Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] is a bacterial copolymer in the polyhydroxyalkanoates (PHAs) family, a next-generation bioplastic. Our research team recently engineered a newly P(3HB-co-3HHx)-producing bacterial strain, Cupriavidus necator PHB-4/pBBR_CnPro-phaCRp. This strain can produce P(3HB-co-2 mol% 3HHx) using crude palm kernel oil (CPKO) as a sole carbon substrate. However, the improvement of P(3HB-co-3HHx) copolymer production by this strain has not been studied so far. Thus, this study aims to enhance the production of P(3HB-co-3HHx) copolymers containing higher 3HHx monomer compositions using response surface methodology (RSM). Three significant factors for P(3HB-co-3HHx) copolymers production, i.e., CPKO concentration, sodium hexanoate concentration, and cultivation time, were studied in the flask scale. As a result, a maximum of 3.6 ± 0.4 g/L of P(3HB-co-3HHx) with 4 mol% 3HHx compositions was obtained using the RSM optimized condition. Likewise, the higher 3HHx monomer composition (5 mol%) was obtained when scaling up the fermentation in a 10L-stirrer bioreactor. Furthermore, the produced polymer's properties were similar to marketable P(3HB-co-3HHx), making this polymer suitable for a wide range of applications.
A simple procedure for recovering biodegradable polymer from bacterial cells has been developed using economical and environmentally friendly solvent or chemicals. Recombinant bacterium, Cupriavidus necator harboring pBBR1MCS-C2 plasmid polyhydroxyalkanoate (PHA) synthase gene was used for the production of copolymer P(3HB-co-3HHx) from crude palm kernel oil (CPKO). NaOH was chosen in this study as it could give high purity and recovery yield. Increase of NaOH concentration had resulted in an increase of the PHA purity, but the recovery yield had decreased. The greater improvement of PHA purity and recovery were achieved by incubating the freeze-dried cells (10-30 g/L) in NaOH (0.1 M) for 1-3 h at 30°C and polishing using 20% (v/v) of ethanol. The treatment caused negligible degradation of the molecular weight of PHA recovered from the bacterial cells. The present review also highlights other extraction methods to provide greater insights into economical and sustainable recovery of PHA from bacterial cells.
The nutrition-versatility of Burkholderia sp. strain USM (JCM 15050) has initiated the studies on the use of this bacterium for polyhydroxyalkanoate (PHA) production. To date, the Burkholderia sp. has been reported to synthesize 3-hydroxybutyrate, 3-hydroxyvalerate and 3-hydroxy-4-methylvalerate monomers. In this study, the PHA biosynthetic genes of this strain were successfully cloned and characterized. The PHA biosynthetic cluster of this strain consisted of a PHA synthase (phaC), β-ketothiolase (phaA), acetoacetyl-CoA reductase (phaB) and PHA synthesis regulator (phaR). The translated products of these genes revealed identities to corresponding proteins of Burkholderia vietnamiensis (99-100 %) and Cupriavidus necator H16 (63-89%). Heterologous expression of phaCBs conferred PHA synthesis to the PHA-negative Cupriavidus necator PHB¯4, confirming that phaCBs encoded functionally active protein. PHA synthase activity measurements revealed that the crude extracts of C. necator PHB¯4 transformant showed higher synthase activity (243 U/g) compared to that of wild-types Burkholderia sp. (151 U/g) and C. necator H16 (180 U/g). Interestingly, the transformant C. necator PHB¯4 harbouring Burkholderia sp. PHA synthase gene accumulated poly(3-hydroxybutyrate-co-4-hydroxybutyrate) with 4-hydroxybutyrate monomer as high as up to 87 mol% from sodium 4-hydroxybutyrate. The wild type Burkholderia sp. did not have the ability to produce this copolymer.
The nucleotide sequences of the second internal transcribed spacer of rDNA were determined for adult worms of Necator americanus originating from Togo (Africa) and Sarawak (Malaysia). The length of the sequences of specimens from Togo (325 bp) were shorter than those from Sarawak (327 bp). There were six fixed genetic differences in the aligned sequences of N. americanus from Sarawak and Togo, excluding one or two polymorphic sites within the sequence of N. americanus from each geographical region. These findings suggest that there is either population variation in the sequence of N. americanus, or that N. americanus from the two countries may represent genetically distinct but morphologically similar (i.e. cryptic) species, however, comparison of the sequence differences among other hookworm species supports the latter conclusion.
