Agricultural residues are rich in bioactive compounds. These residues can be used as an alternate source for the production of different products like biogas, biofuel, mushroom, and tempeh as the raw material in various researches and industries. The use of agro-industrial wastes as raw materials can help to reduce the production cost and also reduce the pollution load from the environment. Agro-industrial wastes are used for manufacturing of biofuels, enzymes, vitamins, antioxidants, animal feed, antibiotics, and other chemicals through solid state fermentation (SSF). A variety of microorganisms are used for the production of these valuable products through SSF processes. Therefore, SSF and their effect on the formation of value-added products are reviewed and discussed.
Protein engineering has been employed to successfully improve organic solvent resistance of enzymes. Exploration of nature’s full potential (how many beneficial positions/beneficial substitutions of the target enzyme) to improve organic solvent resistance of enzymes by a systematic study was performed.
We report the results of screening the previously generated BSLA (Bacillus subtilis lipase A)-SSM (site saturation mutagenesis) library (covering the full natural diversity of BSLA with one amino acid exchange) in presence of three cosolvents. The potential of single amino acid substitution that nature offers to improve the cosolvent resistance of BSLA was determined by analyzing the number of beneficial positions/substitutions, accessibility and chemical compositions.
Lessons learned from analysis of BSLA-SSM library are: (1) 41–59% of BSLA positions with in total 4–10% of all possible substitutions improve the cosolvent resistance against TFE, DOx, and DMSO; (2) charged substitutions are preferred to improve DOx and TFE resistance whereas polar ones are preferred for DMSO; (3) charged substitutions on the surface predominantly improved resistance while polar ones were preferred in buried “regions”. (4) Interestingly, 58–93% of beneficial substitutions led to chemically different amino acids.
The thriving of biodiesel industry has led to produce 10% (v/v) crude glycerol, thus creating an overflow problem. Biofuel production is restricted by Escherichia coli due to its toxicity to bacterial cells. Therefore, a platform chemical and fuel additive 2,3-butanediol (2,3-BD) with low toxicity to microbes could be a promising alternative for biofuel production by recombinant E. coli using glycerol as the sole substrate.
A novel expression system of E. coli was developed to express the dhaD gene encoding glycerol dehydrogenase (GDH) to produce value-added metabolic products through aerobic biotransformation of glycerol. The dhaD gene obtained from Klebsiella pneumoniae SRP2 was expressed in E. coli BL21(DE3)pLysS using an E. coli–K. pneumoniae shuttle vector pJET1.2/blunt consisting of chloramphenicol-resistance gene under the control of the T7lac promotor. RT-PCR analysis and dhaD overexpression confirmed that the 2,3-BD synthesis pathway gene was expressed on RNA and protein levels. Therefore, the recombinant E. coli exhibited a 38.9-fold higher enzyme activity (312.57 units/mg protein), yielding 8.97 g/L 2,3-BD, a 2.4-fold increase with respect to the non-recombinant strain.
The engineered strain E. coli BL21(DE3)pLysS/pJET1.2/blunt-dhaD, carrying the 2,3-BD pathway gene dhaD from our newly isolated Klebsiella pneumoniae SRP2 strain, displayed the best ability to synthesize 2,3-BD from low-cost biomass glycerol. The value of expression of an important glycerol metabolism gene dhaD is the highest ever achieved with an engineered E. coli strain. From these results, the first reported dhaD expression system has paved the way for improvement of 2,3-BD production and is efficient for another heterologous gene expression in E. coli.
Laccases belongs to multinuclear copper-containing oxidase and can act on a variety of aromatic and non-aromatic compounds. Due to their broad substrate specificity, they are considered as a promising candidate in various industrial and biotechnological sectors. They are regarded as a “Green Tool”/“Green Catalyst” in biotechnology. The present review focuses on structure, reaction mechanism, categories, applications, economic feasibility, limitations, and future prospects of fungal laccases. Thus, this review would help in understanding laccases along with the areas, which has not been focused and requires attention. Since past, immense work has been carried out on laccases: yet, new discoveries and application are ever increasing which includes bio-fuel, bio-sensor, fiber board synthesis, bioremediation, clinical, textile industry, food, cosmetics, and many more. Hence, it can be stated that fungal laccase is an enzyme which is “discovered but yet undiscovered”.
Deep eutectic solvents have attracted considerable attention in numerous fields. There is little information on mung bean epoxide hydrolase-catalyzed epoxides in deep eutectic solvent-containing system.
Adding deep eutectic solvents with hydrogen bond donor of acids to phosphate buffer resulted in an obvious decrease in the optical purity of product; nevertheless, the relative slight change was observed by the addition of the deep eutectic solvents with hydrogen bond donor of alcohols and urea. Of the tested deep eutectic solvents, 10% additional amount of choline chloride/trithylene glycol can cause a significant improvement in the enantiopurity of product, from 83.2 ± 1.3% to 87.9 ± 0.3%. Moreover, with the increase in the addition amount of choline chloride/triethylene glycol from 10 to 30%, the enantiomeric excess of product enhanced from 87.9 to 94%, but a decline of product yield was observed. Finally, the evaluation of enzyme stability showed that the additional amount from 10% to 30% was not beneficial to the activity recovery.
In short, adding choline chloride/triethylene glycol contributes to the improvement in the optical purity of (R)-1-phenyl-1, 2-ethanediol in the catalysis of styrene oxide by mung bean epoxide hydrolases; meanwhile, the enzyme immobilization could be essential.
Pretreatment is the key step to overcome the recalcitrance of lignocellulosic biomass making sugars available for subsequent enzymatic hydrolysis and microbial fermentation. During the process of pretreatment and enzymatic hydrolysis as well as fermentation, various toxic compounds may be generated with strong inhibition on cell growth and the metabolic capacity of fermenting strains. Zymomonas mobilis is a natural ethanologenic bacterium with many desirable industrial characteristics, but it can also be severely affected by lignocellulosic hydrolysate inhibitors. In this review, analytical methods to identify and quantify potential inhibitory compounds generated during lignocellulose pretreatment and enzymatic hydrolysis were discussed. The effect of hydrolysate inhibitors on Z. mobilis was also summarized as well as corresponding approaches especially the high-throughput ones for the evaluation. Then the strategies to enhance inhibitor tolerance of Z. mobilis were presented, which include both forward and reverse genetics approaches such as classical and novel mutagenesis approaches, adaptive laboratory evolution, as well as genetic and metabolic engineering. Moreover, this review provided perspectives and guidelines for future developments of robust strains for efficient bioethanol or biochemical production from lignocellulosic materials.
The conversion of CO2 into high value-added products has a very important environmental and economic significance. Microbial electrosynthesis (MES) is a promising technology, which adopts a bioelectrochemical system to transform CO2 into organic chemicals.
