Effective optimization of microalgae-to-bioethanol process systems hinges on an in-depth characterization of key process parameters relevant to the overall bioprocess engineering. One of the such important variables is the biomass particle size distribution and the effects on saccharification levels and bioethanol titres. This study examined the effects of three different microalgal biomass particle size ranges, 35 μm ≤ x ≤ 90 μm, 125 μm ≤ x ≤ 180 μm, and 295 μm ≤ x ≤ 425 μm, on the degree of enzymatic hydrolysis and bioethanol production. Two scenarios were investigated: single enzyme hydrolysis (cellulase) and double enzyme hydrolysis (cellulase and cellobiase). The glucose yield from biomass in the smallest particle size range (35 μm ≤ x ≤ 90 μm) was the highest, 134.73 mg glucose/g algae, while the yield from biomass in the larger particle size range (295 μm ≤ x ≤ 425 μm) was 75.45 mg glucose/g algae. A similar trend was observed for bioethanol yield, with the highest yield of 0.47 g EtOH/g glucose obtained from biomass in the smallest particle size range. The results have shown that the microalgal biomass particle size has a significant effect on enzymatic hydrolysis and bioethanol yield.
Oil palm frond (OPF) juice is a potential industrial fermentation substrate as it has high sugars content and the OPF are readily available daily. However, maximum sugars yield and storage stability of the OPF juice are yet to be determined. This study was conducted to determine the effect of physical pretreatment and storage duration of OPF petiole on sugars yield. Storage stability of OPF juice at different storing conditions was also investigated. It was found that OPF petiole squeezed by hydraulic pressing machine gave the highest sugars recovery at almost 40 g/kg, accounting for a recovery yield of 88%. Storage of OPF petiole up to 72 hrs prior to squeezing reduced the free sugars by 11 g/kg. Concentrated OPF juice with 95% water removal had the best storage stability at both 4 and 30°C, when it was stored for 10 days. Moreover, concentrated OPF syrup prepared by thermal processing did not give any Maillard effect on microbial growth. Based on our results, OPF juice meets all the criteria as a good fermentation substrate as it is renewable, consistently available, and easy to be obtained, it does not inhibit microbial growth and product formation, and it contains no impurities.
Volumetric mass transfer coefficient (kLa) is an important parameter in bioreactors handling viscous fermentations such as xanthan gum production, as it affects the reactor performance and productivity. Published literatures showed that adding an organic phase such as hydrocarbons or vegetable oil could increase the kLa. The present study opted for palm oil as the organic phase as it is plentiful in Malaysia. Experiments were carried out to study the effect of viscosity, gas holdup, and kLa on the xanthan solution with different palm oil fractions by varying the agitation rate and aeration rate in a 5 L bench-top bioreactor fitted with twin Rushton turbines. Results showed that 10% (v/v) of palm oil raised the kLa of xanthan solution by 1.5 to 3 folds with the highest kLa value of 84.44 h(-1). It was also found that palm oil increased the gas holdup and viscosity of the xanthan solution. The kLa values obtained as a function of power input, superficial gas velocity, and palm oil fraction were validated by two different empirical equations. Similarly, the gas holdup obtained as a function of power input and superficial gas velocity was validated by another empirical equation. All correlations were found to fit well with higher determination coefficients.
