The importance and development of industrial biotechnology processing has led to the utilisation of microbial enzymes in various applications. One of the important enzymes is amylase, which hydrolyses starch to glucose. In Malaysia, the use of sago starch has been increasing, and it is presently being used for the production of glucose. Sago starch represents an alternative cheap carbon source for fermentation processes that is attractive out of both economic and geographical considerations. Production of fermentable sugars from the hydrolysis of starches is normally carried out by an enzymatic processes that involves two reaction steps, liquefaction and saccharification, each of which has different temperature and pH optima with respect to the maximum reaction rate. This method of starch hydrolysis requires the use of an expensive temperature control system and a complex mixing device. Our laboratory has investigated the possibility of using amylolytic enzyme-producing microorganisms in the continuous single-step biological hydrolysis of sago flour for the production of a generic fermentation medium. The ability of a novel DNA-recombinated yeast, Saccharomyces cerevisiae strain YKU 107 (expressing alpha-amylase production) to hydrolyse gelatinised sago starch production has been studied with the aim of further utilizing sago starch to obtain value-added products.
Chemostat cultures of NS0 cell lines were carried out at dilution rates ranging from 0.8 d(-1) to 0.2 d(-1). Compared with the control, the viable cell density of the Bcl-2 cell line was approximately 10% higher at 0.8 d(-1) and increased to 55% when the dilution rate was reduced to 0.2 d(-1). As the dilution rate was reduced, the viability of the two cultures diverged reaching a difference of 43% at 0.2 d(-1). The specific growth rate of the control cells was the same as the dilution rate down to a value of 0.6 d(-1). By contrast, the specific growth rate of Bcl-2 cells was parallel to the dilution rate down to a value as low as 0.3 d(-1). For both NS0 cell lines, the G1 cell population decreased, while the S and G2/M cell populations increased as the dilution rate was reduced. The antibody titer of the control cells increased from 7 to 21 microg.ml(-1) as the dilution rate was reduced from 0.8 to 0.2 d(-1). With an initial increase from 2 to 15 microg.ml(-1) as the dilution rate was reduced from 0.8 to 0.4 d(-1), the antibody titer of the Bcl-2 cells remained constant as the dilution rate was further reduced to 0.2 d(-1). A good correlation between specific antibody production rate and the percentage of G2/M cells was observed.
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.
Accumulation of nitrite intermediate in autohydrogenotrophic denitrification process has been a challenging difficulty to tackle. This study showed that further growth of "true denitrifying" bacteria and adaptation to nitrite led to a faster reduction of nitrite than nitrate as a solution to circumvent nitrite accumulation. Moreover, two effective parameters namely pH and bicarbonate dose were optimized in order to achieve a better reduction rate. Sodium bicarbonate dose ranging from 20 to 2000 mg/L and pH in the range of 6.5-8.5 was selected to be examined employing 0.2 g MLVSS/L of reacclimatized denitrifying bacteria. Eleven runs of experiments were designed considering the interactive effect of these two operative parameters. A fairly close reduction time less than 4.5 h (>22.22 mg NO2(-)-N/g MLVSS/h) was gained for the pH range between 7 and 8. The highest specific nitrite reduction rate at 25 mg NO2(-)-N/g MLVSS/h was achieved applying 1000 mg NaHCO3/L at pH 7.5 and 8. The pH was found to be the leading parameter and bicarbonate as the less effective parameter on nitrite reduction removal. Central composite design (CCD) and response surface design (RSM) were employed to develop a model as well as define the optimum condition. Using the experimental data, the developed quadratic model predicted optimum condition at pH 7.8 and sodium bicarbonate dose 1070 mg/L upon which denitrifiers managed to accomplish reduction within 3.5 h and attained the specific degradation rate of 28.57 mg NO2(-)-N/g MLVSS/h.