In this study, Clostridium scatologenes ATCC 25775T, an anaerobic acetogenic bacterium, demonstrated its utility as a biocatalyst in a MES system, for the first time. With the cathodic potential of the MES system decreased from − 0.6 to − 1.2 V (vs. Ag/AgCl), the current density of the MES, and the production of organic chemicals, increased. Combining the genetic analysis and the results of the wet lab experiments, we believe C. scatologenes may accept electrons directly from the cathode to reduce CO2 into organic compounds at a potential of − 0.6 V. The acetic and butyric acid reached a maximum value of 0.03 and 0.01 g/L, respectively, and the maximum value of total coulombic efficiency was about 84%, at the potential of − 0.6 V. With the decrease in cathodic potentials, both direct electron transfer and exogenous electron shuttle, H2 might be adopted for the C. scatologenes MES system. At a potential of − 1.2 V, acetic acid, butyric acid and ethanol were detected in the cathodic chamber, with their maximum values increasing to 0.44, 0.085 and 0.015 g/L, respectively. However, due to the low H2 utilization rate by the C. scatologenes planktonic cell, the total coulombic efficiency of the MES system dropped to 37.8%.
Clostridium scatologenes is an acetogenic bacterium which may fix CO2 through the Wood–Ljungdahl pathway. Under H2 fermentation, C. scatologenes may reduce CO2 to acetic acid, butyric acid and ethanol. It can also be used as the biocatalyst in MES systems.
Oil leakages frequently occur during oil product development and oil transportation. These incidents are a vital factor in water contamination, thus leading to serious environmental destruction. Therefore, superhydrophobic/superoleophilic material is one of the solutions to treat oily wastewater.
This study aimed to develop a simple, fast and low-cost method to treat oily wastewater by synthesizing a new superhydrophobic/superoleophilic corn straw fiber via conventional impregnation. The corresponding results illustrate that abundant homogeneous silica (SiO2) granules evenly accreted on the surface of the prepared fiber were conducive to high surface roughness. Meanwhile, (Heptadecafluoro-1,1,2,2-tetradecyl) trimethoxysilane, a sort of silane coupling agent, could greatly reduce surface free energy by grafting with SiO2 particles on the corn straw fiber surface. The obtained superhydrophobic/superoleophilic corn straw fiber exhibited a water contact angle of 152° and an oil contact angle of 0° for various oils, strongly demonstrating its considerable application as an oil absorbent that can be applied for oil cleanup. In addition, the prepared fiber displayed a great chemical stability and environmental durability.
Due to its high absorption capacity and absorption efficiency, the prepared fiber has great potential as a new oil absorbent for treatment of oily water.
A NAD(P)H-dependent enoate reductase (OYE2p) from Saccharomyces cerevisiae YJM1341 was discovered by genome data mining for asymmetric reduction of (E/Z)-citral to (R)-citronellal with high enantioselectivity.
This enzyme was heterologously expressed in E. coli and characterized for its biocatalytic properties. OYE2p was identified with reduction activities toward a diverse range of ɑ,β-unsaturated compounds bearing conjugated aldehyde, ketone, imide, carboxylic acid and ester.
OYE2p showed the highest specific activity at 40 °C and a pH optimum at 7.0–8.0. The stability of OYE2p was rather pH-independent, and the half-life time values of the enzyme at pH 6.0–8.0 were more than 257 h. With regard to the reduction of (E)-citral and (Z)-citral, OYE2p exhibited different selectivity patterns. (E)-citral was exclusively reduced to (R)-citronellal by OYE2p in ≥ 99% ee, which was independent on pH. OYE2p produced both enantiomers of citronellal from (Z)-citral, but showed (R)-citronellal formation tendency, and the ee value of (R)-citronellal was affected by pH in the reaction system. Accordingly, the ee values for (R)-citronellal formation increased with the increasing levels of E-isomer in the (E/Z)-citral mixture as well as the increase of pH. Under the reaction conditions (30 °C and pH 8.6), using purified OYE2p as catalyst, 200 mM (E/Z)-citral (an approximately 10:9 mixture of geometric E-isomer and Z-isomer) was efficiently converted to (R)-citronellal with 88.8% ee and 87.2% yield.
All these positive features demonstrate high potential of OYE2p for practical synthesis of (R)-citronellal and in asymmetric reduction of activated alkenes.
The toxicity of waste printed circuit boards (PCBs) to bacteria was considered as the major limitation in bioleaching of copper from PCBs. To reduce the toxicity of PCBs, copper extraction from PCBs was investigated using bacteria-free cultural supernatant from some metallurgical microbial consortium, whose predominant organisms were Leptospirillum ferriphilum and Sulfobacillus thermosulfidooxidans.
About 100% copper was recovered in 2 h from 5 g/L PCBs by bacteria-free cultural supernatant. The result indicated that the indirect non-contact mechanism was the predominant mechanism in bioleaching of copper from PCBs. It was not necessary for bacteria to exist in copper extraction. In addition, the role of bacteria was most likely to regenerate Fe3+ as an oxidant. Furthermore, the biooxidation of Fe2+ to Fe3+ was determined as the rate-limited step in bioleaching of copper from PCBs. In addition, the biooxidation activity of bacteria would be strongly inhibited by the toxicity of PCBs.
The separation of bacteria from the PCBs probably was the optimum way to avoid the negative effect of PCBs. Accordingly, a biooxidation–leaching–separation cycle was designed to avoid the toxicity of PCBs. Eventually, 93.4% of copper was recovered in total from 100 g/L PCB concentrates in 9 days.
Due to progress in science and technology, several harmful polycyclic aromatic hydrocarbons are synthesized and released into the environment. In the present investigation, a phenanthrene- and pyrene-degrading white rot fungi Ganoderma lucidum strain CCG1 was isolated from the Janjgir Champa district of Chhattisgarh, India, and then the degradation of phenanthrene and pyrene was estimated by high-performance liquid chromatography.
It was found that G. lucidum able to degrade 99.65% of 20 mg/L of phenanthrene and 99.58% of pyrene in mineral salt broth after 30th day of incubation at 27 °C. G. lucidum produced significant amounts (p < 0.0001) of ligninolytic enzymes (Laccase, lignin peroxidase and manganese peroxidase) in the phenanthrene- and pyrene-containing mineral salt broth. G. lucidum produced maximum 10,788.00 U/L laccase, 3283.00 U/L Lignin peroxidase and 47,444.00 U/L Manganese peroxidase enzymes in the presence of phenanthrene and produced maximum 10,166.00 U/L laccase, 3613.00 U/L lignin peroxidase and 50,977.00 U/L manganese peroxidase enzymes in the presence of pyrene. Therefore, G. lucidum will be a potent phenanthrene and pyrene degrader from the environment.
Although rational genetic engineering is nowadays the favored method for microbial strain improvement, random mutagenesis is still in many cases the only option. Atmospheric and room-temperature plasma (ARTP) is a newly developed whole-cell mutagenesis tool based on radio-frequency atmospheric-pressure glow discharge plasma which features higher mutation rates than UV radiation or chemical mutagens while maintaining low treatment temperatures. It has been successfully applied on at least 24 bacterial and 14 fungal species, but also on plants, dinoflagellates, and other microbial communities for the improvement of tolerance to medium components, to increase cellular growth and production of cellular biomass, to enhance enzyme activity, and to increase the production of various chemicals. Achievements like 385.7% of acetic acid production enhancement in Acetobacter pasteurianus give this new mutagenesis tool a promising future. However, certain questions remain regarding optimal operational conditions, the effects at subcellular levels, and standard operation procedures, which need to be addressed to facilitate applications of ARTP in microbial breeding and other fields such as evolution of enzymes.
