Recovery of medium-chain-length polyhydroxyalkanoates (PHAs) through enzymatic digestion treatments and ultrafiltration

Medium-chain-length (mcl) polyhydroxyalkanoates (PHAs) are biodegradable polyesters accumulated intracellularly as energy resources by bacterial species such as Pseudomonas putida. The most popular method for PHA recovery in the downstream processing is solvent extraction using chloroform and methanol. An alternate method is bioseparation using enzymatic digestion process which eliminates the need for hazardous solvents. This research focuses on an attempt to optimize the recovery of PHAs by solubilisation of non-PHA granules through enzymatic treatments such as; Alcalase (to digest the denatured proteins), sodium dodecyl sulfate (SDS) to assist solubilisation, ethylene diamine tetra acetic acid (EDTA) to complex divalent cations and lysozyme to digest the peptidoglycan wall enveloping the cell. The experiment was designed through Taguchi's design of experiment (DOE) using Qualitek-4 software. The results show that Alcalase enzyme used had the most significant effect on the treatment process and contributed to about 71.5% in terms of process factor importance among the different factors on treatment performance for PHA recovery. It is desired to recover the PHA granules in water suspension after the enzymatic treatment by removing the solubilised non-PHA cell material through crossflow ultrafiltration system and purified through continuous diafiltration process. Final purity of PHA in water suspension obtained using GC analysis is 92.6%, with a nearly 90% recovery, thus concluding that this method is indeed a suitable alternative.     

Extraction of short-chain-length poly-[(R)-hydroxyalkanoates] (scl-PHA) by the “anti-solvent” acetone under elevated temperature and pressure

A novel method was developed for extraction of short-chain-length poly(hydroxyalkanoates) (scl-PHA) from microbial biomass by the well-known “scl-PHA anti-solvent” acetone at elevated temperature and pressure in a closed system combining components for extraction, filtration, and product work-up. Recovery of scl-PHA using this new approach was compared with established methods using chloroform at ambient pressure. The new method performs similar regarding product purity (98.4 vs. 97.7 %) and extraction yield (96.8 % by both methods), and is by far faster than established chloroform extraction (20 min vs. 12 h). Separation of the polymer from acetone is simply achieved by cooling down the acetone solution of scl-PHA, thus allows for a nearly quantitative recovery of the solvent that conveniently can be reused. Characterization of scl-PHA extracted by both methods does not reveal any significant difference in terms of molar mass and thermo analytical parameters.     

Recovery of poly(3‐hydroxybutyrate‐co‐3‐hydroxyhexanoate) from Ralstonia eutropha cultures with non‐halogenated solvents

Reduced downstream costs, together with high purity recovery of polyhydroxyalkanoate (PHA), will accelerate the commercialization of high quality PHA‐based products. In this work, a process was designed for effective recovery of the copolymer poly(hydroxybutyrate‐co‐hydroxyhexanoate) (P(HB‐co‐HHx)) containing high levels of HHx (>15 mol%) from Ralstonia eutropha biomass using non‐halogenated solvents. Several non‐halogenated solvents (methyl isobutyl ketone, methyl ethyl ketone, and butyl acetate and ethyl acetate) were found to effectively dissolve the polymer. Isoamyl alcohol was found to be not suitable for extraction of polymer. All PHA extractions were performed from both dry and wet cells at volumes ranging from 2 mL to 3 L using a PHA to solvent ratio of 2% (w/v). Ethyl acetate showed both high recovery levels and high product purities (up to 99%) when using dry cells as starting material. Recovery from wet cells, however, eliminates a biomass drying step during the downstream process, potentially saving time and cost. When wet cells were used, methyl isobutyl ketone (MIBK) was shown to be the most favorable solvent for PHA recovery. Purities of up to 99% and total recovery yields of up to 84% from wet cells were reached. During polymer recovery with either MIBK or butyl acetate, fractionation of the extracted PHA occurred, based on the HHx content of the polymer. PHA with higher HHx content (17–30 mol%) remained completely in solution, while polymer with a lower HHx content (11–16 mol%) formed a gel‐like phase. All PHA in solution could be precipitated by addition of threefold volumes of n‐hexane or n‐heptane to unfiltered PHA solutions. Effective recycling of the solvents in this system is predicted due to the large differences in the boiling points between solvent and precipitant. Our findings show that two non‐halogenated solvents are good candidates to replace halogenated solvents like chloroform for recovery of high quality PHA. Biotechnol. Bioeng. 2013; 110: 461–470. © 2012 Wiley Periodicals, Inc.     

