Bio-hydrogen production from wastewater using sludge as inoculum is a sustainable approach for energy production. This study investigated the influence of initial pH and temperature on bio-hydrogen production from dairy wastewater using pretreated landfill leachate sludge (LLS) as an inoculum. The maximum yield of 113.2 ± 2.9 mmol H2/g chemical oxygen demand (COD) (12.8 ± 0.3 mmol H2/g carbohydrates) was obtained at initial pH 6 and 37 °C. The main products of volatile fatty acids were acetate and butyrate with the ratio of acetate:butyrate was 0.4. At optimum condition, Gibb's free energy was estimated at -40 kJ/mol, whereas the activation enthalpy and entropy were 65 kJ/mol and 0.128 kJ/mol/l, respectively. These thermodynamic quantities suggest that bio-hydrogen production from dairy wastewater using pretreated LLS as inoculum was effective and efficient. In addition, genomic and bioinformatics analyses were performed in this study.
The α- and γ-mangostins from Garcinia mangostana pericarps (GMP) exhibit antioxidant, anti-bacterial, anti-inflammatory and anti-tumor properties. The extraction yields α- and γ-mangostins are often limited by the presence of the GMP cell walls. Therefore, the extraction and recovery of mangostins from GMP with an Aspergillus niger cellulase-assisted aqueous micellar biphasic system (CA-AMBS) was developed for enhanced yield of mangostins. Effects of the concentration of cellulase, the incubation time and the temperature of the system on the recovery of mangostins were investigated. The optimum condition for the recovery of α- and γ-mangostins was obtained with the addition of 0.5% (w/w) cellulase incubated at 40°C for 2 h. High log partition coefficients of α-mangostins (log Kα 4.79 ± 0.02) and γ-mangostins (log Kγ 4.02 ± 0.02) were achieved. High yields of α-mangostins (73.4%) and γ-mangostins (14.0%) were obtained from the micelle-rich bottom phase with final concentrations of 3.67 mg/mL and 0.70 mg/mL, respectively. The back-extraction of mangostins was performed with the addition of 30% (w/w) of isopropanol and 0.05 M of KCl at pH 9 to the bottom phase of the CA-AMBS. The yields of the α- and γ-mangostins from GMP were considerably enhanced with the CA-AMBS and the direct recovery of mangostins was demonstrated without additional downstream processing steps.
Aqueous two-phase system (ATPS) has been suggested as a promising separation tool in the biotechnological industry. This liquid-liquid extraction technique represents an interesting advance in downstream processing due to several advantages such as simplicity, rapid separation, efficiency, economy, flexibility and biocompatibility. Up to date, a range of biotechnological products have been successfully recovered from different sources with high yield using ATPS-based strategy. In view of the important potential contribution of the ATPS in downstream processing, this review article aims to provide latest information about the application of ATPS in the recovery of various biotechnological products in the past 7 years (2010-2017). Apart from that, the challenges as well as the possible future work and outlook of the ATPS-based recovery method have also been presented in this review article.
In this present study, alcohol/salt liquid biphasic system was used to extract phlorotannin from brown macroalgae. Liquid biphasic system is a new green technology that integrated with various processes into one-step, by concentrating, separating and purifying the bioproduct in a unit operation. The solvent used is non-toxic and there is potential for solvent recovery which is beneficial to the environment. Phlorotannin is a bioactive compound that has gained much attention due to its health beneficial effect. Therefore, the isolation of phlorotannin is lucrative as it contains various biological activities that are capable to be utilised into food and pharmaceutical application. By using 2-propanol/ammonium sulphate system, the highest recovery of phlorotannin was 76.1% and 91.67% with purification factor of 2.49 and 1.59 from Padina australis and Sargassum binderi, respectively. A recycling study was performed and the salt phase of system was recycled where maximum salt recovery of 41.04% and 72.39% could be obtained from systems containing P. australis and S. binderi, respectively. Similar recovery of phlorotannin was observed after performing two cycles of the system, this concludes that the system has good recyclability and eco-friendly.