Poultry droppings from poultry farms and rice husks obtained from rice milling process are generally considered as wastes and discarded in Nigeria. Although many studies have shown that microbial fuel cells (MFCs) can generate electricity from organic wastes, little or no study have examined MFCs for generating electricity from poultry droppings and rice husk as electrode material.
Laboratory-scale double-chamber MFCs were inoculated with concentrations of poultry droppings wastewater and supplied with rice husk charcoal as anode and cathode electrodes for electricity generation. Power outputs and dissolved organic carbon (DOC) removal efficiencies were compared between MFCs using rice husk charcoal (RHCE) as electrode and those using carbon cloth (CCE) as electrodes. The RHCE-MFC 2 containing 477 mg L−1 dissolved organic carbon produced a volumetric power density of 6.9 ± 3.1 W m−3 which was higher than the control and the CCE-MFCs by a factor of 2 and achieved at DOC removal efficiencies of 40 ± 1.2%.
The results suggest that poultry droppings wastewater is a feasible feedstock for generating electricity in MFCs. The findings also suggest that rice husk charcoal is a potentially useful electrode material in MFCs.
Microalgae biomass has garnered significant attention as a renewable energy feedstock and alternative to petroleum-based fuels. The diverse metabolism of green microalgae species additionally provides opportunities for recovery of products for feed, food, nutraceutical, cosmetic, and biopharmaceutical industries. Recently, the concept of using microalgae as part of a biorefinery model has been adopted in place of refinery methods focused on recovering one target product. This has led to producers exploring co-production of high value and high volume products in an effort to improve process economics. With numerous potential products and applications, the biomass source or specific strain must be carefully selected to accumulate extractable levels of the target molecule(s). It is additionally imperative to understand the morphology and metabolism of the selected strain to cost-effectively manage all stages of commercial production. This review will focus specifically on microalgae in the division of Chlorophyta, or green algae and their extracellular matrices (ECM), potential for commercial products, and finally describe a holistic approach for biomolecule extraction and recovery. Additionally, cell disruption and fractionation methods for recovery of biomolecules for commercial products are highlighted along with an alternative method, aqueous enzymatic processing for multiple biomolecule extraction and recovery from green microalgae. An emphasis is placed on connecting the morphological characteristics of microalgae ECM or organelle membranes to implications on separation and purification technologies.
Botryococcus braunii is difficult to cultivate and has a limited amount of substantive scale-up and productivity assessments with conventionally suspended cultivation systems, such as open pond or closed photobioreactors. The biomass concentrations of cultivated microalgal biofilms are much higher than those of suspension cultures, and the attached microalgal cells are easily separated from cultivation media. However, studies on the attached cultivation conditions for B. braunii have been rarely performed.
Herein, an attached cultivation method for B. braunii SAG 807-1 incubation was introduced. The effects of primary culture conditions on growth and hydrocarbon accumulation were investigated. Seed age influenced the biomass and hydrocarbon accumulation in B. braunii, and the highest values were 5.97 and 2.99 g m−2 day−1, respectively, when seed age was 14 days. The appropriate range of initial inoculation density was 7.9–10.1 g m−2. Light intensity was a dominating factor influencing B. braunii’s growth in the attached culture, and the light saturation point was 100–150 μmol m−2 s−1. Periodic illumination in 8:16 light: dark cycle had the highest utilisation of photons at approximately 1.0 g of biomass per mole of photons. The increasing CO2 concentration in aerated gas improved the growth rate, but its concentration should be 1%.
Attached algal cultivation systems have been widely explored. However, the optimised values for aqueous suspension methods may be unnecessary for the attached system. Optimised seed age, inoculum density, CO2 concentration, light intensity and photoperiod can improve the growth and hydrocarbon accumulation of B. braunii SAG807-1 with the attached culture.
Rice straw is one of the abundant lignocellulosic biomass with potential as a feedstock for bioethanol production. To produce ethanol from the biomass biologically, enzymatic hydrolysis is necessary that can effectively degrade rice straw into fermentable sugars such as glucose and xylose. Many researches utilized many kinds of commercial cellulase reagents on the hydrolysis of cellulose. Since these have different enzyme activities, enzyme reagents suitable for each biomass must be selected. In this study, three appropriate cellulase reagents were selected by multivariate analysis technique and then optimized by design of experiments method for efficient hydrolysis of alkali-pretreated rice straw. Moreover, an ethanol production from the treated straw was performed by simultaneous saccharification and fermentation (SSF) with the optimized enzyme cocktail and xylose-fermenting fungus of Mucor circinelloides.
Pretreatment by alkali solution of rice straw resulted in the increase of fermentable sugar content from 56.3 to 80.0%. The desirable commercial enzyme reagents for saccharification of the straw were determined as a combination of “Cellulase Onozuka 3S”, “Cellulase T Amano 4”, and “Pectinase G Amano” by a multivariate analysis based on various cellulolytic activities of each reagent. The optimum mixing ratio was calculated by response surface method based on design of experiment method. The optimized cocktail successfully achieved 75.3 g/L in production of the total fermentable sugar by hydrolysis of alkali-treated rice straw that agreed with the hydrolysis efficiency of 94.1%. SSF of 100 g/L treated rice straw with the optimal cocktail and M. circinelloides under aerobic condition resulted in 30.5 g/L ethanol concentration for 36 h.
The construction of cellulase cocktail by the proposed statistical method enabled efficient hydrolysis of alkali-treated rice straw. SSF process combining the optimized cocktail and a xylose-fermenting fungus could be expected as a promising system for ethanol production from various lignocellulosic biomasses.
Azoreductases are diverse flavoenzymes widely present among microorganisms and higher eukaryotes. They are mainly involved in the biotransformation and detoxification of azo dyes, nitro-aromatic, and azoic drugs. Reduction of azo bond and reductive activation of pro-drugs at initial level is a crucial stage in degradation and detoxification mechanisms. Using azoreductase-based microbial enzyme systems that are biologically accepted and ecofriendly demonstrated complete degradation of azo dyes. Azoreductases are flavin-containing or flavin-free group of enzymes, utilizing the nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate as a reducing equivalent. Azoreductases from anaerobic microorganisms are highly oxygen sensitive, while azoreductases from aerobic microorganisms are usually oxygen insensitive. They have variable pH, temperature stability, and wide substrate specificity. Azo dyes, nitro-aromatic compounds, and quinones are the known substrates of azoreductase. The present review gives an overview of recent developments in the known azoreductase enzymes from different microorganisms, its novel classification scheme, significant characteristics, and their plausible degradation mechanisms.
Biochar cation exchange capacity (CEC) is a key property that is central to biochar environmental applications including the retention of soil nutrients in soil amendment and removal of certain pollutants in water-filtration applications.
This study reports an innovative biochar-ozonization process that dramatically increases the CEC value of biochars by a factor of 2. The ozonized biochars also show great improvement on adsorption of methylene blue by as much as a factor of about 5. In this study, biochar samples treated with and without ozone were analyzed by means of pH and CEC assays, water field capacity measurement, elemental analysis, methylene blue adsorption, and Raman spectroscopy. Gaseous products’ analyses were carried out using an online universal gas analyzer over the duration of ozone treatments, and temperature changes were monitored using a thermal imaging camera. The results demonstrate a doubling of CEC with a concomitantly large drop in pH of the ozonized biochar compared with the untreated sample, brought about by the creation of acidic oxygen-functional groups on biochar surface, which may represent a significant progress toward the viability of employing biochar as a soil amendment for sustainability on Earth.