This research was carried out to evaluate the effects of flavone, myricetin, naringin, catechin, rutin, quercetin, and kaempferol at the concentration of 4.5% of the substrate (dry matter basis) on the rumen microbial activity in vitro. Mixture of guinea grass and concentrate (60 : 40) was used as the substrate. The results showed that all the flavonoids except naringin and quercetin significantly (P < 0.05) decreased the dry matter degradability. The gas production significantly (P < 0.05) decreased by flavone, myricetin, and kaempferol, whereas naringin, rutin, and quercetin significantly (P < 0.05) increased the gas production. The flavonoids suppressed methane production significantly (P < 0.05). The total VFA concentration significantly (P < 0.05) decreased in the presence of flavone, myricetin, and kaempferol. All flavonoids except naringin and quercetin significantly (P < 0.05) reduced the carboxymethyl cellulase, filter paperase, xylanase, and β -glucosidase activities, purine content, and the efficiency of microbial protein synthesis. Flavone, myricetin, catechin, rutin, and kaempferol significantly (P < 0.05) reduced the population of rumen microbes. Total populations of protozoa and methanogens were significantly (P < 0.05) suppressed by naringin and quercetin. The results of this research demonstrated that naringin and quercetin at the concentration of 4.5% of the substrate (dry matter basis) were potential metabolites to suppress methane production without any negative effects on rumen microbial fermentation.
The study is to identify the extraction of intracellular inulinase (exo- and endoinulinase) and invertase as well as optimization medium composition for maximum productions of intra- and extracellular enzymes from Aspergillus niger ATCC 20611. From two different methods for extraction of intracellular enzymes, ultrasonic method was found more effective. Response surface methodology (RSM) with a five-variable and three-level central composite design (CCD) was employed to optimize the medium composition. The effect of five main reaction parameters including sucrose, yeast extract, NaNO₃, Zn⁺², and Triton X-100 on the production of enzymes was analyzed. A modified quadratic model was fitted to the data with a coefficient of determination (R²) more than 0.90 for all responses. The intra-extracellular inulinase and invertase productions increased in the range from 16 to 8.4 times in the optimized medium (10% (w/v) sucrose, 2.5% (w/v) yeast extract, 2% (w/v) NaNO₃, 1.5 mM (v/v) Zn⁺², and 1% (v/v) Triton X-100) by RSM and from around 1.2 to 1.3 times greater than in the medium optimized by one-factor-at-a-time, respectively. The results of bioprocesses optimization can be useful in the scale-up fermentation and food industry.
This study presents the effect of carbon to nitrogen ratio (C/N) (mol/mol) on the cell growth and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) accumulation by Comamonas sp. EB172 in 2 L fermenters using volatile fatty acids (VFA) as the carbon source. This VFA was supplemented with ammonium sulphate and yeast extract in the feeding solution to achieve C/N (mol/mol) 5, 15, 25, and 34.4, respectively. By extrapolating the C/N and the source of nitrogen, the properties of the polymers can be regulated. The number average molecular weight (M n ) of P(3HB-co-3HV) copolymer reached the highest at 838 × 10(3) Da with polydispersity index (PDI) value of 1.8, when the culture broth was supplemented with yeast extract (C/N 34.4). Tensile strength and Young's modulus of the copolymer containing 6-8 mol% 3HV were in the ranges of 13-14.4 MPa and 0.26-0.34 GPa, respectively, comparable to those of polyethylene (PE). Thus, Comamonas sp. EB172 has shown promising bacterial isolates producing polyhydroxyalkanoates from renewable carbon materials.
Lovastatin, a natural byproduct of some fungi, is able to inhibit HMG-CoA (3-hydroxy-3 methyl glutaryl CoA) reductase. This is a key enzyme involved in isoprenoid synthesis and essential for cell membrane formation in methanogenic Archaea. In this paper, experiments were designed to test the hypothesis that lovastatin secreted by Aspergillus terreus in fermented rice straw extracts (FRSE) can inhibit growth and CH4 production in Methanobrevibacter smithii (a test methanogen). By HPLC analysis, 75% of the total lovastatin in FRSE was in the active hydroxyacid form, and in vitro studies confirmed that this had a stronger effect in reducing both growth and CH4 production in M. smithii compared to commercial lovastatin. Transmission electron micrographs revealed distorted morphological divisions of lovastatin- and FRSE-treated M. smithii cells, supporting its role in blocking normal cell membrane synthesis. Real-time PCR confirmed that both commercial lovastatin and FRSE increased (P < 0.01) the expression of HMG-CoA reductase gene (hmg). In addition, expressions of other gene transcripts in M. smithii. with a key involvement in methanogenesis were also affected. Experimental confirmation that CH4 production is inhibited by lovastatin in A. terreus-fermented rice straw paves the way for its evaluation as a feed additive for mitigating CH4 production in ruminants.
