Effect of process variables on supercritical fluid disruption of Ralstonia eutropha cells for poly(R-hydroxybutyrate) recovery

This research focuses on the disruption of the gram-negative bacterium Ralstonia eutropha cells by supercritical CO2 for poly(R-hydroxybutyrate) (PHB) recovery. The variables affecting cell disruption such as drying strategy, type of modifier, and cultivation time, as well as operating pressure, temperature, and repeated release of supercritical CO2 pressure, have been studied. Effect of this disruption technique on PHB molecular mass was also investigated. PHB recovery was examined using a combination of this method and chemical pretreatments. For salt pretreatment, the cells were exposed to 140 mM NaCl and heat (60 degrees C, 1 h). The cells were also exposed to 0.2-0.8% (w/w) NaOH to examine the effect of alkaline pretreatment. Bacterial cells treated in growth phase exhibited less resistance to disruption than nutrient-limited cells in the stationary phase. It was also found that the wet cells could be utilized to recover PHB, but purity of the product was lower than that obtained from freeze-dried cells. Pretreatment with a minimum of 0.4% (w/w) NaOH was necessary to enable complete disruption with two times pressure release. Salt pretreatment was less effective; however, disruption was improved by the application of alkaline shock. The proposed method is economic and comparable with other recovery methods in terms of the percentage of PHB recovery and energy consumption, while it is environmentally more benign.     

高温湿热酸法破壁提取法夫酵母胞内虾青素

法夫酵母是一种能积累虾青素的红酵母, 对其进行破壁是当前虾青素工业化提取生产的瓶颈工艺。研究在高温湿热条件下,低浓度盐酸对法夫酵母破壁而提取其胞内虾青素的工艺。探讨了不同破壁温度、盐酸浓度、液料比与破壁处理时间等因素对法夫酵母破壁后虾青素及类胡萝卜素提取率的影响, 确定了高温湿热酸法破壁提取虾青素的最佳条件为: 饱和蒸汽压力 0.1 MPa (121°C), 盐酸浓度0.5 mol/L, 液料比 (V/W)30 mL/g, 破壁时间2 min。在最佳条件下进行中试放大实验, 可得到虾青素与类胡萝卜素的提取率分别为: (84.8±3.2)%, (93.3±2)%。经优化后的新破壁工艺安全高效, 不会有毒性残留, 具有良好的工业应用前景。     

丝氨酸生产菌的筛选及静息细胞培养系统中L-丝氨酸的合成

从广西隆安县沼气池里的残渣中筛选到一株能以甲醇为唯一碳源生长的MB200菌株。根据常规形态特征、生理生化性状及16S rDNA基因序列分析将其鉴定为甲基杆菌属(M ethylobacteriumsp.)。其最佳生长条件为:温度32℃、pH值8.0、甲醇体积分数1.25%。建立了MB200生成L-丝氨酸的静息细胞培养系统。确定静息细胞培养的条件为:甘氨酸质量浓度为10 g.L-1,甲醇50 g.L-1,菌体质量浓度为30 g.L-1,pH8.9,于摇床250 r.m in-1,32℃静息培养48 h,L-丝氨酸产量为7.2 g.L-1。     

Mass production of poly-β-hydroxybutyric acid by fully automatic fed-batch culture of methylotroph

Summary Fifty-one methylotrophs were checked with respect to their ability of poly--hydroxybutyric acid (PHB) production from methanol. One of them, Pseudomonas sp. K, was chosen from its good growth on a minimum synthetic medium. Optimal temperature and pH for its growth were 30° C and 7.0, respectively. Concentrations of PO ₄ ^3- and NH ₄ ⁺ in the medium should be kept at low levels. PHB formation was stimulated by deficiency of nutrient such as NH ₄ ⁺ , SO ₄ ^2- , Mg²⁺, Fe²⁺ or Mn²⁺. Among them, nitrogen deficiency was chosen from its effectiveness and easiness for PHB accumulation.The microorganism was cultivated to produce a large amount of poly--hydroxybutyric acid (PHB) from methanol by means of microcomputer-aided fully automatic fed-batch culture technique. During the cultivation, temperature, dissolved oxygen concentration (DO), and methanol concentration in the culture broth were maintained at 30° C 2.5±0.5 ppm and 0.5±0.2 g/l, respectively. Other nutrients, nitrogen source and mineral ions, were also controlled to maintain their initial concentrations in the medium during cell growth phase. When the high cell concentration was achieved (160 g/l), feedings of ammonia and minerals were stopped and only methanol was supplied successively to accumulate PHB. At 175 h, high concentration of PHB (136 g/l) was obtained and total cell concentration became 206 g/l. DO must be maintained above the critical level during the PHB formation phase, too. PHB yield from methanol (g PHB/g methanol) was 0.18 and the maximum PHB content reached 66% of dry weight. Solid PHB produced by the strain had the melting point of 176° C and the average molecular weight of 3.0x10⁵.     