This biochar-ozonization process technology has the potential to effectively convert conventional biochars into surface-oxygenated products with dramatically higher CEC values.
Cellulases played an important role in the production of bioenergy and bio-products. Cellulases from bacteria with some special characteristics drew great attention due to its fast growth speed, wide adaption to harsh environment, and production of multi-function cellulases.
An endoglucanase gene egls from Bacillus velezensis A4 was cloned and expressed in Escherichia coli BL21 (DE3). The recombinant enzyme Egls was partially purified using aqueous two-phase system. The highest recovery rate of the enzyme was 90.39% at PEG 4000 (25% w/w), phosphate buffer 8.08% (w/w) (pH 6.0), and NaCl (5% w/w). The enzyme molecular weight was 55 KD estimated by zymogram. The optimal pH and temperature of recombinant enzyme Egls were pH 6.0 and 55 °C, respectively. The enzyme was stable at pH range of 5.0–7.0 at 55 °C for 60 min. The enzyme exhibited Km, Vmax, Kcat values as 63.38 mg/ml, 55.6 mg/min, and 3.93 × 103/S, respectively. The addition of 10 mM of Mg2+, Mn2+, or 5% (w/w) of Triton-X 100 in the reaction system enhanced the enzyme activity significantly. The enzyme showed both endoglucanase and exoglucanase activity.
An endoglucanase gene egls from B. velezensis A4 was cloned and expressed in E. coli BL21 (DE3). The recombinant enzyme Egls was purified by aqueous two-phase system and characterized. The enzyme can be applied for the efficient pretreatment of lignocellulosic biomass for bioenergy and bio-products production.
Compared with shake flask, bioreactor offers a relatively stable and controllable extracellular environment for cell growth and metabolism. Meanwhile, parallel micro-bioreactor system could well meet the screening flux requirement in the process of high-throughput strain screening. In this study, a self-developed 8-parallel micro-bioreactor system with non-invasive optical biosensors was introduced to substitute the traditional shake flask.
Optical pH and DO biosensors could be well applied for the process monitoring and controlling in the cultures of commonly used microorganisms through maintaining constant temperature and bioreactor shading treatment. Subsequently, 8-parallel micro-bioreactor system was adopted in the rescreening procedure of high-throughput screening process, and it significantly increased the feasibility of scaling up a cultivating system without any sacrifice on the screening flux. As a result, a designated mutant strain Lactobacillus paracasei S4 was obtained, which presented an improvement of 18.9% on titer value of l-lactic acid. Moreover, the yield also increased from 0.903 ± 0.005 to 0.932 ± 0.013 g/g.
In this study, the micro-bioreactor system proved to be applicable and effective in the rescreening procedure of high-throughput screening process. The adopted approach provided a robust tool for screening the strain with high l-lactic acid producing performance.
Aspergillus niger is a highly important industrial microorganism because of its amazing capacity to produce citric acid (CA). To explore the metabolic mechanism and physiological phenotype associated with high CA productivity, the transcriptomes of high CA-producing A. niger YX-1217 and degenerative strain YX-1217G were investigated using A. niger ATCC1015 as a control.
These strains showed distinct transcriptional differences in CA production. By contrast, the genes encoding glycoside hydrolases, aspartyl endoproteases, and carboxypeptidases were unusually upregulated in CA-producing strain YX-1217, which involved the carbohydrate hydrolysis and polypeptide degradation pathways, and should be related to its powerful capacity to utilize cornmeal fluidified liquid as raw material for the production of CA. In central metabolism of YX-1217, gene 9.735.1, which encodes glyceraldehyde 3-phosphate dehydrogenase, and two transcriptionally outstanding genes, 6000119 (An15g01920) and 3.2152.1 (An08g10920) that encode citrate synthase, were upregulated, thereby ensuring CA accumulation. In addition, a relatively strong electron transport chain, a regeneration system for NAD+/NADP+, and an efficient resistance mechanism may have contributed to the high CA production rate of YX-1217.
These comparisons have shed light on the mechanism underlying high CA yield in A. niger YX-1217 as well as provide insights into the development of novel strains that produce other organic acids.
Methanol has attracted interest as a substrate for improvement of product titers and yields of bioprocesses, because methanol has a more reduced state than sugars do and can be renewably synthesized from abundant natural gas supplies. Our aim was to engineer methylotrophic Pichia pastoris to convert methanol into value-added lovastatin more productively.
A strengthened biosynthetic pathway of lovastatin was constructed through the assembly of three modules with increasing module-specific antibiotic stress in the methylotrophic yeast P. pastoris. The resulting strain (P. p/LV_V#9) produced 287.5 ± 2.0 mg/L lovastatin in a 5-L bioreactor from methanol. The production was further improved by identification and overexpression of a statin pump protein, TapA, a membrane protein capable of lovastatin efflux out of the cell. A TapA-overexpressing strain, P. p/LV_V#9-TapA, produced 419.0 ± 9.5 mg/L lovastatin from methanol: 46% more than P. p/LV_V#9 did, and 520% more relative to the strain (P. p/LV_SC) with single-copy genes.
A methylotrophic yeast strain producing 419.0 ± 9.5 mg/L lovastatin was constructed by optimization of biosynthetic gene dosages and coexpression of a statin pump protein; these results proved that P. pastoris is a promising chassis organism for natural-product biosynthesis. A membrane protein, TapA was found to perform the function of exporting intracellular lovastatin and enhanced lovastatin production.
Benzofuran and its derivatives contain central pharmacophores and are important structures in medicinal chemistry. Chemical synthesis of benzofuran rings often requires expensive catalysts and stringent operational conditions. Biosynthesis is recognized as a promising way to save energy and produce valuable compounds. Dioxin biodegradation pathways can form several benzofuran derivatives, and these pathways may be a better choice for further synthesis of important biological compounds. 2-Hydroxy-4-(3′-oxo-3′H-benzofuran-2′-yliden)but-2-enoic acid (HOBB), a benzofuran derivative, can be biosynthesized from dibenzofuran (DBF) through co-metabolic degradation in a lateral dioxygenation pathway.
Efficient biosynthesis of HOBB was observed using whole cells of Pseudomonas putida strain B6-2. After cultivation in LB medium containing biphenyl, the cells were suspended to an OD600 of 5 to conduct biosynthesis in the presence of 0.5-mM DBF at pH 7 for 8 h. The bacterial cells were used twice to degrade approximately 0.70-mM DBF, and in batch process, accumulated about 0.29-mM HOBB. HOBB could be easily purified from the reaction with ethyl acetate using the neutral-acid extraction method, and 13.58 ± 0.31 mg of HOBB was obtained from 22.49 ± 0.74-mg DBF with an overall production yield of 60.4% (w/w). The product HOBB, which is a yellow powder, could be detected and identified by LC–MS, GC–MS, and NMR.
In this study, a new biological route was developed to produce HOBB from DBF using whole cells of P. putida B6-2 (DSM 28064). The biosynthesis of HOBB may contribute to studies of the DBF lateral pathway and provide a new green route for synthesizing benzofuran derivatives with pharmacological activities.