Reporter gene activity under the regulation of the oil palm metallothionein-like gene, MT3-A promoter was assessed in prokaryotes. Vector constructs containing MT3-A promoter with (W1MT3-A) and without (W2MT3-A) five prime untranslated region (5'-UTR) fused to ß-glucuronidase (GUS) gene in pCAMBIA 1304 vector were produced. 5'-rapid amplification of cDNA ends (RACE) using mRNA isolated from Escherichia coli and Agrobacterium tumefaciens harboring W1MT3-A confirmed that fusion transcripts of MT3-A 5'-UTR-GUS were successfully produced in both bacteria. Competitive PCR and GUS fluorometric assay showed changes in the level of GUS gene transcripts and enzyme activity in response to increasing concentrations of Cu²+ and Zn²+. The application of Cu²+ increased GUS activity and GUS mRNA level in both bacteria. In E. coli, a high level of GUS activity driven by W1MT3-A and W2MT3-A was observed in treatment with 25 μM Cu²+ resulting in an increase in the GUS mRNA level to 7.2 and 7.5 x 10⁻⁴ pmol/μl respectively, compared to the control (5.1 x 10⁻⁴ pmol/μl). The lowest GUS activity and GUS mRNA level were obtained for W1MT3-A and W2MT3-A in the presence of 100 μM Cu²+ in both bacteria compared to the control (without Cu²+). The application of different Zn²+ concentrations resulted in a strong decrease in the GUS activity and GUS mRNA level in E. coli and A. tumefaciens. These findings showed that the oil palm MT3-A promoter is functional in prokaryotes and produced detectable GUS transcripts and enzyme activities. This promoter may potentially be used in prokaryotic systems which require metal inducible gene expression.
A comparative study on the stability and potential of alginate and pectin based beads for production of poultry probiotic cells using MRS medium in repeated batch fermentation was conducted. The bead cores, made of three types of materials, i.e., ca-alginate, ca-pectinate and ca-alginate/pectinate, were compared. The effect of single and double layer coatings using chitosan and core material, respectively, on the bead stability and cell production were also studied. The pectin based beads were found to be more stable than that of the alginate beads and their stability was further improved by coating with chitosan. The cell concentration in pectin based beads was comparable to that in the alginate beads. On the other hand, pectin based beads gave significantly lower cell concentration in the growth medium for the initial fermentation cycles when compared to the alginate beads. In conclusion, pectin was found to be potential encapsulation material for probiotic cell production owing to its stability and favourable microenvironment for cell growth.
Ectoine production using inexpensive and renewable biomass resources has attracted great interest among the researchers due to the low yields of ectoine in current fermentation approaches that complicate the large-scale production of ectoine. In this study, ectoine was produced from corn steep liquor (CSL) and soybean hydrolysate (SH) in replacement to yeast extract as the nitrogen sources for the fermentation process. To enhance the bacterial growth and ectoine production, biotin was added to the Halomonas salina fermentation media. In addition, the effects addition of surfactants such as Tween 80 and saponin on the ectoine production were also investigated. Results showed that both the CSL and SH can be used as the nitrogen source substitutes in the fermentation media. Higher amount of ectoine (1781.9 mg L-1) was produced in shake flask culture with SH-containing media as compared to CSL-containing media. A total of 2537.0 mg L-1 of ectoine was produced at pH 7 when SH-containing media was applied in the 2 L batch fermentation. Moreover, highest amount of ectoine (1802.0 mg L-1) was recorded in the SH-containing shake flask culture with addition of 0.2 μm mL-1 biotin. This study demonstrated the efficacy of industrial waste as the nutrient supplement for the fermentation of ectoine production.