Extractive disruption process integration using ultrasonication and an aqueous two‐phase system for protein recovery from Chlorella sorokiniana

Microalgae emerge as the most promising protein sources for aquaculture industry. However, the commercial proteins production at low cost remains a challenge. The process of harnessing microalgal proteins involves several steps such as cell disruption, isolation and extraction. The discrete processes are generally complicated, time‐consuming and costly. To date, the notion of integrating microalgal cell disruption and proteins recovery process into one step is yet to explore. Hence, this study aimed to investigate the feasibility of applying methanol/potassium ATPS in the integrated process for proteins recovery from Chlorella sorokiniana. Parameters such as salt types, salt concentrations, methanol concentrations, NaCl addition were optimized. The possibility of upscaling and the effectiveness of recycling the phase components were also studied. The results showed that ATPS formed by 30% (w/w) K₃PO₄ and 20% (w/w) methanol with 3% (w/w) NaCl addition was optimum for proteins recovery. In this system, the partition coefficient and yield were 7.28 and 84.23%, respectively. There were no significant differences in the partition coefficient and yield when the integrated process was upscaled to 100‐fold. The recovered phase components can still be recycled effectively at fifth cycle. In conclusions, this method is simple, rapid, environmental friendly and could be implemented at large scale.     

Comparison of different solvents for extraction of polyhydroxybutyrate from Cupriavidus necator

Polyhydroxybutyrate (PHBs) have attracted much attention due to their biodegradability and biocompatibility properties. The main drawback to the commercial production of them is their high cost. The recovery of PHB from bacterial cytoplasm significantly increases total processing costs. Efficient, economical, and environment‐friendly extraction of PHB from cells is required for its industrial production. In the present study, several nonhalogenated organic solvents (ethylene carbonate, dimethyl sulfoxide, dimethyl formamide, hexane, propanol, methanol, and acetic acid) were examined for their efficacy regarding recovery at different temperatures from culture broth containing Cupriavidus necator cells. The highest recovery percentage (98.6%) and product purity (up to 98%) were seen to be those of ethylene carbonate‐assisted extraction at 150°C within 60 min of incubation time. Average molecular weight of the recovered PHB (1.3 × 10⁶) was not significantly affected by the extraction solvent and conditions. The melting point of PHB extracted using ethylene carbonate was measured to be 176.2°C with an enthalpy of fusion of 16.8% and the corresponding degree of crystallinity of 59.2%. NMR and GC analyses confirmed that the extracted biopolymer was PHB. The presented strategy can help researchers to reduce the cost to obtain the final product.     

An efficient method for the extraction of astaxanthin from the red yeast Xanthophyllomyces dendrorhous

This study investigated an efficient method for the extraction of astaxanthin from the red yeast Xanthophyllomyces dendrorhous. The extraction process comprised three steps: (1) cultivating the yeast; (2) treating the yeast culture suspension with microwaves to destroy the cell walls and microbodies; and (3) drying the yeast and extracting the astaxanthin pigment using ethanol, methanol, acetone, or a mixture of the three as the extraction solvent. Ultimately, various treatment tests were performed to determine the conditions for optimal pigment extraction, and the total carotenoid and astaxanthin contents were quantified. A frequency of 2,450 MHz, an output of 500 watts, and irradiation time of 60 s were the most optimum conditions for yeast cell wall destruction. Furthermore, optimal pigment extraction occurred when using a cell density of 10 g/l at 30 C over 24 h, with a 10% volume of ethanol.     