A glucose concentration is an important factor for a fed-batch process of Saccharomyces cerevisiae. Therefore, it is necessary to be controlled under a critical concentration to avoid overflow metabolism and to gain high productivity of biomass. In the study, 2D fluorescence spectroscopy was applied for an online monitoring and controlling of the yeast cultivations to attain the pure oxidative metabolism.
The characteristic of the NADH intensity can effectively identify the metabolic switch between oxidative and oxidoreductive states. Consequently, the feed rate was regulated using the single signal based on the fluorescence intensity of NADH. With this closed-loop control of the glucose concentration, a biomass yield was obtained at 0.5 gbiomass/gglucose. In addition, ethanol production could be avoided during the controlled feeding phase.
The fluorescence sensor with a single signal of the NADH fluorescence intensity has potential to control a glucose concentration under the critical value in real time. Therefore, this achievement of the feedback control is promising to build up a compact and economical fluorescence sensor with the specific wavelength using light-emitting diodes and photodiodes. The sensor could be advantageous to the bioprocess monitoring because of a cost-effective and miniaturized device for routine analysis.
A mixed feeding strategy (co-feeding of complex carbon sources with methanol) has become a common practice for process development in Pichia pastoris to increase cell biomass and enzyme production levels. However, in some cases mixed feeding did not have a significant impact or even had a negative effect on specific enzyme productivity. We hypothesized that this may be due to a bottleneck in the protein secretion pathway caused by too strong protein expression as a result of mixed feeding operation.
Using glucose oxidase (Gox) as a model protein, the individual and synergistic effects of co-feeding of sorbitol or yeast extract (YE) with methanol and Hac1p overexpression on the secretory expression of Gox were investigated both in shake flasks and in a laboratory fermenter. The results showed that YE is superior to sorbitol in terms of stimulating protein expression and cell growth. Moreover, separate applications of the mixed feeding strategy and secretory pathway engineering only achieved limited success in enhancing Gox levels, while the combined use of the two strategies acted synergistically, leading to 297% increase of Gox production and the final enzyme titer reached 787.4 U/mL in GSgox-Pp on 1-L fermenter.
Co-feeding of YE combined with secretion pathway engineering significantly improved glucose oxidase secretion, which can be also applied to improve secretory expression of other foreign proteins in P. pastoris system.
Saccharopolyspora erythraea (S. erythraea) is a Gram-positive erythromycin–producing filamentous bacterium. The lack of comprehensive S. erythraea genome-scale metabolic models (GEMs) hinders the efficiency of metabolic engineering as well as fermentation process optimization.
In this study, the GEMs model of S. erythraea iZZ1342 was reconstructed according to the latest genome annotations, omics databases, and literatures. Compared with the previous S. erythraea model—GSMR, the new model iZZ1342 presented great improvements both on scope and coverage in the number of reactions, metabolites, and annotated genes. In detail, the number of unique reactions in iZZ1342 was increased from 1482 to 1684, the number of metabolites was increased from 1546 to 1614, and the number of unique genes was increased from 1272 to 1342. We also added 1441 gene-protein-reaction associations in iZZ1342 which lacks in the previous model to overcome the limitation in the application of strain designing. Compared with the transcriptomics data obtained from the published literature, 86.3% ORFs and 92.9% reactions in iZZ1342 can be verified. The results of the sensitivity analysis showed the similar trend in the E. coli GEMs. The prediction of growth on available 27 kinds of carbon sources and 33 kinds of nitrogen sources showed the accuracy rate was 77.8 and 87.9%, respectively. Compared with the physiological data obtained from chemostat cultivation, the simulation results showed good consistency. The correlation coefficient between the 13C-labeled experiment data and the flux simulation result was 0.97. All the above results showed that the iZZ1342 model has good performance. Furthermore, four genes are in the range of successful knockout by comparing these targets with the results which have been earlier published.
The new model iZZ1342 improved significantly in model size and prediction performance, which will lay a good foundation to study the systematic metabolic engineering of S. erythraea system in vivo.
Laccase, a multicopper oxidase that catalyzes the oxidation of phenols, aromatic amines, and benzenethiols, has attracted much attention in applications of organic synthesis, bioremediation, and pulp/textile bleaching. However, free laccases cannot be recycled and are easily inactivated in diverse environmental conditions. Enzyme immobilization is a promising strategy to improve stability, resistance to extreme conditions, and reusability of laccase.
In this study, amino-functionalized magnetic Fe3O4 nanoparticles were synthesized for co-immobilization of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) and laccase by glutaraldehyde cross-linking method. The magnetic nanoparticles were characterized with FTIR, XRD and VSM. Cyclic voltammetry was carried out to verify electrochemical behaviors of the co-immobilized laccase and TEMPO nanoparticles. When the co-immobilized laccase and TEMPO nanoparticles were used to decolorize acid fuchsin, the maximum decolorization rate of 77.41% was obtained with the ratio of TEMPO to laccase being 0.3 mM/g:120 U/g.
The co-immobilized nanoparticles retained above 50% residual activity after eight cycles of operation, which presented an approach to develop a co-immobilized laccase and mediator system for potential industrial application.
Tubular photobioreactors (PBRs) have a great potential in large-scale biomass cultivation and mixers in tubular PBRs have been intensively investigated to achieve high biomass productivity. However, mixers increase not only biomass yield, but also energy consumption. To evaluate performances on increasing light/dark (L/D) cycles and energy consumption of adding a mixer simultaneously, a new parameter named as efficiency of L/D cycle enhancement is introduced. Discrete double inclined ribs, intensively studied in heat transfer, are introduced to tubular PBRs in this work. The number of ribs in a cross section is discussed. These tubular PBRs are investigated in terms of the flow structure, L/D cycle frequency and efficiency of L/D cycle enhancement by computational fluid dynamics. The numerical results show that the increment of L/D cycle frequency caused by the discrete double inclined ribs is larger than the increment of energy consumption caused by the ribs under a wide range of incident light intensity. In general, the increasing of rib length ratio results in a decrease of efficiency and the PBR with two pairs of ribs performs the best. Based on the general trends, a PBR with two pairs of ribs and of which the rib length ratio is 5 is recommended for further studies.
This review describes the biodegradation of Lindane (γ-hexachlorocyclohexane, γ-HCH) from the diverse sources. Environmental degradation of γ-HCH has been described in terms of integrated biological approaches such as metagenomics, cloning, phytoremediation, nanobiodegradation, and biosrfactants, genes and enzymes responsible for γ-HCH degradation and exploration of new strains of γ-HCH-degrading microbes from different environmental sources. Metagenomics-based approaches help in the identification and isolation of new genes from the uncultivable sources and provide insights for future research. There is potential in the elucidation of pathways of degradation of persistent organic pollutants (POPs) from environment by the microorganisms. This is possible by means of new/improved microbial species. The behavior of isolated strains and the microorganisms when present in community is altogether different. Therefore, there is a need to develop new technology which will identify the minor component of the microbial community involved in degradation because the minor part might have profound effect on degradation. This is mediated by the biological activity of the microbial system.