Polyhydroxyalkanoates (PHA) are naturally occurring biopolymers, obtained from microorganisms. Properties like biodegradability and biocompatibility make PHA a part of today's commercial polymer industry. However, the production cost of PHA has been a great barrier to extend its application to large scale production. Substrates and usage of pure cultures constitute the main reason for its high production cost. On the other hand, rapid industrialization i.e., industrial sectors such as sugar, pulp and paper, fruit and food processing, dairies, slaughterhouses, and poultries, has resulted in the generation of the huge quantity of wastes. Consequently, becoming large source of environmental pollution and health hazard. This review emphasizes on the usage of various waste feedstocks obtained from industrial and agricultural industries as an alternate substrate for PHA production. As these waste materials are rich in organic material and also microbes, they can be the good starting material for PHA production. Additionally, advantages and economic importance of mixed cultures and also PHA applications are discussed. Future prospects and challenges in PHA production from waste feedstocks are also highlighted.
Xylanase enzyme degrades linear polysaccharide β-1,4 xylan and the hemicellulose of the plant cell wall. There is a growing demand in finding a cost-effective alternative for industrial scale production of xylanase with high purity for pharmaceutical applications. In this study, an alcohol/salt aqueous biphasic system (ABS) was adopted to recover xylanase from the Bacillus subtilis fermentation broth. The effects of several ABS parameters such as types and concentrations of alcohols and salts (i.e., sulphate, phosphate, and citrate), amount of crude loading and pH of the system on the recovery of xylanase were investigated. Partition coefficient of xylanase (KE), selectivity (S) and yield (YT) of xylanase in top phase of the ABS were measured. Highest KE (6.58 ± 0.05) and selectivity (4.84 ± 0.33) were recorded in an ABS of pH 8 composed of 26% (w/w) 1-propanol, 18% (w/w) ammonium sulphate. High YT of 71.88% ± 0.15 and a purification fold (PFT) of 5.74 ± 0.33 were recorded with this optimum recovery of xylanase using alcohol/salt ABS. The purity of xylanase recovered was then qualitatively verified with sodium dodecyl sulphate (SDS) gel electrophoresis. The SDS profile revealed the purified xylanase was successfully obtained in the top phase of the one-step 1-propanol/sulphate ABS with a distinct single band.
Three newly discovered H2 producing bacteria namely Clostridium perfringens strain JJC, Clostridium bifermentans strain WYM and Clostridium sp. strain Ade.TY originated from landfill leachate sludge have demonstrated highly efficient H2 production. The maximum H2 production attained from these isolates are in the descending order of strain C. perfringens strain JJC > C. bifermentans strain WYM > Clostridium sp. strain Ade.TY with yield of 4.68 ± 0.12, 3.29 ± 0.11, and 2.87 ± 0.10 mol H2/mol glucose, respectively. The result has broken the conventional theoretical yield of 4 mol H2/mol glucose. These isolates were thermodynamically favourable with Gibbs free energy between -33 and -35 kJ/mol (under process conditions: pH 6, 37 °C and 5 g/L glucose). All three isolates favour butyrate pathway for H2 production with the ratio of acetate and butyrate of 0.77, 0.65 and 0.80 for strain JJC, WYM and Ade.TY, respectively. This study reported provides a new insight on the potential of unique bacteria in H2 production.
Previously, a unique co-compost produced by composting empty fruit bunch with anaerobic sludge from palm oil mill effluent, which contributed to establishing a zero-emission industry in Malaysia. Little was known about the bacterial functions during the composting process and fertilization capacity of this co-compost. We isolated 100 strains from the co-compost on 7 types of enumeration media and screened 25 strains using in vitro tests for 12 traits, grouping them according to three functions: plant growth promoting (fixation of nitrogen; solubilization of phosphorus, potassium, and silicate; production of 3-indoleacetic acid, ammonia, and siderophore), biocontrolling (production of chitinase and anti-Ganoderma activity), and composting (degradation of lignin, xylan, and cellulose). Using 16S rRNA gene sequence analysis, 25 strains with strong or multi-functional traits were found belong to the genera Bacillus, Paenibacillus, Citrobacter, Enterobacter, and Kosakonia. Furthermore, several strains of Citrobacter sedlakii exhibited a plant growth-stimulation in vivo komatsuna plant cultivation test. In addition, we isolated several multifunctional strains; Bacillus tequilensis CE4 (biocontrolling and composting), Enterobacter cloacae subsp. dissolvens B3 (plant growth promoting and biocontrolling), and C. sedlakii CESi7 (plant growth promoting and composting). Some bacteria in the co-compost play significant roles during the composting process and plant cultivation after fertilization, and some multifunctional strains have potential for use in accelerating the biodegradation of lignocellulosic biomass, protecting against Ganoderma boninense infection, and increasing the yield of palm oil.
Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [(P(3HB-co-4HB)] copolymer receives attention as next generation biomaterial in medical application. However, the exploitation of the copolymer is still constrained since such copolymer has not yet successfully been performed in industrial scale production. In this work, we intended to establish pilot production system of the copolymer retaining the copolymer quality which has recently discovered to have novel characteristic from lab scale fermentation. An increase of agitation speed has significantly improved the copolymer accumulation efficiency by minimizing the utilization of substrates towards cell growth components. This is evidenced by a drastic increase of PHA content from 28 wt% to 63 wt% and PHA concentration from 3.1 g/L to 6.5 g/L but accompanied by the reduction of residual biomass from 8.0 g/L to 3.8 g/L. Besides, fermentations at lower agitation and aeration have resulted in reduced molecular weight and mechanical strength of the copolymer, suggesting the role of sufficient oxygen supply efficiency in improving the properties of the resulting copolymers. The KLa-based scale-up fermentation was performed successfully in maintaining the yield and the quality of the copolymers produced without a drastic fluctuation. This suggests that the scale-up based on the KLa values supported the fermentation system of P(3HB-co-4HB) copolymer production in single-stage using mixed-substrate cultivation strategy.
Amauroderma rugosum is a wild mushroom species widely distributed in tropics and is classified under the class of Basidiomycetes. Basidiomycetes are well-known for their abilities of producing lignocellulolytic enzymes such as lignin peroxidase (LiP), laccase (Lac) and manganese peroxidase (MnP). Different factors such as nutrient sources, incubation period and agitation affect the production of lignocellulolytic enzymes. The A. rugosum produced LiP in the medium supplemented with potato dextrose broth (PDB), 0.5% yeast and 1.0% saw dust at 26.70±3.31 U/mL. However, the LiP activity was increased to 106.32±5.32 U/mL when supplemented with 150 μm of copper (CuSO4). The aqueous two-phase system (ATPS) is a simple, rapid and low cost method for primary extraction and recovery of LiP. A total of 25 systems made from five different molecular weights of polyethylene glycol (PEG)/dipotassium hydrogen phosphate (K2HPO4) were tested. PEG 600 produced the highest top phase purification factor (PFT) of 1.33±0.62 with yield of 72.18±8.50%. The optimization of the ATPS parameters, such as volume ratio VR, pH and crude enzyme loading are the factors controlling the phase partition. Our results showed that significant improvement (PFT of 6.26±2.87 with yield of 87.31±3.14%) of LiP recovery can be achieved by optimized the parameters.
Polyhydroxyalkanoates (PHAs), a class of renewable and biodegradable green polymers, have gained attraction as a potential substitute for the conventional plastics due to the increasing concern towards environmental pollution as well as the rapidly depleting petroleum reserve. Nevertheless, the high cost of downstream processing of PHA has been a bottleneck for the wide adoption of PHAs. Among the options of PHAs recovery techniques, aqueous two-phase extraction (ATPE) outshines the others by having the advantages of providing a mild environment for bioseparation, being green and non-toxic, the capability to handle a large operating volume and easily scaled-up. Utilizing unique properties of thermo-responsive polymer which has decreasing solubility in its aqueous solution as the temperature rises, cloud point extraction (CPE) is an ATPE technique that allows its phase-forming component to be recycled and reused. A thorough literature review has shown that this is the first time isolation and recovery of PHAs from Cupriavidus necator H16 via CPE was reported. The optimum condition for PHAs extraction (recovery yield of 94.8% and purification factor of 1.42 fold) was achieved under the conditions of 20 wt/wt % ethylene oxide-propylene oxide (EOPO) with molecular weight of 3900 g/mol and 10 mM of sodium chloride addition at thermoseparating temperature of 60°C with crude feedstock limit of 37.5 wt/wt %. Recycling and reutilization of EOPO 3900 can be done at least twice with satisfying yield and PF. CPE has been demonstrated as an effective technique for the extraction of PHAs from microbial crude culture.