An Efficient RNA Extraction Method for Estimating Gut Microbial Diversity by Polymerase Chain Reaction

An extraction method was developed to recover high-quality RNA from rumen digesta and mouse feces for phylogenetic analysis of metabolically active members of the gut microbial community. Four extraction methods were tested on different amounts of the same samples and compared for efficiency of recovery and purity of RNA. Trizol extraction after bead beating produced a higher quantity and quality of RNA than a similar method using phenol/chloroform. Dissociation solution produced a 1.5- to 2-fold increase in RNA recovery compared with phosphate-buffered saline during the dissociation of microorganisms from rumen digesta or fecal particles. The identity of metabolically active bacteria in the samples was analyzed by sequencing 87 amplicons produced using bacteria-specific 16S rDNA primers, with cDNA synthesized from the extracted RNA as the template. Amplicons representing the major phyla encountered in the rumen (Firmicutes, 43.7%; Proteobacteria, 28.7%; Bacteroidetes, 25.3%; Spirochea, 1.1%, and Synergistes, 1.1%) were recovered, showing that development of the RNA extraction method enables RNA-based analysis of metabolically active bacterial groups from the rumen and other environments. Interestingly, in rumen samples, about 30% of the sequenced random 16S rRNA amplicons were related to the Proteobacteria, providing the first evidence that this group may have greater importance in rumen metabolism than previously attributed by DNA-based analysis.     

Fermentative production of rhamnolipid and purification by adsorption chromatography

Glycolipids are one of the major classes of biosurfactants in which the rhamnolipids are best studied. The present work investigates the optimization of inoculum age and batch time for maximizing the yield of rhamnolipid from Pseudomonas aeruginosa (MTCC 2453). The yield and titer of rhamnolipids were maximum in the fermentation batch with an inoculum age of 24?hr. Batch time studies were performed on biomass production, rhamnolipid production, and sunflower oil utilization. The maximum yield of rhamnolipid was achieved at 96?hr when the culture cells were in the late exponential/early stationary phase. At optimum substrate concentration, maximum yield of 10.8?g/L was achieved. Further, downstream processing of crude rhamnolipid from broth using organic solvent extraction and subsequent purification using adsorption chromatography was done. In this study, chromatographic method was developed for purification of rhamnolipid by adsorption phenomena with more than 88.7% purity and 86.5% recovery. The present study provides new perspective on concepts involving separation by adsorption. Further antimicrobial properties and surfactant properties were studied for rhamnolipid production.     

The recovery of poly(3-hydroxybutyrate) by using dispersions of sodium hypochlorite solution and chloroform

Summary To take advantage of both differential digestion by hypochlorite and solvent extraction, we used dispersions of sodium hypochlorite solution and chloroform in the recovery of microbial PHB. The treatment with hypochlorite alone caused such severe degradation and the molecular weight decreased drastically with increasing hypochlorite concentration. However, using the dispersion, the degradation of PHB was markedly diminished owing to theshielding effect of chloroform. In this case, we could obtain PHB of above 97% purity with a number average molecular weight of 1,000,000 comparable to the original molecular weight of 1,200,000.     

Cost-effective recovery and purification of polyhydroxyalkanoates by selective dissolution of cell mass

Highly efficient separation and purification of polyhydroxyalkanoates (PHAs) from PHA-containing cell mass is essential to production of the bioplastics from renewable resources in a cost-effective, environmentally friendly way. Based on selective dissolution of non-PHA cell mass (NPCM) by protons in aqueous solution and crystallization kinetics of PHA biopolymers, a simple process is developed and demonstrated to recover PHAs from cell mass to high purity (>97 wt %) with high yield (>95 wt %). The average molecular weight of biopolyesters is controlled, which follows an exponential function of process severity, a combined factor of processing conditions. Compared with conventional chemical treatment such as sequential surfactant and hypochlorite treatment, this new technology substantially reduces the chemical cost for PHA recovery and purification from PHA-containing cell mass.     