An attempt was made to isolate selenite-reducing bacteria from a contaminated lake that receives industrial effluents and domestic sewage. The isolated dominant bacterial strain AJK3 was identified as Bacillus cereus, based on biochemical characterization and 16S rDNA sequencing. The time dependent selenium removal at different selenite concentrations monitored with ICP-AES indicates the substantial selenite reduction capability of the isolated strain. The selenium nanoparticles produced during the bacterial reduction of selenite were analyzed with UV–visible spectroscopy, X-ray diffraction, transmission electron microscopy, zeta potential measurement, Fourier transform infrared spectroscopy and Raman spectroscopy.
The nanoparticle synthesis was confirmed from the red colour emergence in culture broth and wide UV–vis peaks. The produced nanoparticles were polydisperse, spherical, size varied from 50 to 150 nm and the mean particle size was about 93 nm. The amorphous nature of the generated nanoparticles was confirmed from the Raman spectroscopy, XRD and SAED patterns. The IR data and zeta potential values substantiated the protein capping of the produced nanoparticles.
Thus, the present study suggests that the isolated bacterial strain can be exploited as a prospective, renewable, natural, nanofactory for the bacteriogenic synthesis of nanoparticles. Also, the study has application in bioremediation of selenite from the contaminated environment.
Thermotolerant lignocellulolytic enzymes have become a subject of interest in industrial processes due to their ability to degrade lignocellulosic polysaccharides. Development of cost-effective, large-scale screening for production of desirable enzymes by thermophilic fungi is a challenge. The present investigation focused on isolating, screening, and identifying industrially relevant thermophilic producers of lignocellulolytic enzymes from various locations in the Warangal district, Telangana, India.
Fifteen thermophilic fungi were isolated from soil on their ability to grow at 50 °C and were screened for their activity of cellulase, hemicellulase, and lignin degradation based on holo zone around colonies. The appearance of the black color zone of diffusion in esculin agar is a positive indication for the β-glucosidases activity test. Out of fifteen isolates, Aspergillus fumigatus JCM 10253 have shown as a potential producer of extracellular enzymes for lignocelluloses degradation showing higher activity for cellulase (EI 1.50) as well as β-glucosidase (4 mg/mL), simultaneously for xylanase (EI 1.18) by plate assay methods. A. fumigatus JCM 10253 was selected for extracellular hydrolytic enzymes production under solid-state fermentation. Maximum CMCase (26.2 IU/mL), FPase (18.2 IU/mL), β-glucosidase (0.87 IU/mL), and xylanase (2.6 IU/mL) activities were obtained after incubation time of 144 h at 50 °C. The thermostability of crude cellulase showed the optimum activity at 60 °C and for FPase, β-glucosidase, and xylanase at 50 °C which recommended that the enzymes have a potentially significant role in the biofuel industries.
The high titer production of active enzymes that cleave different β-1,4-glycosidic bonds still remains a challenge and is the major bottleneck for the lignocellulosic conversion. In particular, the finding of thermostable enzymes which would allow the development of more robust processes is a major goal in this field.
Marigold petals, the current commercial source for lutein and zeaxanthin are harvested through a labor-intensive operation with downstream purification requiring multiple processing steps involving various harsh solvents. Lutein and zeaxanthin are in-demand carotenoids due to their significant role in human eye health. A possible alternative source for lutein and zeaxanthin, distillers dried grain with solubles (DDGS), shows promise and contains a yet-to-be quantified amount of the desired carotenoids. The US corn industry produces an abundant, and in some cases excess, annual supply of DDGS. The large volume of DDGS produced could serve as a significant source for lutein and zeaxanthin recovery and provides an additional market stream for the growing carotenoid industry. This paper demonstrates one of the first quantitative reports regarding the concentration of lutein and zeaxanthin in DDGS. Using Soxhlet extraction, followed by purification with centrifugal partition chromatography, it was determined that 36.09 ± 16.87 µg lutein and 15.48 ± 6.13 µg zeaxanthin could be purified from all extractives retained in the oleoresin per gram of DDGS. As compared to lutein and zeaxanthin present in corn, this is a three to fivefold increase indicating that these compounds become concentrated during the dry grind process. Recovery of lutein and zeaxanthin from DDGS, a low-value stream by-product of corn ethanol industry, results in a new revenue stream and would add value to a common US commodity.
Aldo–keto reductase (AKR) or alcohol dehydrogenases (ADH)-mediated stereoselective reduction of prochiral carbonyl compounds is an efficient way of preparing single enantiomers of chiral alcohols. However, steric hindrance of substrate affects the catalytic performance of enzymes. The present study aims to discover and identify AKRs/ADHs capable of catalyzing highly stereoselective reduction of sterically hindered ketones.
Five AKRs from different microorganisms (CaCR, ScCR, KmCR, CPR-C1, and CPR-C2) were identified through gene mining, and overexpressed in recombinant Escherichia coli BL21(DE3). The specific activity and stereoselectivity of the AKRs were further evaluated towards various ketoesters and heterocyclic ketones, which are sterically bulky and are valuable in industrial applications. Each purified enzyme exhibited catalytic activity to one or more of the tested substrates. Among the enzymes, ScCR showed a broader substrate spectrum compared to the others. Regarding Km values related to substrate association, we also provided insights into the specificity and preference of certain enzymes. Consequently, enantiopure (R)-methyl mandelate, ethyl (R)-mandelate, ethyl (R)-2-hydroxy-4-phenylbutyrate, and (S)-N-benzyl-3-pyrrolidinol (> 99%e.e.) were obtained through the identified AKRs.
The stereospecific AKRs were obtained through gene mining, which possesses the potential for application in the preparation of important optically active alcohols.
3-Hydroxypropionic acid (3-HP) is one of the top value-added chemicals that can be produced from renewable resources. Crude glycerol, a cheap feedstock, is the foremost by-product generated from biodiesel industries. In this study, Klebsiella pneumonia was engineered for efficient 3-HP production from glycerol.
Three metabolically engineered K. pneumoniae strains were constructed and the strain JJQ02 (ΔldhAΔdhaT) overexpressing aldehyde dehydrogenase from E. coli was the most suitable for 3-HP production. This strain produced 61.9 g/L of 3-HP (a yield of 0.58 mol/mol) in 38 h in a 5-L fermentor by control of the aeration rate. Deletion of ldhA and dhaT effectively eliminated lactate formation and reduced 1,3-propanediol production, but more 2,3-butanediol was accumulated due to redox balance. The fed-batch process was successfully scaled-up in a 300-L bioreactor where the 3-HP production level was 54.5 g/L (a yield of 0.43 mol/mol) in 51 h.
Deficiency of both lactate dehydrogenase and 1,3-propanediol oxidoreductase as well as control of aeration significantly enhanced 3-HP production, and the successful scale-up of the fed-batch process in a 300-L bioreactor indicated a great potential to industrial production of 3-HP.
Shewanella genus is famous for applications like electron transfer in microbe fuel cells and bioremediation of heavy metals through the Mtr pathway. A potential way to enhance the electron genesis ability of Shewanella is to express exogenous mtr genes via recombinant DNA technology. Thus, to design and develop expression vectors capable of replicating in Shewanella and enhance the genetic toolbox of the same is important.
In this study, a plasmid construct with a replication origin, repB, and pLacI promoter is reported for the first time to drive the expression of green fluorescent protein in S. oneidensis MR-1. Based on the same vector, the Mtr pathway genes mtrA, mtrC, and mtrCAB were also successfully expressed in MR-1. The recombinant strains had higher ferric reductase activity compared to the wild type. The highest enzymatic activity of 508.33 U/L in genetic Shewanella with mtrC gene is obtained, which is 1.53-fold higher than that of wild strain. The plasmids were stable up to 90 generations.