Nowadays, the pretreatment of wastewater prior to discharge is very important in various industries as the wastewater without any treatment contains high organic pollution loads that would pollute the receiving waterbody and potentially cause eutrophication and oxygen depletion to aquatic life. The reuse of seafood wastewater discharge in microalgae cultivation offers beneficial purposes such as reduced processing cost for wastewater treatment, replenishing ground water basin as well as financial savings for microalgae cultivation. In this paper, the cultivation of Chlorella vulgaris with an initial concentration of 0.01 ± 0.001 g⋅L-1 using seafood sewage discharge under sunlight and fluorescent illumination was investigated in laboratory-scale without adjusting mineral nutrients and pH. The ability of nutrient removal under different lighting conditions, the metabolism of C. vulgaris and new medium as well as the occurrence of auto-flocculation of microalgae biomass were evaluated for 14 days. The results showed that different illumination sources did not influence the microalgae growth, chemical oxygen demand (COD) and biochemical oxygen demand (BOD) significantly. However, the total nitrogen (total-N) and total phosphorus (total-P) contents of microalgae were sensitive to the illumination mode. The amount of COD, BOD, total-N and total-P were decreased by 88%, 81%, 95%, and 83% under sunlight mode and 81%, 74%, 79%, and 72% under fluorescent illumination, respectively. Furthermore, microalgae were auto-flocculated at the final days of cultivation with maximum biomass concentration of 0.49 ± 0.01 g⋅L-1, and the pH value had increased to pH 9.8 ± 0.1 under sunlight illumination.
D-optimal design was employed to optimize the mixture of cross-linking agents formulation: microbial transglutaminase (MTGase) and ribose, and the processing parameters (i.e. incubation and heating time) in the mixture in order to obtain combined-cross-linked bovine serum albumin gels that have high gel strength, pH close to neutral and yet medium in browning. Analysis of variance (ANOVA) showed that the contribution of quadratic term to the model over the linear was significant for pH and L* value, whereas linear model was significant for gel strength. Optimization study using response surface methodology (RSM) was performed to the mixture components and process variables and the optimum conditions obtained were: MTGase of 1.34-1.43 g/100 mL, ribose of 1.07-1.16 g/100 mL, incubation time of 5 h at 40 degrees C and heating time of 3 h at 90 degrees C.
This study focused on kinetics of levan yield by Bacillus subtilis M, in a 150 L stirred tank bioreactor under controlled pH conditions. The optimized production medium was composed of (g/L): commercial sucrose 100.0, yeast extract 2.0, K2HPO4 3.0 and MgSO4⋅7H2O 0.2; an increase in both carbohydrates consumption and cell growth depended on increasing the size of the stirred tank bioreactor from 16 L to 150 L. The highest levansucrase production (63.4 U/mL) and levan yield of 47 g/L was obtained after 24 h. Also, the specific levan yield (Yp/x) which reflects the cell productivity increased with the size increase of the stirred tank bioreactor and reached its maximum value of about 29.4 g/g cells. These results suggested that B. subtilis M could play an important role in levan yield on a large scale in the future. Chemical modifications of B. subtilis M crude levan (CL) into sulfated (SL), phosphorylated (PL), and carboxymethylated levans (CML) were done. The difference in CL structure and its derivatives was detected by FT-IR transmission spectrum. The cytotoxicity of CL and its derivatives were evaluated by HepGII, Mcf-7 and CaCo-2. In general most tested levans forms had no significant cytotoxicity effect. In fact, the carboxymethylated and phosphrylated forms had a lower anti-cancer effect than CL. On the other hand, SL had the highest cytotoxicity showing SL had a significant anti-cancer effect. The results of cytotoxicity and cell viability were statistically analyzed using three-way ANOVA.