Polyhydroxyalkanoates (PHAs) are produced in microbes as a source of carbon and energy storage. They are biodegradable and have properties similar to synthetic plastics, which make them an interesting alternative to petroleum-based plastics. In this study, a refined method of recovering PHA from Cupriavidus necator biomass was proposed by incorporating the use of the yellow mealworm (the larval phase of the mealworm beetle, Tenebrio molitor) as partial purification machinery, followed by washing of the fecal pellets with distilled water and sodium hydroxide. The PHA contents of the cells used in this study were 55wt% (produced from palm olein) and 60 wt% (produced from waste animal fats). The treatment of distilled water and NaOH further increased the purity of PHA to 94%. In parallel, analysis of the 16S rRNA metagenomic sequencing of the mealworm gut microbiome has revealed remarkable changes in the bacterial diversity, especially between the mealworms fed with cells produced from palm olein and waste animal fats. This biological recovery of PHA from cells is an attempt to move towards a green and sustainable process with the aim of reducing the use of harmful solvents and strong chemicals during polymer purification. The results obtained show that - purities of >90%, without a reduction in the molecular weight, can be obtained through this integrative biological recovery approach. In addition, this study has successfully shown that the cells, regardless of their origins, were readily consumed by the mealworms, and there is a correlation between the feed type and the mealworm gut microbiome.
This study aimed to enhance production of polyhydroxybutyrate P(3HB) by a newly engineered strain of Cupriavidus necator NSDG-GG by applying response surface methodology (RSM). From initial experiment of one-factor-at-a-time (OFAT), glucose and urea were found to be the most significant substrates as carbon and nitrogen sources, respectively, for the production of P(3HB). OFAT experiment results showed that the maximum biomass, P(3HB) content, and P(3HB) concentration of 8.95 g/L, 76 wt%, and 6.80 g/L were achieved at 25 g/L glucose and 0.54 g/L urea with an agitation rate of 200 rpm at 30 °C after 48 h. In this study, RSM was applied to optimize the three key variables (glucose concentration, urea concentration, and agitation speed) at a time to obtain optimal conditions in a multivariable system. Fermentation experiments were conducted in shaking flask by cultivation of C. necator NSDG-GG using various glucose concentrations (10-50 g/L), urea concentrations (0.27-0.73 g/L), and agitation speeds (150-250 rpm). The interaction between the variables studied was analyzed by ANOVA analysis. The RSM results indicated that the optimum cultivation conditions were 37.70 g/L glucose, 0.73 g/L urea, and 200 rpm agitation speed. The validation experiments under optimum conditions produced the highest biomass of 12.84 g/L, P(3HB) content of 92.16 wt%, and P(3HB) concentration of 11.83 g/L. RSM was found to be an efficient method in enhancing the production of biomass, P(3HB) content, and P(3HB) concentration by 43, 21, and 74%, respectively.
Hookworm infections are still endemic in low and middle income tropical countries with greater impact on the socioeconomic and public health of the bottom billion of the world's poorest people. In this study, a real-time polymerase chain reaction (PCR) coupled with high resolution melting-curve (HRM) analysis was evaluated for an accurate, rapid and sensitive tool for species identification focusing on the five human hookworm species.
The potential of plant oils as sole carbon sources for production of P(3HB-co-3HHx) copolymer containing a high 3HHx monomer fraction using the recombinant Cupriavidus necator strain Re2160/pCB113 has been investigated. Various types and concentrations of plant oils were evaluated for efficient conversion of P(3HB-co-3HHx) copolymer. Crude palm kernel oil (CPKO) at a concentration of 2.5 g/L was found to be most suitable for production of copolymer with a 3HHx content of approximately 70 mol%. The time profile of these cells was also examined in order to study the trend of 3HHx monomer incorporation, PHA production and PHA synthase activity. (1)H NMR and (13)C NMR analyses confirmed the presence of P(3HB-co-3HHx) copolymer containing a high 3HHx monomer fraction, in which monomers were not randomly distributed. The results of various characterization analyses revealed that the copolymers containing a high 3HHx monomer fraction demonstrated soft and flexible mechanical properties.