We have demonstrated an expression system based on pLacI promoter and repB ori in Shewanella. Consequently, the combination of repB and pLacI will have great potential in Shewanella to turn on expression of different genes constitutively.
Saccharomyces cerevisiae is one of the most important industrial microorganisms. A robust genome editing tool is vital for both fundamental research and applications. To save the time and labor consumed in the procedure of genome editing, a self-cloning CRISPR/Cpf1 system (scCRISPR/Cpf1), in which a self-cleaving plasmid and PCR-generated site-specific crRNA fragment were included, was developed.
Using scCRISPR/Cpf1 as the genetic tool, simple and fast singleplex and multiplex genomic integration of in vivo assembled DNA parts were investigated. Moreover, we validate the applicability of scCRISPR/Cpf1 for cell factory development by creating a patchoulol production strain through two rounds of iterative genomic integration. The results showed that scCRISPR/Cpf1 enables singleplex and tripleplex genomic integration of in vivo assembled DNA parts with efficiencies of 80 and 32%, respectively. Furthermore, the patchoulol production strain was successfully and rapidly engineered and optimized through two rounds of iterative genomic integration by scCRISPR/Cpf1.
scCRISPR/Cpf1 allows for CRISPR/Cpf1-facilitated genome editing by circumventing the step to clone a site-specific crRNA plasmid without compromising efficiency in S. cerevisiae. This method enriches the current set of tools available for strain engineering in S. cerevisiae.
For the purification of alcohols derived from microbial fermentations, extensive processing is required. Adsorption is described as one of the most cost-effective and efficient techniques for the separation of water and purification of alcohols. Biobased sorbents (called biosorbents) are advantageous for dehydration of alcohols as they can be developed from cost-effective feedstocks such as waste agricultural biomass or byproducts, have adsorption capacities at par with chemical adsorbents, and can be safely disposed. Alternatively, the spent adsorbents can be reused for fuel or energy production. Agricultural byproducts are low cost and abundantly available materials containing cellulose, hemicellulose, proteins, and lignin as their constituents. Biosorbents have the capability to adsorb water by the polar interaction of their hydroxyl, carboxyl, carbonyl, and amine groups with water molecules. The pore size distribution and thermal stability of biosorbents are also industrially relevant features. They are a promising option to be used in industries for dehydration of alcohols. This paper reviews adsorptive purification of bioalcohols with a focus on using biosorbents, and describes their structure, global availability, water adsorption mechanism, and the use of biosorbents in liquid phase and vapor phase adsorption systems for the purification of ethanol, butanol, and other higher alcohols.
The continuous escalation in wastewater production with declining dependency on conventional resources as a result of rapid urbanization, increasing global water scarcity and growing population have initiated many researchers to look out for efficient means of utilizing wastewater. In this regard, an effectual process economizing approach has been achieved, upon utilizing the discharged wastewater from validamycin A recovery process in fermentation medium as a replacement of costly tap water.
In this study, wastewater was successfully used as a fermentation medium for the production of validamycin A in plant-scale bioreactor. Moreover, a new strain Streptomyces hygroscopicus K2509 was screened showing a good production capability in this low-cost culture environment and showed maximum validamycin A production and productivity of 21.23 g/L and 0.29 g/L h, respectively. Execution of this study has managed the effective utilization of wastewater by reducing 12.42% production cost per kilogram of validamycin A in an environmental friendly way.
The novel successful approach for using process wastewater even on being executed in plant-scale bioreactor was proved cost effective. In short, the presented effective way of utilizing wastewater in study will definitely serve as potentially cheap fermentation medium upon replacing tremendously used expensive tap water.
Bioremediation is the use of biological interventions for mitigation of the noxious effects caused by pollutants in the environment including wastewater. It is very useful approach for a variety of applications in the area of environmental protection. It has become an attractive alternative to the conventional cleanup technologies that employ plants and their associated microorganisms to remove, contain, or render harmless environmental contaminants.
Three parallel hydroponic treatment systems (each 2 m long × 0.75 m wide × 0.65 m deep) and one control unit were filled with brewery wastewater to an effective depth of 0.5 m. Two sets of floating polystyrene platform were used for each treatment unit to support vetiver tillers for conducting bioremediation study. The wastewater was fed to the hydroponic treatment units at hydraulic loading rate of 10 cm d−1 and hydraulic residence time of 5 days. Influent and effluent samples were collected once a month for 7 months, and analyzed to determine the various parameters relating to the water quality including plant growth and nutrient analyses.
Vetiver grass grew and established with well-developed root and shoots in the hydroponics under fluctuations of brewery wastewater loads and showed phytoremedial capacity to remove pollutants. Removal efficiencies for BOD5 and COD were significant (p < 0.05), up to 73% (748–1642 mg l−1 inlet), and up to 58% (835–2602 mg l−1 inlet), respectively. Significant removal efficiencies (p < 0.05) ranged from 26 to 46% (14–21 mg l−1 inlet) for TKN, 28–46% (13–19 mg l−1 inlet) for NH4 +-N, 35–58% (4–11 mg l−1 inlet) for NO3 −-N, and 42–63% (4–8 mg l−1 inlet) for PO4 −3-P were recorded. Nutrient accumulation in the samples harvested were varied between 7.4 and 8.3 g N kg−1 dry weight and 6.4–7.5 g P kg−1 dry weight in the hydroponic treatment units during the study period.
This study has shown suitability of vetiver grass for organics and nutrient removal in the bioremediation of brewery wastewater using hydroponics technique in addition to production of valuable biomass. Bioremediation using hydroponics is green and environmentally sustainable approach that offers promising alternative for wastewater treatment in developing countries including Ethiopia.
Enhancing cellulase production in Trichoderma reesei is of great interest for an economical biorefinery. Artificial transcription factors are a potentially powerful molecular strategy for improving cellulase production in T. reesei. In this study, enhanced transcriptional activators XYR1VP, ACE2VP, and ACE1VP were constructed by linking the C terminus of XYR1, ACE2, or ACE1 with an activation domain of herpes simplex virus protein VP16. T. reesei transformants TXYR1VP, TACE2VP, and TACE1VP showed improved cellulase and/or xylanase production. TXYR1VP has a cellulase-free phenotype but with significantly elevated xylanase production. Xylanase I and xylanase II activities [U/(mg biomass)] increased by 51% and 80%, respectively, in TXYR1VP in comparison with parental strain RUT C30. The filter paper activity of TACE2VP in the Avicel-based medium increased by 52% compared to that of RUT C30. In the Avicel-based medium, TACE1VP manifested an 80% increase in FPase activity and a 50% increase in xylanase activity as compared to those of RUT C30. Additionally, when pretreated corn stover was hydrolyzed, crude enzymes produced from TACE1VP yielded a greater glucose release than did the enzymes produced by parental strain RUT C30.
Crude glycerol is a main by-product from biodiesel production, and efficient utilization of crude glycerol will bring significant economic and environmental benefits. However, the complex compositions of crude glycerol may impair the cellular growth and inhibit the crude glycerol consumption. Therefore, it is necessary to find a simple method to treat the crude glycerol and release the inhibition on cell metabolism.
