="Italic">phaCAB</Emphasis> genes from thermophilic <Emphasis Type="Italic">Caldimonas manganoxidans</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis> for poly(3-hydroxybutyrate) (PHB) production

PHB biosynthesis pathway, consisting of three open reading frames (ORFs) that encode for β-ketothiolase (phaA _Cma , 1179 bp), acetoacetyl-CoA reductase (phaB _Cma , 738 bp), and PHA synthase (phaC _Cma , 1694 bp), of Caldimonas manganoxidans was identified. The functions of PhaA, PhaB, and PhaC were demonstrated by successfully reconstructing PHB biosynthesis pathway of C. manganoxidans in Escherichia coli, where PHB production was confirmed by OD₆₀₀, gas chromatography, Nile blue stain, and transmission electron microscope (TEM). The protein sequence alignment of PHB synthases revealed that phaC _Cma shares at least 60% identity with those of class I PHB synthase. The effects of PhaA, PhaB, and PhaC expression levels on PHB production were investigated. While the overexpression of PhaB is found to be important in recombinant E. coli, performances of PHB production can be quantified as follows: PHB concentration of 16.8 ± 0.6 g/L, yield of 0.28 g/g glucose, content of 74%, productivity of 0.28 g/L/h, and Mw of 1.41 MDa.     

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.     

Arachidonic acid-rich oil production by Mortierella alpina with different gas distributors

Arachidonic acid (ARA)-rich oil production by Mortierella alpina is a high oxygen demand and shear-sensitive process. In the aerobic fermentation process, oxygen supply is usually a limiting factor owing to the low solubility of oxygen in the fermentation broth. Two kinds of perforated ring gas distributors and a novel microporous ceramic membrane gas distributor were designed and applied to improve oxygen supply. With the decrease of the orifice diameter of perforated ring gas distributors, dry cell weight (DCW), lipids concentration, and ARA content in total fatty acid increased from 17.86 g/L, 7.08 g/L, and 28.08 % to 25.67 g/L, 11.94 g/L, and 36.99 %, respectively. Furthermore, the effect of different dissolved oxygen (DO) on ARA-rich oil production with membrane gas distributor was also studied. The maximum DCW, lipid concentration, and ARA content using membrane gas distributor with DO controlled at 40 % reached 29.67 g/L, 16.74 g/L, and 49.53 %, respectively. The ARA titer increased from 1.99 to 8.29 g/L using the membrane gas distributor to substitute the perforated ring gas distributor. In the further experiment, a novel tubular titanium metal membrane gas distributor was successfully applied in a 7,000 L bioreactor and the results demonstrated that membrane gas distributor was industrially practical.     

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.     

In silico aided metabolic engineering of Klebsiella oxytoca and fermentation optimization for enhanced 2,3-butanediol production

Klebsiella oxytoca naturally produces a large amount of 2,3-butanediol (2,3-BD), a promising bulk chemical with wide industrial applications, along with various byproducts. In this study, the in silico gene knockout simulation of K. oxytoca was carried out for 2,3-BD overproduction by inhibiting the formation of byproducts. The knockouts of ldhA and pflB genes were targeted with the criteria of maximization of 2,3-BD production and minimization of byproducts formation. The constructed K. oxytoca ΔldhA ΔpflB strain showed higher 2,3-BD yields and higher final concentrations than those obtained from the wild-type and ΔldhA strains. However, the simultaneous deletion of both genes caused about a 50 % reduction in 2,3-BD productivity compared with K. oxytoca ΔldhA strain. Based on previous studies and in silico investigation that the agitation speed during 2,3-BD fermentation strongly affected cell growth and 2,3-BD synthesis, the effect of agitation speed on 2,3-BD production was investigated from 150 to 450 rpm in 5-L bioreactors containing 3-L culture media. The highest 2,3-BD productivity (2.7 g/L/h) was obtained at 450 rpm in batch fermentation. Considering the inhibition of acetoin for 2,3-BD production, fed-batch fermentations were performed using K. oxytoca ΔldhA ΔpflB strain to enhance 2,3-BD production. Altering the agitation speed from 450 to 350 rpm at nearly 10 g/L of acetoin during the fed-batch fermentation allowed for the production of 113 g/L 2,3-BD, with a yield of 0.45 g/g, and for the production of 2.1 g/L/h of 2,3-BD.     

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.     

Improvement of kojic acid production in Aspergillus oryzae B008 mutant strain and its uses in fermentation of concentrated corn stalk hydrolysate

A strain designated M866, producing kojic acid with a high yield, was obtained by combining induced mutation using ion beam implantation and ethyl methane sulfonate treatment of a wild type strain of Aspergillus oryzae B008. The amount of kojic acid produced by the strain M866 in a shaking flask was 40.2 g/L from 100 g/L of glucose, which was 1.7 times higher than that produced by wild strain (23.58 g/L). When the mixture of glucose and xylose was used as carbon source, the resulting kojic acid production was raised with the increasing of glucose ratios in the mixture. With concentrations of glucose at 75 g/L and xylose at 25 g/L mixed in the medium, the production of kojic acid reached 90.8 %, which was slightly lower than with glucose as the sole source of carbon. In addition, the kojic acid fermentation of the concentrated hydrolysate from corn stalk was also investigated in this study, the maximum concentration of kojic acid accumulated at the end of the fermentation was 33.1 g/L and this represents the yield based on reducing sugar consumed and the overall productivity of 0.36 g/g and 0.17 g/L/h, respectively.