Preparation,statistical optimization and characterization of poly(3-hydroxybutyrate) fermented by Cupriavidus necator utilizing various hydrolysates of alligator weed (Alternanthera philoxeroides) as a sole carbon source

Alligator weed (Alternanthera philoxeroides) is a stoloniferous, amphibious and perennial herb which has invaded many parts of the world and led to serious environmental and ecological problems. In order to exploit cheap carbon source for poly(3-hydroxybutyrate) (PHB) production, alligator weed hydrolysates were prepared by acid and enzyme treatment and used for PHB production via Cupriavidus necator. The bacterium utilized alligator weed enzymatic hydrolysate and produced the PHB concentration of 3.8 ± 0.2 g/L at the conditions of pH 7.0, 27.5°C, 1.5 g/L of nitrogen source, and 25 g/L of carbon source, this exceeded the value of 2.1 ± 0.1 g/L from acid hydrolysate media at the same conditions. In order to obtain the optimum conditions of PHB production, response surface methodology was employed which improved PHB content. The optimum conditions for PHB production are as follows: carbon source, 34 g/L; nitrogen source, 2 g/L; pH, 7; temperature, 28°C. After 72 hr of incubation, the bacterium produced 8.5 g/L of dry cell weight and 4.8 g/L of PHB. The PHB was subjected to Fourier transform infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC), and Molecular weight analysis and found the melting temperature, number average molecular mass, and polydispersity were 168.20°C, 185 kDa, and 2.1, respectively.     

Selective extraction of free astaxanthin from Haematococcus culture using a tandem organic solvent system

A novel tandem solvent process of dodecane and methanol was developed for the selective extraction of free astaxanthin from red encysted Haematococcus culture. The process consists of dodecane extraction for astaxanthin mixture from the culture (stage 1) and methanol extraction for free astaxanthin from the dodecane extract (stage 2). In the first stage, astaxanthin mixture was directly extracted to dodecane from the culture broth without cell harvest process, followed by a rapid separation of the dodecane extract and the culture medium containing cell debris by simple settling. In the second stage, free astaxanthin was selectively collected to methanol from the dodecane extract, accompanied with saponification of astaxanthin-esters by the addition of NaOH to methanol. During saponification, use of the optimum NaOH concentration (0.02 M) and low temperature (4 degrees C) reaction minimized the degradation of free astaxanthin, resulting in a total recovery yield of free astaxanthin of over 85%. The free-astaxanthin-containing methanol extract was also simply separated from dodecane by gravity settling, after which the astaxanthin-free dodecane was effectively recycled to the first stage, yielding a stable extractability of astaxanthin mixture during repeated extraction. Our results indicate the potential of the proposed tandem solvent process as an alternative extraction technology for the high-value antioxidant Haematococcus astaxanthin.     

High-cell-density production of poly-β-hydroxybutyrate (PHB) from methanol by Methylobacterium extorquens: production of high-molecular-mass PHB

Methylobacterium extorquens ATCC 55366 was successfully cultivated at very high cell densities in a fed-batch fermentation system using methanol as a sole carbon and energy source and a completely minimal culture medium for the production of poly--hydroxybutyrate (PHB). Cell biomass levels were between 100 g/l and 115 g/l (dry weight) and cells contained between 40% and 46% PHB on a dry-weight basis. PHB with higher molecular mass values than previously reported for methylotrophic bacteria was obtained under certain conditions. Shake-flask and fermentor experiments showed the importance of adjusting the mineral composition of the medium for improved biomass production and higher growth rates. High-cell-density cultures were obtained without the need for oxygen-enriched air; once the oxygen transfer capacity of the fermentor was reached, methanol was thereafter added in proportion to the amount of available dissolved oxygen, thus preventing oxygen limitation. Controlling the methanol concentration at a very low level (less than 0.01 g/l), during the PHB production phase, led not only to prevention of oxygen limitation but also to the production of very high-molecular-mass PHB, in the 900–1800 kDa range. Biomass yields relative to the total methanol consumed were in the range 0.29–0.33 g/g, whereas PHB yields were in the range 0.09–0.12 g/g. During the active period of PHB synthesis, PHB yields relative to the total methanol consumed were between 0.2 g/g and 0.22 g/g. M. extorquens ATCC 55366 appears to be a promising organism for industrial PHB production.     