The coexisting mechanism of a synthetic bacterial community (SBC) was investigated to better understand how to manage microbial communities. The SBC was constructed with three kinds of phenol-utilizing bacteria, Pseudomonas sp. LAB-08, Comamonas testosteroni R2, and Cupriavidus sp. P-10, under chemostat conditions supplied with phenol as a sole carbon and energy source. Population densities of all strains were monitored by real-time quantitative PCR (qPCR) targeting the gene encoding the large subunit of phenol hydroxylase. Although the supply of phenol was stopped to allow perturbation in the SBC, all of the strains coexisted and the degradation of phenol was maintained for more than 800 days. The qPCR analyses showed that strains LAB-08 and R2 became dominant simultaneously, whereas strain P-10 was a minor population. This phenomenon was observed before and after the phenol-supply stoppage. The kinetic parameters for phenol of the SBC changed before and after the phenol-supply stoppage, which suggests a change in functional roles of strains in the SBC. Transcriptional levels of phenol hydroxylase and catechol dioxygenases of three strains were monitored by reverse-transcription qPCR (RT-qPCR). The RT-qPCR analyses revealed that all strains shared phenol and survived independently before the phenol-supply stoppage. After the stoppage, strain P-10 would incur the cost for degradation of phenol and catechol, whereas strains LAB-08 and R2 seemed to be cheaters using metabolites, indicating the development of the metabolic network. These results indicated that it is important for the management and redesign of microbial communities to understand the metabolism of bacterial communities.
Saccharomyces cerevisiae has a long-standing history of biotechnological applications even before the dawn of modern biotechnology. The field is undergoing accelerated advancement with the recent systems and synthetic biology approaches. In this review, we highlight the recent findings in the field with a focus on omics studies of S. cerevisiae to investigate its stress tolerance in different industries. The latest advancements in S. cerevisiae systems and synthetic biology approaches for the development of genome-scale metabolic models (GEMs) and molecular tools such as multiplex Cas9, Cas12a, Cpf1, and Csy4 genome editing tools, modular expression cassette with optimal transcription factors, promoters, and terminator libraries as well as metabolic engineering. Omics data analysis is key to the identification of exploitable native genes/proteins/pathways in S. cerevisiae with the optimization of heterologous pathway implementation and fermentation conditions. Through systems and synthetic biology, various heterologous compound productions that require non-native biosynthetic pathways in a cell factory have been established via different strategies of metabolic engineering integrated with machine learning.
Human amnion epithelial cells (HAECs) hold great promise in tissue engineering for regenerative medicine. Large numbers of HAECs are required for this purpose. Hence, exogenous growth factor is added to the culture medium to improve epithelial cells proliferation. The aim of the present study was to determine the effects of epidermal growth factor (EGF) on the proliferation and cell cycle regulation of cultured HAECs. HAECs at P1 were cultured for 7 days in medium containing an equal volume mix of HAM's F12: Dulbecco's Modified Eagles Medium (1:1) supplemented with different concentrations of EGF (0, 5, 10, 20, 30 and 50 ng/ml EGF) in reduced serum. Morphology, growth kinetics and cell cycle analysis using flow cytometry were assessed. Quantitative gene expression for cell cycle control genes, pluripotent transcription factors, epithelial genes and neuronal genes were also determined. EGF enhanced HAECs proliferation with optimal concentration at 10 ng/ml EGF. EGF significantly increased the proportion of HAECs at S- and G2/M-phase of the cell cycle compared to the control. At the end of culture, HAECs remained as diploid cells under cell cycle analysis. EGF significantly decreased the mRNA expression of p21, pRb, p53 and GADD45 in cultured HAECs. EGF also significantly decreased the pluripotent genes expression: Oct-3/4, Sox2 and Nanog; epithelial genes expression: CK14, p63, CK1 and Involucrin; and neuronal gene expression: NSE, NF-M and MAP 2. The results suggested that EGF is a strong mitogen that promotes the proliferation of HAECs through cell cycle regulation. EGF did not promote HAECs differentiation or pluripotent genes expression.
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.
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.
The feasibility of using palm oil fractions as cheap and abundant sources of raw material for the synthesis of amino acid surfactants was investigated. Of a number of enzymes screened, the best results were obtained with the immobilized enzyme, Lipozyme. The effects of temperature, solvent, incubation period, fatty substrate/amino acid molar ratio, enzyme amount, and water removal on the reactions were analyzed and compared to those on reactions with free fatty acids and pure triglycerides as fatty substrates. All reactions were most efficient when carried out at high temperatures (70-80 degrees C) in hexane as a solvent. However, while reactions with free fatty acids proceeded better when a slight excess of the free fatty acids over the amino acids was used, reactions with triglycerides and palm oil fractions were best performed at equimolar ratios. Also, the addition of molecular sieves slightly enhanced reactions with free fatty acids but adversely affected reactions with triglycerides and palm oil fractions. Although reactions with palm oil fractions took longer (6 d) to reach equilibrium compared to reactions with free fatty acids (4 d) and pure triglycerides (4 d), better yields were obtained. Such lipase-catalyzed transacylation of palm oil fractions with amino acids is potentially useful in the production of mixed medium- to long-chain surfactants for specific applications.