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.
The partitioning behavior of immunoglobulin G (IgG) in the aqueous two-phase system (ATPS) composed of poly(ethylene glycol) (PEG) and phosphate was studied. The parameters of ATPS exhibiting the pronounced effects on the partitioning behavior of IgG include phase composition, PEG molecular weight, and the addition of sodium chloride (NaCl). The accumulation of IgG at the interface of the ATPS increased drastically as the tie-line length (TLL) was increased. This trend was correlated with a linear relationship relating the natural logarithm of interfacial partition coefficient (ln G) to the difference of PEG concentration between the top phase and the bottom phase (Δ[PEG]), and a good fit was obtained. An attempt was made to correlate the natural logarithm of partition coefficient (ln K) to the presence of NaCl with the proposed linear relationship, ln K = α″ ln [Cl(-)] + β″. The proposed relationship, which serves as a better description of the underlying mechanics of the protein partitioning behavior in the polymer-salt ATPS, provides a good fit (r(2) > 0.95) for the data of IgG partitioning. An optimum recovery of 99.97% was achieved in an ATPS (pH 7.5) composed of 14.0% (w/w) PEG 1450, 12.5% (w/w) phosphate and 5.0% (w/w) NaCl.
The feasible use of aqueous two-phase systems (ATPSs) to establish a viable protocol for the recovery of laccase from processed Hericium erinaceus (Bull.:Fr.) Pers. fruiting bodies was evaluated. Cold-stored (4.00±1.00°C) H. erinaceus recorded the highest laccase activities of 2.02±0.04 U/mL among all the processed techniques. The evaluation was carried out in twenty-five ATPSs, which composed of polyethylene glycol (PEG) with various molecular weights and potassium phosphate salt solution to purify the protein from H. erinaceus. Optimum recovery condition was observed in the ATPS which contained 17% (w/w) PEG with a molecular weight of 8000 and 12.2% (w/w) potassium phosphate solution, at a volume ratio (VR) of 1.0. The use of ATPS resulted in one-single primary recovery stage process that produced an overall yield of 99% with a purification factor of 8.03±0.46. The molecular mass of laccases purified from the bottom phase was in the range of 55-66 kDa. The purity of the partitioned laccase was confirmed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Aqueous two-phase system (ATPS) extractive bioconversion provides a technique which integrates bioconversion and purification into a single step process. Extractive bioconversion of gamma-cyclodextrin (γ-CD) from soluble starch with cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) enzyme derived from Bacillus cereus was evaluated using polyethylene glycol (PEG)/potassium phosphate based on ATPS. The optimum condition was attained in the ATPS constituted of 30.0% (w/w) PEG 3000 g/mol and 7.0% (w/w) potassium phosphate. A γ-CD concentration of 1.60 mg/mL with a 19% concentration ratio was recovered after 1 h bioconversion process. The γ-CD was mainly partitioned to the top phase (YT=81.88%), with CGTase partitioning in the salt-rich bottom phase (KCGTase=0.51). Repetitive batch processes of extractive bioconversion were successfully recycled three times, indicating that this is an environmental friendly and a cost saving technique for γ-CD production and purification.
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.
Purification of cyclodextrin glycosyl transferase (CGTase) from Bacillus cereus using polyethylene glycol (PEG)-potassium phosphates aqueous two-phase flotation (ATPF) system was studied in this paper. The effects of varying PEG molecular weight, tie-line length (TLL) value, volume ratio (VR), pH value, crude concentration and gas nitrogen flotation time were investigated. The optimal condition for purification of CGTase was attained at 18.0% (w/w) PEG 8000, 7.0% (w/w) potassium phosphates, VR of 3.0, 20% (w/w) crude load at pH 7, and 80 min nitrogen flotation time at a flow rate of 5 L/min. With this optimal condition, purification factor (PFT) of 21.8 and a yield (YT) of 97.1% were attained. CGTase was successfully purified in a single downstream processing step using the ATPF.