Mangrove derived microorganisms constitute a rich bioresource for bioprospecting of bioactive natural products. This study explored the antioxidant potentials of Streptomyces bacteria derived from mangrove soil. Based on 16S rRNA phylogenetic analysis, strain MUM292 was identified as the genus Streptomyces. Strain MUM292 showed the highest 16S rRNA gene sequence similarity of 99.54% with S. griseoruber NBRC12873T. Furthermore, strain MUM292 was also characterized and showed phenotypic characteristics consistent with Streptomyces bacteria. Fermentation and extraction were performed to obtain the MUM292 extract containing the secondary metabolites of strain MUM292. The extract displayed promising antioxidant activities, including DPPH, ABTS, and superoxide radical scavenging and also metal-chelating activities. The process of lipid peroxidation in lipid-rich product was also retarded by MUM292 extract and resulted in reduced MDA production. The potential bioactive constituents of MUM292 extract were investigated using GC-MS and preliminary detection showed the presence of pyrazine, pyrrole, cyclic dipeptides, and phenolic compound in MUM292 extract. This work demonstrates that Streptomyces MUM292 can be a potential antioxidant resource for food and pharmaceutical industries.
Corn was inoculated with Lactobacillus plantarum and Propionibacterium freudenreichii subsp. shermanii either independently or as a mixture at ensiling, in order to determine the effect of bacterial additives on corn silage quality. Grain corn was harvested at 32-37% of dry matter and ensiled in a 4 L laboratory silo. Forage was treated as follows: bacterial types: B0 (without bacteria-control), B1 (L. plantarum), B2 (P. freudenreichii subsp. shermanii), and B3 (combination of L. plantarum and P. freudenreichii subsp. shermanii). Each 2 kg of chopped forage was treated with 10 mL of bacterial culture and allowed to ferment for 27 days. The first experiment determined the most suitable wavelength for detection of bacteria (490 nm and 419 nm for B1 and B2, resp.) and the preferable inoculation size (1 × 105 cfu/g). The second experiment analysed the effect of B1 and B2 applied singly or as a mixture on the fermentation characteristics and quality of corn silage. L. plantarum alone increased crude protein (CP) and reduced pH rapidly. In a mixture with P. freudenreichii, the final pH was the lowest compared to other treatments. As a mixture, inclusion of bacteria resulted in silage with lower digestibility than control. Corn silage treated with L. plantarum or P. freudenreichii either alone or mixed together produced desirable silage properties; however, this was not significantly better than untreated silage.
Carotenoids produced by microbial sources are of industrial and medicinal importance due to their antioxidant and anticancer properties. In the current study, optimization of β-carotene production in M. circinelloides strain 277.49 was achieved using response surface methodology (RSM). Cerulenin and ketoconazole were used to inhibit fatty acids and the sterol biosynthesis pathway, respectively, in order to enhance β-carotene production by diverting metabolic pool towards the mevalonate pathway. All three variables used in screening experiments were found to be significant for the production of β-carotene. The synergistic effect of the C/N ratio, cerulenin, and ketoconazole was further evaluated and optimized for superior β-carotene production using central composite design of RSM. Our results found that the synergistic combination of C/N ratios, cerulenin, and ketoconazole at different concentrations affected the β-carotene productions significantly. The optimal production medium (std. order 11) composed of C/N 25, 10 μg/mL cerulenin, and 150 mg/L ketoconazole, producing maximum β-carotene of 4.26 mg/L (0.43 mg/g) which was 157% greater in comparison to unoptimized medium (1.68 mg/L, 0.17 mg/g). So, it was concluded that metabolic flux had been successfully redirected towards the mevalonate pathway for enhanced β-carotene production in CBS 277.49.