Lignocellulosic biomass dedicated to bioethanol production usually contains pentoses and inhibitory compounds such as furfural that are not well tolerated by Saccharomyces cerevisiae. Thus, S. cerevisiae strains with the capability of utilizing both glucose and xylose in the presence of inhibitors such as furfural are very important in industrial ethanol production. Under the synergistic conditions of transaldolase (TAL) and alcohol dehydrogenase (ADH) overexpression, S. cerevisiae MT8-1X/TAL-ADH was able to produce 1.3-fold and 2.3-fold more ethanol in the presence of 70 mM furfural than a TAL-expressing strain and a control strain, respectively. We also tested the strains' ability by mimicking industrial ethanol production from hemicellulosic hydrolysate containing fermentation inhibitors, and ethanol production was further improved by 16% when using MT8-1X/TAL-ADH compared to the control strain. Transcript analysis further revealed that besides the pentose phosphate pathway genes TKL1 and TAL1, ADH7 was also upregulated in response to furfural stress, which resulted in higher ethanol production compared to the TAL-expressing strain. The improved capability of our modified strain was based on its capacity to more quickly reduce furfural in situ resulting in higher ethanol production. The co-expression of TAL/ADH genes is one crucial strategy to fully utilize undetoxified lignocellulosic hydrolysate, leading to cost-competitive ethanol production.
The ability of oleaginous yeast Lipomyces starkeyi to efficiently produce lipids when cultivated on sap extracted from felled oil palm trunk (OPT) as a novel inexpensive renewable carbon source was evaluated. OPT sap was found to contain approximately 98 g/L glucose and 32 g/L fructose. Batch fermentations were performed using three different OPT sap medium conditions: regular sap, enriched sap, and enriched sap at pH 5.0. Under all sap medium conditions, the cell biomass and lipid production achieved were approximately 30 g/L and 60% (w/w), respectively. L. starkeyi tolerated acidified medium (initial pH ≈ 3) and produced considerable amounts of ethanol as well as xylitol as by-products. The fatty acid profile of L. starkeyi was remarkably similar to that of palm oil, one of the most common vegetable oil feedstock used in biodiesel production with oleic acid as the major fatty acid followed by palmitic, stearic and linoleic acids.
Gallic acid (GA) is a hydrophilic polyphenol which is noteworthy for strong antioxidant capacity. The drawbacks of conventional extraction approaches such as time-consuming and high processing cost are often viewed as a hurdle to extract GA from plant sources in industrial scale. Aqueous two-phase system (ATPS) is a separation approach which can be employed as an alternative to the conventional approaches. The partition behaviour of GA in an alcohol/salt ATPS was investigated in this study to aid the development of industrial scale ATPS to extract GA from natural sources. The separation of GA was characterized by determining the types of alcohol and salt, phase composition, sample load, pH of the system and addition of adjuvants applied in the alcohol/salt ATPS construction. The hydrophilic GA was targeted to the salt-rich phase of the alcohol/salt ATPS with a partition coefficient (KGA) of 25.00 ± 0.00. The optimum condition of ATPS for the maximum partition of GA was achieved in ATPS comprised of 24% (w/w) 1-propanol and 22% (w/w) phosphate salt at pH 8 with 5% (w/w) of 1 mg/mL sample loading and 2% (w/w) NaCl addition. The findings suggest that ATPS can be applied for separation of GA from various natural sources.
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.