In this paper, a linear relationship is proposed relating the natural logarithm of partition coefficient, ln K for protein partitioning in poly (ethylene glycol) (PEG)-phosphate aqueous two-phase system (ATPS) to the square of tie-line length (TLL(2)). This relationship provides good fits (r(2) > 0.98) to the partition of bovine serum albumin (BSA) in PEG (1450 g/mol, 2000 g/mol, 3350 g/mol, and 4000 g/mol)-phosphate ATPS with TLL of 25.0-50.0% (w/w) at pH 7.0. Results also showed that the plot of ln K against pH for BSA partitioning in the ATPS containing 33.0% (w/w) PEG1450 and 8.0% (w/w) phosphate with varied working pH between 6.0 and 9.0 exhibited a linear relationship which is in good agreement (r(2) = 0.94) with the proposed relationship, ln K = α' pH + β'. These results suggested that both the relationships proposed could be applied to correlate and elucidate the partition behavior of biomolecules in the polymer-salt ATPS. The influence of other system parameters on the partition behavior of BSA was also investigated. An optimum BSA yield of 90.80% in the top phase and K of 2.40 was achieved in an ATPS constituted with 33.0% (w/w) PEG 1450 and 8.0% (w/w) phosphate in the presence of 8.5% (w/w) sodium chloride (NaCl) at pH 9.0 for 0.3% (w/w) BSA load.
The present study aimed to optimize the conditions for the production of adventitious roots from Eurycoma longifolia Jack, an important medicinal woody plant, in bioreactor culture. The effects of the type and concentration of auxin on root growth were studied, as well as the effects of the NH4(+):NO3(-) ratio on adventitious root growth and the production of phenolics and flavonoids. Approximately 5 g L(-1) fresh weight of adventitious roots was inoculated into a 3 L balloon-type bubble bioreactor, which contained 2 L 3/4 MS medium supplemented with 30 g L(-1) sucrose and cultures were maintained in the dark for 7 weeks at 24 ± 1°C. Higher concentrations of IBA (7.0 and 9.0 mg L(-1)) and NAA (5.0 mg L(-1)) enhanced the biomass and accumulation of total phenolics and flavonoids. The adventitious roots were thin, numerous, and elongated in 3/4 MS medium supplemented with 5.0 and 7.0 mg L(-1) IBA, whereas the lateral roots were shorter and thicker with 5.0 mg L(-1) NAA compared with IBA treatment. The optimum biomasses of 50.22 g L(-1) fresh weight and 4.60 g L(-1) dry weight were obtained with an NH4(+):NO3(-) ratio of 15:30. High phenolic and flavonoid productions (38.59 and 11.27 mg L(-1) medium, respectively) were also obtained with a ratio of 15:30. Analysis of the 2,2-diphenyl-1-picrylhydrazyl (DPPH)-scavenging activity indicated higher antioxidant activity with an NH4(+):NO3(-) ratio of 30:15. These results suggest that balloon-type bubble bioreactor cultures are suitable for the large-scale commercial production of E. longifolia adventitious roots which contain high yield of bioactive compounds.
Metabolic engineering is a research field that focuses on the design of models for metabolism, and uses computational procedures to suggest genetic manipulation. It aims to improve the yield of particular chemical or biochemical products. Several traditional metabolic engineering methods are commonly used to increase the production of a desired target, but the products are always far below their theoretical maximums. Using numeral optimisation algorithms to identify gene knockouts may stall at a local minimum in a multivariable function. This paper proposes a hybrid of the artificial bee colony (ABC) algorithm and the minimisation of metabolic adjustment (MOMA) to predict an optimal set of solutions in order to optimise the production rate of succinate and lactate. The dataset used in this work was from the iJO1366 Escherichia coli metabolic network. The experimental results include the production rate, growth rate and a list of knockout genes. From the comparative analysis, ABCMOMA produced better results compared to previous works, showing potential for solving genetic engineering problems.