Cyanobacteria are widely distributed Gram-negative bacteria with a long evolutionary history and the only prokaryotes that perform plant-like oxygenic photosynthesis. Cyanobacteria possess several advantages as hosts for biotechnological applications, including simple growth requirements, ease of genetic manipulation, and attractive platforms for carbon neutral production process. The use of photosynthetic cyanobacteria to directly convert carbon dioxide to biofuels is an emerging area of interest. Equipped with the ability to degrade environmental pollutants and remove heavy metals, cyanobacteria are promising tools for bioremediation and wastewater treatment. Cyanobacteria are characterized by the ability to produce a spectrum of bioactive compounds with antibacterial, antifungal, antiviral, and antialgal properties that are of pharmaceutical and agricultural significance. Several strains of cyanobacteria are also sources of high-value chemicals, for example, pigments, vitamins, and enzymes. Recent advances in biotechnological approaches have facilitated researches directed towards maximizing the production of desired products in cyanobacteria and realizing the potential of these bacteria for various industrial applications. In this review, the potential of cyanobacteria as sources of energy, bioactive compounds, high-value chemicals, and tools for aquatic bioremediation and recent progress in engineering cyanobacteria for these bioindustrial applications are discussed.
Lower concentration of glucose was often obtained from enzymatic hydrolysis process of agricultural residue due to complexity of the biomass structure and properties. High substrate load feed into the hydrolysis system might solve this problem but has several other drawbacks such as low rate of reaction. In the present study, we have attempted to enhance glucose recovery from agricultural waste, namely, "sago hampas," through three cycles of enzymatic hydrolysis process. The substrate load at 7% (w/v) was seen to be suitable for the hydrolysis process with respect to the gelatinization reaction as well as sufficient mixture of the suspension for saccharification process. However, this study was focused on hydrolyzing starch of sago hampas, and thus to enhance concentration of glucose from 7% substrate load would be impossible. Thus, an alternative method termed as cycles I, II, and III which involved reusing the hydrolysate for subsequent enzymatic hydrolysis process was introduced. Greater improvement of glucose concentration (138.45 g/L) and better conversion yield (52.72%) were achieved with the completion of three cycles of hydrolysis. In comparison, cycle I and cycle II had glucose concentration of 27.79 g/L and 73.00 g/L, respectively. The glucose obtained was subsequently tested as substrate for bioethanol production using commercial baker's yeast. The fermentation process produced 40.30 g/L of ethanol after 16 h, which was equivalent to 93.29% of theoretical yield based on total glucose existing in fermentation media.
Citric acid (CA) has a high demand due to its various uses in the food and pharmaceutical industries. However, the natural supply of CA is minimal compared to its growing industrial demand. The increasing demand for CA can be fulfilled by using biotechnological processes. This study utilized liquid state bioconversion by Aspergillus niger for CA production using sugarcane molasses as the primary substrate. Sugarcane molasses which is agricultural waste consists of significant proportion of organic matters such as lipids and carbohydrates. This makes sugarcane molasses as a potential and alternative source of producing CA at a lower cost. In this study, statistical optimization was applied to improve CA production using submerged fermentation in shake flasks. Aspergillus niger was cultured in potato dextrose agar. Then, inoculum spores were introduced into the fermentation media for a specific duration according to the experimental design from Central Composite Design (CCD) tool under Response Surface Methodology (RSM) in Design Expert 6.0 software. Three parameters were chosen to be optimized at 32⁰C i.e.agitation rate (160, 80, 200 rpm), substrate concentration (47, 60, 73%) and fermentation time (24, 72, 120 h). High Performance Liquid Chromatography (HPLC)and Fourier-transform infrared spectroscopy(FTIR) analyses were conducted to measure CA yield. The optimization study showed that the media incubated for 72 hours with a substrate concentration of 60% and an agitation speed of 180 rpm produced the highest CA yield(21.2 g/L).The analysis of variance (ANOVA) also showed that CCD quadratic model was significant with P-value< 0.0104 and R2is0.8964.