Two methods of large-scale extraction of an antibiotic produced by Myxococcus coralloides.

Two methods for the isolation of an antibiotic produced by Myxococcus coralloides have been developed: the chloroform extraction method and the charcoal adsorption method. The recovery of antibiotic in each case was 25% and 30%, respectively, by the two methods. Although the two methods yield a relative low recovery, the charcoal adsorption method seems more attractive and promising due to its simplicity and economic advantages.     

An efficient method for the application of PHA‐poor solvents to extract polyhydroxybutyrate from Cupriavidus necator

There are many published studies presenting ethanol and acetone as PHAs‐poor solvents, where these two solvents are shown to dissolve <2% (w/v) of PHAs at low temperatures. In this study, the suitability of ethanol and acetone for the recovery of PHB at different temperatures (from room temperature to near boiling point) in Cupriavidus necator was investigated. Experiments were performed using response surface methodology to examine the effects of different temperatures and heating incubation times on recovery percentage using the two solvents. The highest recovery percentage (92.3%) and product purity (up to 99%) were obtained with ethanol‐assisted extraction at 76°C for 32 min of incubation time. Under these conditions the extracted PHB exhibited a molecular mass of 1.2 × 10⁶. The present strategy showed that at temperatures near its boiling point, ethanol, as a nonhalogenated solvent, represents a good alternative to halogenated solvents, like chloroform, when PHB recovery is concerned. DSC analysis showed good thermal properties for ethanol‐ and acetone‐extracted biopolymers. GC and ¹H NMR analysis confirmed the extracted biopolymer to be polyhydroxybutyrate of good purity. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1480–1486, 2016     

Potential Aqueous Two‐Phase Processes for the Primary Recovery of Colored Protein from Microbial Origin

The primary recovery of c‐phycocyanin and b‐phycoerythrin from Spirulina maxima and Porphyridium cruentum, respectively, using an established extraction strategy was selected as a practical model system to study the generic application of polyethylene glycol (PEG)‐phosphate aqueous two‐phase systems (ATPS). The generic practical implementation of ATPS extraction was evaluated for the recovery of colored proteins from microbial origin. A comparison of the influence of system parameters, such as PEG molecular mass, concentration of PEG as well as salt, system pH and volume ratio, on the partition behavior of c‐phycocyanin and b‐phycoerythrin was carried out to determine under which conditions target colored protein and contaminants concentrate to opposite phases. One‐stage processes are proposed for the primary recovery of the colored proteins. PEG1450‐phosphate ATPS extraction (volume ratio (V_R) equal to 0.3, tie‐line length (TLL) of 34 % w/w and system pH 7.0) for the recovery of c‐phycocyanin from Spirulina maxima resulted in a primary recovery process that produced a protein purity of 2.1 ± 0.2 (defined as the relationship of 620 nm to 280 nm absorbance) and a product yield of 98 % [w/w]. PEG1000‐phosphate ATPS extraction (i.e., V_R = 1.0, PEG 1000, TLL 50 % w/w and system pH 7.0) was preferred for the recovery of b‐phycoerythrin from Porphyridium cruentum, which resulted in a protein purity of 2.8 ± 0.2 (defined as the relationship of 545 nm to 280 nm absorbance) and a product yield of 82 % [w/w]. The purity of c‐phycocyanin and b‐phycoerythrin from the crude extract increased 3‐ and 4‐fold, respectively, after ATPS. The results reported herein demonstrated the benefits of the practical generic application of ATPS for the primary recovery of colored proteins from microbial origin as a first step for the development of purification processes.     