Recovery of poly-3-hydroxybutyrate from Alcaligenes eutrophus by surfactant–chelate aqueous system

A new method for the recovery of poly(3-hydroxybutyrate) (PHB) from Alcaligenes eutrophus was reported. This process involved the use of a surfactant–chelate aqueous solution. The key factors that influenced the purity, recovery rate and Mv of recovered PHB were investigated. The purity and recovery rate were determined by the amount of surfactant, the ratio of chelate to dry biomass, pH value, temperature and treatment time, whereas the Mv was affected by pH value and temperature. The optimal recovery conditions were a 0·12:1 surfactant-to-dry biomass ratio, a 0·08:1 chelate-to-dry biomass ratio, a pH value of 13, a 50°C temperature and a 10-min treatment time. Under such conditions, a purity of 98·7%, a recovery rate of 93·3% and a Mv of 316000 were obtained. The original Mv was 402000.     

Production of poly-β-hydroxybutyrate from methanol: characterization of a new isolate of Methylobacterium extorquens

Summary Poly--hydroxybutyric acid (PHB) and similar bacterial polyesters are promising candidates for the development of environment-friendly, totally biodegradable plastics. The use of methanol, one of the cheapest noble substrates available, may help to reduce the cost of producing such bioplastics. As a first step, a culture collection of 118 putative methylotrophic microorganisms was obtained from various soil samples without any laboratory enrichment step to favour culture diversity. The most promising culture was selected based on rapidity of growth and PHB accumulation and later identified as Methylobacterium extorquens. This isolate was obtained from soml contaminated regularly with used oil products for some 40 years. Concentrations of methanol greater than 8 g/l affected growth significantly and the methanol concentration was optimal at 1.7 g/l. PHB concentrations averaged 25–30% (w/v) of dry weight under non-optimized conditions. Controlling methanol concentration, using an open-loop configuration, led to biomass levels of 9–10 g/l containing 30–33% PHB while preventing methanol accumulation. The new isolate was also able to produce the co-polymer PHB/poly--hydroxyvalerate (PHV) using the mixture methanol + valerate. The PHV-to-PHB ratio was about 0.2 at the end of the fermentation. An average molecular mass varying between 2 and 3 × 10⁵ Da was obtained for three PHB samples using two different measurement methods.Publication number NRCC No. 33672 Offprint requests to: D. Groleau     

Growth and formation of poly (hydroxybutyric acid) by Methylobacterium rhodesianum at methanol concentrations of above 25 g/I

Methylobacterium rhodesianum MB 126 was cultivated using extended cultures without outflow. The feeding regime was based on the pH-regulated synchronous dosages of ammonia, methanol, phosphatc and trace elements according to supposed stoichiometric relations. The acidity of the culture medium was kept constant at pH 6.8, whereas the dissolved oxygen concentration was adjusted at 80% of saturation by autoregulation of the stirrer speed. However, besides testing technical conditions, two types of fermentations were discovered which are described in this paper. Firstly, although at the beginning of the bioprocesses the impeller speed increased up to 2,000 rpm, a decrease of dissolved oxygen down to zero was unavoidable. Secondly, methanol was accumulated temporarily up to 44 g/l and 26 g/l at 23 h of fermentation time and without inhibition of growth at least up to 30 g/l or PHB production. During this accumulation of the carbon substrate, exponential growth phases were detected showing growth rates of μ = 0.20/h and 0.21/h. But then, phases of retarded growth followed, whereas the methanol disappeared either continuously or after a steady level. In the course of a 54-h fermentation period, the synthesized PHB amounted to a content of above 50% of cell dry mass. From this data, a volumetric productivity of 0.4 g PHB/lxh was estimated. Moreover, the growth related yield coefficients were calculated to Y_X/MeOH = 0.21 and Y_X/MeOH = 0.14, whereas the product related yield coefficients amounted to Y_PHB/MeOH = 0.12 and Y_PHB/MeOH = 0,09. Since the shift down of growth rates as well as the production of PHB agreed in time with partial oxygen limitation (40% oxygen saturation), the competition observed between the tricarboxylic acid cycle and PHB synthesis was discussed. Summarizing the results, it was concluded that the frequently described inhibitory effect of methanol of above 2 g/l seems to be rather an effect of experimentally chosen conditions than of a general physiological phenomenon. Therefore, it could be demonstrated that the toxicity of methanol could be overcome if it was not dosed at different times but simultaneously with other medium components.     