A therapeutic approach for treating diabetes is to decrease thepost-prandial hyperglycaemia. This is done by retarding the absorption of glucose through the inhibition of carbohydrate hydrolyzing enzymes, α-amylaseand α-glucosidase, in the digestive tract. Inhibition of both enzymes helpsto reduce the glucose level in the blood of a diabetic patient. This study was aimed to investigate the production of α-glucosidase and α-amylase inhibitors from local fruit wastes (honeydew skin, banana peel, and pineapple skin) using solid state fermentation. Each of the fruit wastes was fermented with three different types of white rot fungus Phenarochaete chrysosporium(PC), Panus tigrinusM609RQY(M6) andRO209RQY(RO2)for 7 days. Sampling was carried out starting from day 4 to day 7 to determine the enzyme inhibition activity. The samples were extracted using water prior to enzyme analysis. Most of the fruit samples showed varying degree of percentage inhibition activity depending on the sampling time. Extract of fermented banana peels with RO2 on day 4 showed the higherα-glucosidase inhibition (56.57±0.32%), followed byhoneydew extract fermented with the same fungus on the same day (39.68±0.05%). Extracts of each fruit wastesample fermented with PCshowed the least α-glucosidase inhibition (below 15%). Meanwhile for α-amylase inhibition activity, the extract from fermented honeydew skins with PCon day7 showed the highest inhibition activity i.e.98.29±0.63%. The least inhibition activity (43.37±0.54%) was observed in the extract from honeydew skins fermented withM6 on day 5. All positive resultsshowed that fruit wastes could be the alternative sourcesfor antidiabetic agent especially for α-amylase and α-glucosidase inhibitors.
A xylanase gene (xyn2) from Trichoderma reesei ATCC 58350 was previously cloned and expressed in Kluyveromyces lactis GG799. The production of the recombinant xylanase was conducted in a developed medium with an optimised batch and with fed-batches that were processed with glucose. The glucose served as a carbon source for cell growth and as an inducer for xylanase production. In a 1-L batch system, a glucose concentration of 20 g L(-1) and 80 % dissolved oxygen were found to provide the best conditions for the tested ranges. A xylanase activity of 75.53 U mL(-1) was obtained. However, in the batch mode, glucose depletions reduced the synthesis of recombinant xylanase by K. lactis GG799. To maximise the production of xylanase, further optimisation was performed using exponential feeding. We investigated the effects of various nitrogen sources combined with the carbon to nitrogen (C/N) molar ratio on the production of xylanase. Of the various nitrogen sources, yeast extract was found to be the most useful for recombinant xylanase production. The highest xylanase production (110.13 U mL(-1)) was measured at a C/N ratio of 50.08. These conditions led to a 45.8 % increase in xylanase activity compared with the batch cultures. Interestingly, the further addition of 500 g L(-1) glucose led to a 6.2-fold increase (465.07 U mL(-1)) in recombinant xylanase activity. These findings, together with those of the exponential feeding strategy, indicate that the composition of the C/N molar ratio has a substantial impact on recombinant protein production in K. lactis.
This review is focused on the production of microbial lipases by high cell density fermentation. Lipases are among the most widely used of the enzyme catalysts. Although lipases are produced by animals and plants, industrial lipases are sourced almost exclusively from microorganisms. Many of the commercial lipases are produced using recombinant species. Microbial lipases are mostly produced by batch and fed-batch fermentation. Lipases are generally secreted by the cell into the extracellular environment. Thus, a crude preparation of lipases can be obtained by removing the microbial cells from the fermentation broth. This crude cell-free broth may be further concentrated and used as is, or lipases may be purified from it to various levels. For many large volume applications, lipases must be produced at extremely low cost. High cell density fermentation is a promising method for low-cost production: it allows a high concentration of the biomass and the enzyme to be attained rapidly and this eases the downstream recovery of the enzyme. High density fermentation enhances enzyme productivity compared with the traditional submerged culture batch fermentation. In production of enzymes, a high cell density is generally achieved through fed-batch operation, not through perfusion culture which is cumbersome. The feeding strategies used in fed-batch fermentations for producing lipases and the implications of these strategies are discussed. Most lipase-producing microbial fermentations require oxygen. Oxygen transfer in such fermentations is discussed.