Efficient production of medium-chain-length poly(3-hydroxyalkanoates) from octane by Pseudomonas oleovorans: economic considerations

Poly(3-hydroxyalkanoates) (PHA) have the potential to become a biodegradable alternative for conventional plastics. In order to produce PHA at competitive costs in comparison with commonly used plastics, efficient PHA production systems will have to be developed. Poly(3-hydroxybutyrate) fermentations are well developed and in actual use on an industrial scale; medium-chain-length PHA (mcl-PHA) production is less well described, although the vast majority of all PHA known today are mcl-PHA. This paper compares and describes mcl-PHA production systems with respect to the volumetric productivity, the cellular PHA content and the polymer yield on carbon substrates. Nitrogen was shown to be the most effective limitation to trigger PHA formation in P. oleovorans after different nutrient limitations had been compared. By using an economic model for the calculation of PHA production costs, we show that it should be possible to produce octane-based mcl-PHA on a large scale (more than 1000 tonnes/year) at costs below U.S. $ 10 kg⁻¹. Received: 4 April 1997 / Accepted: 20 May 1997     

Evaluation of six methods for extraction and purification of viral DNA from urine and serum samples

The sensitivity and reliability of PCR for diagnostic and research purposes require efficient unbiased procedures of extraction and purification of nucleic acids. One of the major limitations of PCR-based tests is the inhibition of the amplification process by substances present in clinical samples. This study used specimens spiked with a known amount of plasmid pBKV (ATCC 33-1) to compare six methods for extraction and purification of viral DNA from urine and serum samples based on recovery efficiency in terms of yield of DNA and percentage of plasmid pBKV recovered, purity of extracted DNA, and percentage of inhibition. The most effective extraction methods were the phenol/chloroform technique and the silica gel extraction procedure for urine and serum samples, respectively. Considering DNA purity, the silica gel extraction procedure and the phenol/chloroform method produced the most satisfactory results in urine and serum samples, respectively. The presence of inhibitors was overcome by all DNA extraction techniques in urine samples, as evidenced by semiquantitative PCR amplification. In serum samples, the lysis method and the proteinase K procedure did not completely overcome the presence of inhibitors.     

Evaluation of green solvents: Oil extraction from oleaginous yeast Lipomyces starkeyi using cyclopentyl methyl ether (CPME)

Cyclopentyl methyl ether (CPME) was evaluated for extracting oil or triacylglycerol (TAG) from wet cells of the oleaginous yeast Lipomyces starkeyi. CPME is a greener alternative to chloroform as a potential solvent for oil recovery. A monophasic system of CPME and biphasic system of CPME:water (1:0.7) performed poorly having the lowest TAG extraction efficiency and TAG selectivity compared to other monophasic systems of hexane and chloroform and the biphasic Bligh and Dyer method (chloroform:methanol:water). Biphasic systems of CPME:water:alcohol (methanol/ethanol/1‐propanol) were tested and methanol achieved the best oil extraction efficiency compared to ethanol and 1‐propanol. Different biphasic systems of CPME:methanol:water were tested, the best TAG extraction efficiency and TAG selectivity achieved was 9.9 mg/mL and 64.6%, respectively, using a starting ratio of 1:1.7:0.6 and a final ratio of 1:1:0.8 (CPME:methanol:water). Similar results were achieved for the Bligh and Dyer method (TAG extraction efficiency of 10.2 mg/mL and TAG selectivity of 66.0%) indicating that the biphasic CPME system was comparable. The fatty acid profile remained constant across all the solvent systems tested indicating that choice of solvent was not specific for any certain fatty acid. This study was able to demonstrate that CPME could be used as an alternative solvent for the extraction of oil from the wet biomass of oleaginous yeast. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1096–1103, 2017     

Downstream processing of human antibodies integrating an extraction capture step and cation exchange chromatography