A new halogen-free and environmental-friendly method using water and ethanol is developed as an alternative for the recovery of polyhydroxyalkanoates (PHA) from recombinant Cupriavidus necator in comparison to the established chloroform extraction method. After optimisation, our results showed that the halogen-free method produced a PHA with 81% purity and 96% recovery yield, in comparison to the chloroform extraction system which resulted in a highly pure PHA with 95% yield. Although the purity of the PHA using the new method is lower, the molecular weight of the extracted PHA is not compromised. This new method can be further developed as an alternative and more environmental-friendly method for industrial application.
A combination of palm olein (POl) and fructose was used as carbon source for the biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] by recombinant Cupriavidus necator Re2058/pCB113. Cultures grown using 5g/L PO alone as carbon source produced cell dry weight (CDW) of 5.13g/L, 67% PHA/CDW and accumulated a copolymer containing 27mol% 3HHx in shake flask cultures. When cultures were grown in 5g/L fructose alone as the carbon source they produced CDW of 2.32g/L, 11% PHA/CDW and accumulated only poly(3-hydroxybutyrate) [P(3HB)] homopolymer. When the cells were cultured in 5g/L POl in combination with 7g/L fructose, CDW of 7.41g/L and 80% PHA/CDW was obtained with 17mol% 3HHx monomer fraction. Biosynthesis was carried out using a 13L fermenter to study the accumulation of 3HHx monomer fraction in the bacterial cells at different time point. The molecular weights of P(3HB-co-3HHx) with 4-15mol% 3HHx monomer were in the range between 5.47-6.85×105Da, which were at least two fold higher than previously reported values. Interestingly, the increase in Mwof the copolymer along with the increase in 3HHx molar fraction was observed. The viscoelastic property of the copolymer further confirmed the increase in Mw.
Two female and one male adult hookworms were recovered from a female patient in Thailand. Based on gross and microscopic morphology, the three hookworms are members of Necator americanus. Phylogenetic reconstruction based on partial NADH dehydrogenase subunit 1 (nad1) mitochondrial gene sequences shows that these hookworms belong to the same genetic lineage as N. americanus adult worm from Zhejiang, China. The male and female hookworms were genetically distinct, belonging to two different nad1-haplotypes. This is the first report targeting the nad1 gene on the identification and genetic characterization of the human hookworms originated from infected patient. The nad1 gene marker is useful for species and higher taxa differentiation of hookworms.
The cost of polyhydroxyalkanoates (PHAs) can be reduced by improving their productivity and recovery. In this study, we attempted to obtain a high cell density culture from a 13 L bioreactor and subsequently improved the recently developed biological recovery process using mealworms to obtain the PHA granules. A cell dry weight of 161 g/L containing 68-70 wt% P(3HB) was obtained. The freeze-dried cells contained a significant amount of mineral salts from the culture medium which reduced the cells' palatability for the mealworms. A simple washing procedure with water was sufficient to remove the residual mineral salts and this improved the cells' consumption by up to 12.5% of the mealworms' body weight. As a result, one kilogram of mealworms consumed 125 g of the washed cells daily and 87.2 g of feacal pellets were recovered, which was almost twice the weight of the unwashed cells. In addition, it also improved the purity of the PHA in the faecal pellets to a value <90% upon washing with water to remove the water-soluble compounds. This study has demonstrated a significant improvement in the production and recovery of PHA. In addition, the resulting mealworms showed a significant increase in protein content up to 79% and a decrease in fat content down to 8.3% of its dry weight.
Special features of the Japanese ocean include its ranges of latitude and depth. This study is the first to examine the diversity of Class I and II PHA synthases (PhaC) in DNA samples from pelagic seawater taken from the Japan Trench and Nankai Trough from a range of depths from 24 m to 5373 m. PhaC is the key enzyme in microorganisms that determines the types of monomer units that are polymerized into polyhydroxyalkanoate (PHA) and thus affects the physicochemical properties of this thermoplastic polymer. Complete putative PhaC sequences were determined via genome walking, and the activities of newly discovered PhaCs were evaluated in a heterologous host.