Oil palm fronds are the most abundant lignocellulosic biomass in Malaysia. In this study, fronds were tested as the potential renewable biomass for ethanol production. The soaking in aqueous ammonia pretreatment was applied, and the fermentability of pretreated fronds was evaluated using simultaneous saccharification and fermentation. The optimal pretreatment conditions were 7 % (w/w) ammonia, 80 °C, 20 h of pretreatment, and 1:12 S/L ratio, where the enzymatic digestibility was 41.4 % with cellulase of 60 FPU/g-glucan. When increasing the cellulase loading in the hydrolysis of pretreated fronds, the enzymatic digestibility increased until the enzyme loading reached 60 FPU/g-glucan. With 3 % glucan loading in the SSF of pretreated fronds, the ethanol concentration and yield based on the theoretical maximum after 12 and 48 h of the SSF were 7.5 and 9.7 g/L and 43.8 and 56.8 %, respectively. The ethanol productivities found at 12 and 24 h from pretreated fronds were 0.62 and 0.36 g/L/h, respectively.
This study aimed to enhance the crystallizability of bio-based succinic acid for its efficient recovery while maintaining the end product at the highest purity. Immobilization of Actinobacillus succinogenes was initially evaluated based on three different carriers: volcanic glass, clay pebbles, and silica particles. The adsorption capacity of metabolites with a low concentration (10 g/L) and a high concentration (40 g/L) was investigated. It was demonstrated that clay pebbles adsorbed the least succinic acid (
Sago hampas is a starch-based biomass from sago processing industries consisted of 58% remaining starch. This study has demonstrated the bioconversion of sago hampas to volatile fatty acids (VFAs) by Clostridium beijerinckii SR1 via anaerobic digestion. Higher total VFAs were obtained from sago hampas (5.04 g/L and 0.287 g/g) as compared to commercial starch (5.94 g/L and 0.318 g/g). The physical factors have been investigated for the enhancement of VFAs production using one-factor-at-a-time (OFAT). The optimum condition; 3% substrate concentration, 3 g/L of yeast extract concentration and 2 g/L of ammonium nitrate enhanced the production of VFAs by 52.6%, resulted the total VFAs produced is 7.69 g/L with the VFAs yield of 0.451 g/g. VFAs hydrolysate produced successfully generated 273.4 mV of open voltage circuit and 61.5 mW/m2 of power density in microbial fuel cells. It was suggested that sago hampas provide as an alternative carbon feedstock for bioelectricity generation.
The present study focused on lipopeptide biosurfactant production by Streptomyces sp. PBD-410L in batch and fed-batch fermentation in a 3-L stirred-tank reactor (STR) using palm oil as a sole carbon source. In batch cultivation, the impact of bioprocessing parameters, namely aeration rate and agitation speed, was studied to improve biomass growth and lipopeptide biosurfactant production. The maximum oil spreading technique (OST) result (45 mm) which corresponds to 3.74 g/L of biosurfactant produced, was attained when the culture was agitated at 200 rpm and aeration rate of 0.5 vvm. The best aeration rate and agitation speed obtained from the batch cultivation was adopted in the fed-batch cultivation using DO-stat feeding strategy to further improve the lipopeptide biosurfactant production. The lipopeptide biosurfactant production was enhanced from 3.74 to 5.32 g/L via fed-batch fermentation mode at an initial feed rate of 0.6 mL/h compared to that in batch cultivation. This is the first report on the employment of fed-batch cultivation on the production of biosurfactant by genus Streptomyces.