Poly(3-Hydroxybutyrate) Production with High Productivity and High Polymer Content by a Fed-Batch Culture of Alcaligenes latus under Nitrogen Limitation

Alcaligenes latus has been known to produce poly(3-hydroxybutyrate) (PHB) in a growth-associated manner even under nutrient-sufficient conditions. However, the PHB content obtained by fed-batch culture was always low, at ca. 50%, which makes the recovery process inefficient. In this study, the effect of applying nitrogen limitation on the production of PHB by A. latus was examined. In flask and batch cultures, the PHB synthesis rate could be increased considerably by applying nitrogen limitation. The PHB content could be increased to 87% by applying nitrogen limitation in batch culture, which was considerably higher than that typically obtainable (50%) under nitrogen-sufficient conditions. In fed-batch culture, cells were first cultured by the DO-stat feeding strategy without applying nitrogen limitation. Nitrogen limitation was applied at a cell concentration of 76 g (dry cell weight)/liter, and the sucrose concentration was maintained within 5 to 20 g/liter. After 8 h of nitrogen limitation, the cell concentration, PHB concentration, and PHB content reached 111.7 g (dry cell weight)/liter, 98.7 g/liter, and 88%, respectively, resulting in a productivity of 4.94 g of PHB/liter/h. The highest PHB productivity, 5.13 g/liter/h, was obtained after 16 h.     

Recovery of polyhydroxybutyrate (PHB) from Cupriavidus necator biomass by solvent extraction with 1,2‐propylene carbonate

An integrated procedure for the recovery of polyhydroxybutyrate (PHB) produced by Cupriavidus necator based on the extraction with 1,2‐propylene carbonate was evaluated. The effect of temperature (100–145°C) and contact time (15–45 min), precipitation period, and biomass pretreatments (pH shock and/or thermal treatments) on PHB extraction efficiency and polymer properties was evaluated. The highest yield (95%) and purity (84%) were obtained with the combination of a temperature of 130°C and a contact time of 30 min, with a precipitation period of 48 h. Under these conditions, PHB had a molecular weight of 7.4×10⁵, which was the highest value obtained. Lower values (2.2×10⁵) were obtained for higher temperatures (145°C), while lower temperatures resulted in incomplete extraction yields (45–54%). No further yield improvement was achieved with the pH/heat pretreatments, but the polymer's molecular weight was increased to 1.3×10⁶. The PHB physical properties were not significantly affected by any of the tested procedures, as shown by the narrow ranges obtained for the glass transition temperature (4.8–5.0°C), melting temperature (170.1–180.1°C), melting enthalpy (77.8–88.5 J/g) and crystallinity (55–62%). 1,2‐Propylene carbonate was shown to be an efficient solvent for the extraction of PHB from biomass. The precipitation procedure was found to highly influence the polymer recovery and its molecular weight. Although polymer molecular weight and purity were improved by applying pH/heat pretreatment to the biomass, the procedure involves the use of large amounts of chemicals, which increases the recovery costs and makes the process environmentally unfriendly.     

Recovery of Poly-β-Hydroxybutyrate from Estuarine Microflora

Poly-β-hydroxybutyrate (PHB) is a uniquely procaryotic endogenous storage polymer whose metabolism has been shown to reflect environmental perturbations in laboratory monocultures. When hydrolyzed for 45 min in 5% sodium hypochlorite, PHB can be isolated from estuarine detrital microflora in high yield and purified free from non-PHB microbial components. Lyophilization of frozen estuarine samples shortens the exposure time to NaOCl necessary for maximal recovery. Lyophilized samples of hardwood leaves, Vallisneria, and the aerobic upper millimeter of estuarine muds yielded PHB. The efficiency of incorporation of sodium [1-¹⁴C]acetate into PHB is very high and is stimulated by aeration. PHB was not recovered from the anaerobic portions of sediments unless they were aerated for a short time. Levels of PHB in the detrital microbial community do not correlate with the microbial biomass as measured by the extractible lipid phosphate, suggesting that PHB-like eucaryotic endogenous storage materials may more accurately reflect the metabolic status of the population than its biomass.     