A simple, efficient and economical method for the recovery of P(3HB-co-3HHx) was developed using various chemicals and parameters. The initial content of P(3HB-co-3HHx) in bacterial cells was 50-60 wt%, whereas the monomer composition of 3HHx used in this experiments was 3-5 mol%. It was found that sodium hydroxide (NaOH) was the most effective chemical for the recovery of biodegradable polymer. High polyhydroxyalkanoate purity and recovery yield both in the range of 80-90 wt% were obtained when 10-30 mg/ml of cells were incubated in NaOH at the concentration of 0.1 M for 60-180 min at 30 °C and polished using 20 % (v/v) of ethanol.
Palm kernel oil, palm olein, crude palm oil and palm acid oil were used for the synthesis of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] by a mutant strain of Wautersia eutropha (formerly Ralstonia eutropha) harboring the Aeromonas caviae polyhydroxyalkanoate (PHA) synthase gene. Palm kernel oil was an excellent carbon source for the production of cell biomass and P(3HB-co-3HHx). About 87% (w/w) of the cell dry weight as P(3HB-co-3HHx) was obtained using 5 g palm kernel oil/l. Gravimetric and microscopic analyses further confirmed the high PHA content in the recombinant cells. The molar fraction of 3HHx remained constant at 5 mol % regardless of the type and concentration of palm oil products used. The small amount of 3HHx units was confirmed by 13C NMR analysis. The number average molecular weight (M(n)) of the PHA copolymer produced from the various palm oil products ranged from 27 0000 to 46 0000 Da. The polydispersity was in the range of 2.6-3.9.
PHA synthase (PhaC) is the key enzyme in the production of biodegradable plastics known as polyhydroxyalkanoate (PHA). Nevertheless, most of these enzymes are isolated from cultivable bacteria using traditional isolation method. Most of the microorganisms found in nature could not be successfully cultivated due to the lack of knowledge on their growth conditions. In this study, a culture-independent approach was applied. The presence of phaC genes in limestone soil was screened using primers targeting the class I and II PHA synthases. Based on the partial gene sequences, a total of 19 gene clusters have been identified and 7 clones were selected for full length amplification through genome walking. The complete phaC gene sequence of one of the clones (SC8) was obtained and it revealed 81% nucleotide identity to the PHA synthase gene of Chromobacterium violaceum ATCC 12472. This gene obtained from uncultured bacterium was successfully cloned and expressed in a Cupriavidus necator PHB(-)4 PHA-negative mutant resulting in the accumulation of significant amount of PHA. The PHA synthase activity of this transformant was 64 ± 12 U/g proteins. This paper presents a pioneering study on the discovery of phaC in a limestone area using metagenomic approach. Through this study, a new functional phaC was discovered from uncultured bacterium. Phylogenetic classification for all the phaCs isolated from this study has revealed that limestone hill harbors a great diversity of PhaCs with activities that have not yet been investigated.
Aquitalea sp. USM4 (JCM 19919) was isolated from a freshwater sample at Lata Iskandar Waterfall in Perak, Malaysia. It is a rod-shaped, gram-negative bacterium with high sequence identity (99%) to Aquitalea magnusonii based on 16S rRNA gene analysis. Aquitalea sp. USM4 also possessed a PHA synthase gene (phaC), which had amino acid sequence identity of 77-78% to the PHA synthase of Chromobacterium violaceum ATCC12472 and Pseudogulbenkiania sp. NH8B. PHA biosynthesis results showed that wild-type Aquitalea sp. USM4 was able to accumulate up to 1.5 g/L of poly(3-hydroxybutyrate), [P(3HB)]. The heterologous expression of the PHA synthase gene of Aquitalea sp. USM4 (phaCAq) in Cupriavidus necator PHB(-)4 had resulted in PHA accumulation up to 3.2 g/L of P(3HB). It was further confirmed by (1)H nuclear magnetic resonance (NMR) analysis that Aquitalea sp. USM4 and C. necator PHB(-)4 transformant were able to produce PHA containing 3-hydroxyvalerate (3HV), 4-hydroxybutyrate (4HB) and 3-hydroxy-4-methylvalerate (3H4MV) monomers from suitable precursor substrates. Interestingly, relatively high PHA synthase activity of 863 U/g and 1402 U/g were determined in wild-type Aquitalea sp. USM4 and C. necator PHB(-)4 transformant respectively. This is the first report on the member of genus Aquitalea as a new PHA producer as well as in vitro and in vivo characterization of a novel PHA synthase from Aquitalea sp. USM4.