In this paper we explore an alternative process for the purification of human antibodies from a Chinese hamster ovary (CHO) cell supernatant comprising a ligand-enhanced extraction capture step and cation exchange chromatography (CEX). The extraction of human antibodies was performed in an aqueous two-phase system (ATPS) composed of dextran and polyethylene glycol (PEG), in which the terminal hydroxyl groups of the PEG molecule were modified with an amino acid mimetic ligand in order to enhance the partition of the antibodies to the PEG-rich phase. This capture step was optimized using a design of experiments and a central composite design allowed the determination of the conditions that favor the partition of the antibodies to the phase containing the PEG diglutaric acid (PEG-GA) polymer, in terms of system composition. Accordingly, higher recovery yields were obtained for higher concentrations of PEG-GA and lower concentrations of dextran. The highest yield experimentally obtained was observed for an ATPS composed of 5.17% (w/w) dextran and 8% (w/w) PEG-GA. Higher purities were however predicted for higher concentrations of both polymers. A compromise between yield and purity was achieved using 5% dextran and 10% PEG-GA, which allowed the recovery of 82% of the antibodies with a protein purity of 96% and a total purity of 63%, determined by size-exclusion chromatography. ATPS top phases were further purified by cation exchange chromatography and it was observed that the most adequate cation exchange ligand was carboxymethyl, as the sulfopropyl ligand induced the formation of multi-aggregates or denatured forms. This column allowed the elution of 89% of the antibodies present in the top phase, with a protein purity of 100% and a total purity of 91%. The overall process containing a ligand-enhanced extraction step and a cation exchange chromatography step had an overall yield of 73%.     

En route to economical eco‐friendly solvent system in enhancing sustainable recovery of poly(3‐hydroxybutyrate‐co‐4‐hydroxybutyrate) copolymer

Separation of poly(3‐hydroxybutyrate‐co‐4‐hydroxybutyrate) [P(3HB‐co‐4HB)] from bacterial cell matter is a critical step in the downstream process with respect to material quality and eco‐balance as P(3HB‐co‐4HB) is widely used for biomedical applications. Therefore, an efficient and eco‐based extraction of P(3HB‐co‐4HB) using a combination of NaOH and Lysol in digesting the non‐polymeric cell material (NPCM) digestion is developed. The NaOH and Lysol show synergistic influence on the copolymer extraction at a high purity and recovery of 97 and 98 wt% respectively. The optimized cell digestion method was found applicable to a vast batch of cells containing copolymers from various 4HB monomer compositions. At the largest extraction volume of 100 L, P(3HB‐co‐4HB) with a purity of 89 wt% was extracted with a maximum recovery of 90 wt%. The method developed has also eliminated the cell pretreatment step. The extraction method developed in this research has not only produced an economic and efficient copolymer recovery but has also retained the copolymer quality, in term of its molecular weight and thermal properties. It demonstrates a practical and promising downstream processing method in recovering the copolymer effectively from the bacterial biomass.     

Simple and efficient genomic DNA extraction protocol for molecular characterisation of Phytophthora colocasiae causing taro leaf blight

Genomic DNA extraction protocol with relatively high quantity and purity is prerequisite for the successful molecular identification and characterisation of plant pathogens. Conventional DNA extraction methods are often time-consuming and yield only very poor quantity of genomic DNA for samples with higher mycelial age. In our laboratory, we have aimed at establishing an efficient DNA isolation procedure, exclusively for the oomycete pathogen Phytophthora colocasiae causing serious leaf blight disease in taro. For this a phenol free protocol was adopted, which involves SDS/Proteinase K-based inactivation of protein contaminants, extraction of nucleic acids using chloroform: isoamyl alcohol and later precipitation of genomic DNA using isopropanol and sodium acetate. The purity of the isolated DNA was analysed by A_260/280 and A_260/230 spectrophotometric readings and confirmed by restriction digestion with restriction enzyme Eco RI. In this study, a comparative assessment was done with CTAB method and the commercial genomic DNA purification kit (Thermo Fisher Scientific, Fermentas, EU). The extracted DNA was found to be suitable for further downstream applications like ITS amplification of the rDNA ITS region and PCR amplification with species-specific primers.     