Simplified two-stage method to B-phycoerythrin recovery from Porphyridium cruentum

A simplified two-stage method for B-phycoerythrin (BPE) recovery from Porphyridium cruentum was developed. The proposed method involved cell disruption by sonication and primary recovery by aqueous two-phase partition. The evaluation of two different methods of cell disruption and the effect of increasing concentration of cell homogenate from P. cruentum culture upon aqueous two-phase systems (ATPS) performance was carried out to avoid the use of precipitation stages. Cell disruption by sonication proved to be superior over manual maceration since a five time increase in the concentration of B-phycoerythrin release was achieved. An increase in the concentration of crude extract from disrupted P. cruentum cells loaded to the ATPS (from 10 to 40%, w/w) proved to be suitable to increase the product purity and benefited the processing of highly concentrated disrupted extract. Kinetics studies of phase separation performed suggested the use of batch settlers with height/diameter (H/D) ratio less than one to reduce the necessary time for the phases to separate. The proposed ATPS stage comprising of 29% (w/w) polyethylene glycol (PEG) 1000g/mol, 9% (w/w) potassium phosphate, tie-line length (TLL) of 45% (w/w), volume ratio (V(R)) of 4.5, pH 7.0 and 40% (w/w) crude extract loaded in a batch settler with H/D ratio of 0.5 proved to be efficient for the recovery of 90% of B-phycoerythrin at the top PEG-rich phase. The purity of B-phycoerythrin increased up to 4.0 times after the two-stage method. The results reported here demonstrate the potential implementation of a strategy to B-phycoerythrin recovery with a purity of 3.2 (estimated by the absorbance relation of 545-280nm) from P. cruentum.     

PHB accumulation in Nostoc muscorum under different carbon stress situations

Use of algae for intracellular poly-β-hydroxybutyrate (PHB) accumulation for bioplastic production offers an opportunity in economic efficiency by reduced costs. The cyanobacterium Nostoc muscorum is a PHB accumulator which presents a great potential as raw material supplier because of short generation cycles. Here, we examined a range of experimental conditions including different growth conditions of phosphate-starved cells with the addition of external carbon sources. The highest, absolute PHB accumulation was measured in a phosphate-starved medium with 1% (w/w) glucose and 1% (w/w) acetate. PHB accumulated inside algae cells. After 23 days of growth in phosphate-starved medium, 1 L of culture contained up to 145.1 mg PHB. The highest PHB accumulation based on the cell dry weight was in an experiment with aeration and CO₂ addition. The intracellular level of PHB was up to 21.5% cell dry weight after 8 days.     

Control of molecular weight of poly-β-hydroxybutyric acid produced in fet-batch culture of Protomonas extorquens

Summary To control molecular weight of poly--hydroxybutyric acid (PHB) produced in a fedbatch culture of Protomonas extorquens, the effects of cultural temperature, pH, molar ratio of methanol and ammonia, and concentration of methanol in the medium on polymerization were inverstigated. Change of methanol concentration affected average molecular weight of PHB. When the cultivation was carried out at 0.05 g/l of methanol, average molecular weight of PHB reached above 8×10⁵. On the other hand, in the case of 32 g/l of methanol average molecular weight of PHB was less than 0.5×10⁵. Although every sample had a wide molelcular weight distribution, it became possible to control voluntarily average molecular weight of PHB.     

Mass production of poly-β-hydroxybutyric acid by fed-batch culture with controlled carbon/nitrogen feeding

Summary The effect of the ratio of methanol to ammonia, in the feeding solution the C/N ratio, on microbial PHB production was investigated. A constant C/N ratio regulated both the PHB content and the specific rate of PHB production. The results indicated that to produce the maximum PHB effectively in a short time the C/N ratio should be controlled automatically according to the increase in PHB content. Variation of the PHB content was estimated by tracing the timecourse of CO₂ concentration in exhaust gas. When the cell concentration reached 70 g/l, C/N ratio was gradually increased from the initial C/N ratio of 10 (mol methanol/mol ammonia). At 121 h, concentration of PHB reached 136 g/l, which was the maximum level so far obtained. The reaction time was considerably shortened compared with our previous study (175 h). Furthermore, PHB concentration reached 149 g/l at 170 h and total cell concentration became 233 g/l. PHB yield from methanol was 0.20 (g PHB/g methanol), which was also superior to the previous result of 0.18.