Polyhydroxyalkanoates (PHAs), a family of biodegradable and renewable biopolymers show a huge potential as an alternative to conventional plastics. Extractive bioconversion (in situ product recovery) is a technique that integrates upstream fermentation and downstream purification. In this study, extractive bioconversion of PHAs from Cupriavidus necator H16 was performed via a thermo-separating aqueous two-phase system to reduce the cost and environmental impacts of PHAs production. Key operating parameters, such as polymer concentration, temperature, and pH, were optimized. The strategy achieved a yield and PF of 97.6% and 1.36-fold, respectively at 5% EOPO 3900 concentration, 30 °C fermentation temperature and pH 6. The PHAs production process was also successfully scaled up in a 2 L bioreactor. To the best of our knowledge, this is the first report on extractive fermentation of PHAs from Cupriavidus necator utilizing a thermo-separation system to achieve a better productivity and purity of the target product.
It has been tested and proven that vaccination is still the best strategy to combat infectious diseases. However, to date, there are still no vaccines against human soil-transmitted helminthic diseases, despite their high prevalence globally, particularly in developing countries and rural areas with tropical climates and poor sanitation. The development of vaccines against helminths is riddled with obstacles. Helminths have a complex life cycle, multiple stages within the same host with stage-specific antigen expression, and the ability to regulate host immune reactions to evade the immune response. These elements contribute to the main challenge of helminthic vaccines: the identification of effective vaccine candidates. Therefore, this article reviews the current progress and potential future direction of soil-transmitted helminthic vaccines, particularly against Trichuris trichiura, Ascaris lumbricoides, Strongyloides stercoralis, Necator americanus and Ancylostoma duodenale. The study design employed was a systematic review, using qualitative meta-summary synthesis. Preclinical studies and clinical trials on the development of protein subunit vaccines against the five soil-transmitted helminths were searched on PubMed and Scopus. Effectiveness was indicated by a reduction in worm burden or larval output, an increase in specific IgG levels, or an increase in cytokine production. Our findings show that only the hookworm vaccine against N. americanus is in the clinical trial phase, while the rest is still in exploratory research and pre-clinical development phase.
Factors influencing poly(3-hydroxybutyrate) P(3HB) production by Cupriavidus necator CCUG52238(T) utilizing oil palm frond (OPF) juice were clarified in this study. Effects of initial medium pH, agitation speed, and ammonium sulfate (NH(4))(2)SO(4) concentration on the production of P(3HB) were investigated in shake flasks experiments using OPF juice as the sole carbon source. The highest P(3HB) content was recorded at pH 7.0, agitation speed of 220 rpm, and (NH(4))(2)SO(4) concentration at 0.5 g/L. By culturing the wild-type strain of C. necator under the aforementioned conditions, the cell dry weight (CDW) and P(3HB) content obtained were 9.31 ± 0.13 g/L and 45 ± 1.5 wt.%, respectively. This accounted for 40% increment of P(3HB) content compared to the nonoptimized condition. In the meanwhile, the effect of dissolved oxygen tension (DOT) on P(3HB) production was investigated in a 2-L bioreactor. Highest CDW (11.37 g/L) and P(3HB) content (44 wt.%) were achieved when DOT level was set at 30%. P(3HB) produced from OPF juice had a tensile strength of 40 MPa and elongation at break of 8% demonstrated that P(3HB) produced from renewable and cheap carbon source is comparable to those produced from commercial substrate.