Growth and polyhydroxybutyrate production by Ralstonia eutropha in emulsified plant oil medium

Polyhydroxyalkanoates (PHAs) are natural polyesters synthesized by bacteria for carbon and energy storage that also have commercial potential as bioplastics. One promising class of carbon feedstocks for industrial PHA production is plant oils, due to the high carbon content of these compounds. The bacterium Ralstonia eutropha accumulates high levels of PHA and can effectively utilize plant oil. Growth experiments that include plant oil, however, are difficult to conduct in a quantitative and reproducible manner due to the heterogeneity of the two-phase medium. In order to overcome this obstacle, a new culture method was developed in which palm oil was emulsified in growth medium using the glycoprotein gum arabic as the emulsifying agent. Gum arabic did not influence R. eutropha growth and could not be used as a nutrient source by the bacteria. R. eutropha was grown in the emulsified oil medium and PHA production was measured over time. Additionally, an extraction method was developed to monitor oil consumption. The new method described in this study allows quantitative, reproducible R. eutropha experiments to be performed with plant oils. The method may also prove useful for studying growth of different bacteria on plant oils and other hydrophobic carbon sources.     

Extractability of polyhydroxyalkanoate synthesized by <Emphasis Type="Italic">Bacillus flexus</Emphasis> cultivated in organic and inorganic nutrient media

Bacillus flexus was isolated from local soil sample and identified by molecular methods. In inorganic nutrient medium (IM) containing sucrose as carbon source, yield of biomass and polyhydroxyalkanoate (PHA) were 2 g/l and 1 g/l (50% of biomass), respectively. Substitution of inorganic nitrogen by peptone, yeast extract or beef extract resulted in biomass yields of 4.1, 3.9 and 1.6 g/l, respectively. Corresponding yields of PHA in biomass was 30%, 40% and 44%. Cells subjected to change in nutrient condition from organic to inorganic, lacked diaminopimelic acid in the cell wall and the concentration of amino acids also decreased. Under these conditions the extractability of the polymer from the cells by hot chloroform or mild alkali hydrolysis was 86–100% compared to those grown in yeast extract or peptone (32–56%). The results demonstrated that growth, PHA production and the composition of cell wall of B. flexus are influenced by the organic or inorganic nutrients present in the growth medium. Cells grown in inorganic medium lysed easily and this can be further exploited for easier recovery of the intracellular PHA.     

RNASwift: A rapid,versatile RNA extraction method free from phenol and chloroform

RNASwift is an inexpensive, versatile method for the rapid extraction of RNA. Existing RNA extraction methods typically use hazardous chemicals including phenol, chloroform and formamide which are often difficult to completely remove from the extracted RNA. RNASwift uses sodium chloride and sodium dodecyl sulphate to lyse the cells and isolate the RNA from the abundant cellular components in conjunction with solid phase extraction or isopropanol precipitation to rapidly purify the RNA. Moreover, the purified RNA is directly compatible with downstream analysis. Using spectrophotometry in conjunction with ion pair reverse phase chromatography to analyse the extracted RNA, we show that RNASwift extracts and purifies RNA of higher quality and purity in comparison to alternative RNA extraction methods. The RNASwift method yields approximately 25 μg of RNA from only 10⁸Escherichia coli cells. Furthermore, RNASwift is versatile; the same simple reagents can be used to rapidly extract RNA from a variety of different cells including bacterial, yeast and mammalian cells. In addition to the extraction of total RNA, the RNASwift method can also be used to extract double stranded RNA from genetically modified E. coli in higher yields compared to alternative methods.     

Improved detergent-based recovery of polyhydroxyalkanoates (PHAs)

Extracting polyhydroxyalkanoate (PHA) polymer from bacterial cells often involves harsh conditions, including use of environmentally harmful solvents. We evaluated different detergents under various conditions to extract PHA from Ralstonia eutropha and Escherichia coli cells. Most detergents tested recovered highly pure PHA polymer from cells in amounts that depended on the percentage of polymer present in the cell. Detergents such as linear alkylbenzene sulfonic acid (LAS-99) produced a high yield of high purity polymer, and less detergent was needed compared to the amount of SDS to produce comparable yields. LAS-99 also has the advantage of being biodegradable and environmentally safe. Chemical extraction of PHA with detergents could potentially minimize or eliminate the need to use harsh organic solvents, thus making industrial PHA production a cleaner technology process.