The simply purified crude glycerol by activated carbon can be used as the carbon source to produce succinate in two-stage fermentation by the engineered Escherichia coli strain, MLB (ldhA −, pflB −) expressing phosphoenolpyruvate carboxykinase. In the flask experiments, succinate production from crude glycerol without treatment was less than that from pure glycerol. However, in the experiments of 1.5-L bioreactor, little succinate was produced in crude glycerol. The simply purified crude glycerol was used as carbon source for succinate production, and the glycerol consumption and succinate production were enhanced greatly. The succinate produced from the simply purified crude glycerol reached 566.0 mM, which was about ten times higher as that of non-purified one (50.3 mM). The succinate yield of the anaerobic stage achieved 0.97 mol/mol, which was 97% of the theoretical yield.
The treatment of crude glycerol by activated carbon could effectively release the inhibition on the glycerol consumption and succinate production of the engineered E. coli strains, so that the fermentation result of the treated crude glycerol was similar as the pure glycerol. The results showed that the metabolically engineered E. coli strains have great potential to produce succinate from crude glycerol.
Charge heterogeneity is one of the most critical quality attributes of antibodies, which has strong influence on drug’s biological activity and safety. Finding out the key components that affecting charge variants is of great significance for establishing a competitive culture process. In this study, we first illustrated uridine’s great impacts on antibody charge heterogeneity in CHO cell fed-batch cultures.
Uridine was beneficial to cell growth and the maintenance of cell viability, which made IVCC increased by 50% and the final titer improved by 64%. However, uridine had great influences on mAb’s charge variants. In uridine added cultures, the acidic variant levels were about 9% lower than those in control cultures, while the basic variant levels were about 6% higher than those in control cultures. Further investigation found that the decrease of aggregates and glycated forms were responsible for the reduction of acidic variants. What’s more, uridine decreased the lysine variant levels.
Uridine’s addition to fed-batch promoted cell growth and the final titer, in the meanwhile, uridine decreased the acidic variants dramatically. Therefore, feeding uridine is an efficient way to control the generation of acidic charge variants in up-stream process. These findings provide new ideas and guidance for the control and optimization of antibody charge heterogeneity in culture process developments.
Biological conversion of plant biomass into commercially valuable products is one of the highly studied subjects in the last two decades. Studies were continuously being conducted to understand and develop efficient enzymes for the breakdown and conversion of plant cell-wall components into valuable commercial products. Naturally, plant cell-wall components are differentially esterified to protect from the invading microorganisms. However, during the process of evolution, microorganisms have developed special set of enzymes to de-esterify the plant cell-wall components. Among the carbohydrate-active enzymes (CAZy), carbohydrate esterases stand first during the process of enzymatic conversion of plant biomass, as these enzymes de-esterify the plant biomass and make it accessible for the hydrolytic enzymes such as cellulases, hemicellulases, ligninolytic and pectinases. In this article, we have extensively discussed about the structural and functional properties of pectin methyl esterases, feruloyl, cinnamoyl and glucuronoyl esterases which are required for the de-esterification of pectin and lignin–carbohydrate complexes. Pectin esterases are classified among CE8, CE12, CE13 and CE15 carbohydrate esterase class of CAZy database. Whereas, lignin–carbohydrate complex de-esterifying enzymes are classified among CE1 (feruloyl esterase) and CE15 (glucuronoyl esterase) classes. Understanding the structural and functional abilities of pectin and lignin–carbohydrate esterases will significantly aid in developing efficient class of de-esterases for reducing the recalcitrant nature of plant biomass. These efficient de-esterases will have various applications including pretreatment of plant biomass, food, beverage, pulp and paper, textile, pharmaceutical and biofuel industries.
The recovery characteristics of phase-forming polymers are essential for aqueous two-phase systems (ATPS) to recycle in bioseparation engineering.
A new thermo-responsive copolymer (PVBAm) is suggested based on N-vinylcaprolactam, acrylamide, and butyl methacrylate. Together with another thermo-responsive polymer, poly (N-isopropyl acrylamide) (PN), it has been applied to form a recyclable ATPS. PVBAm and PN were designed to obtain structures and molecular weights allowing a lower critical solution temperature (LCST). By polymerization optimization, both PN and PVBAm were obtained with recoveries 98.5% and 95% above their LCST (i.e., PN 32.5 °C and PVBAm 40.5 °C), respectively, which allows each ATPS phase to be effectively recycled. The recycled ATPS based on PVBAm and PN was applied to the partitioning of vitamin B12. Under optimized conditions (5% PVBAm/3.5 %PN ATPS, in the presence of 0.8 M KCl, pH 4.0), the partition coefficient of vitamin B12 reached a value of 5.81.
The new ATPS based on the thermo-responsive copolymer PVBAm/PN possessed appropriate recycling characteristics regarding LCST, as well as recovery and phase separation characteristics.
Cellular physiological responses, which are often obscured by inferences from population-level data, are of great importance in cell biology. Microfluidics has emerged as an important tool for biological research on a small scale, reaching even the single-cell level.
In this work, a flow-focusing microdroplet generator was developed to produce monodisperse microdroplets with high stability. Individual B. coagulans cells were encapsulated in the microdroplets and cultured offline. The specific growth rate of B. coagulans at the single-cell level was analyzed, and the growth of B. coagulans in the droplets showed good consistency with that in flasks, with a correlation coefficient of 0.98. The morphological heterogeneity and its potential relationship with the production of lactic acid by B. coagulans were evaluated using a microscopic imaging method.
We have demonstrated a single-cell monitoring methodology based on microdroplets. This approach has great potential for studying a range of behavioral and physiological features of bacteria at the single-cell level.
Grape is the largest fruit crop worldwide and the grape pomace is an important solid waste generated from pressing and fermentation processes in wine industries. Wine industry residues are rich in bioactive compounds and, in this case, the utilization of grape by-products for alternative uses has been a focus of research. The aim of the present project is to present the primary benefits of winemaking by-products to new products focusing on grape pomace, as well as to discover novel applications in food industry, cosmetics, pharmaceutical, agricultural, livestock fields and in energy recovery systems. Moreover, new green technologies for valuable components recovery will be summarized. Recognizing emerging technologies, researchers would have the opportunity to promote development of value-added products and high-quality applications in different markets and sectors recycling of winery by-products or even side streams. This study presents the main bioactive components of grape pomace, along with new current extraction pathways, targeting the decrease of negative environmental impact in parallel to functional added value applications.
Despite years of concerted research efforts, an industrial-scale technology has yet to emerge for production and conversion of algal biomass into biofuels and bioproducts. The objective of this review is to explore the ways of possible integration of biology, ecology and engineering for sustainable large algal cultivation and biofuel production systems. Beside the costs of nutrients, such as nitrogen and phosphorous, and fresh water, upstream technologies which are not ready for commercialization both impede economic feasibility and conflict with the ecological benefits in the sector. Focusing mainly on the engineering side of chemical conversion of algae to biodiesel has also become obstacle. However, to reduce the costs, one potential strategy has been progressing steadily to synergistically link algal aquaculture to the governmentally mandated reduction of nitrogen and phosphorous concentrations in municipal wastewater. Recent research also supports the suppositions of scalability and cost reduction. Noticeably, less is known of the economic impact of conversion of the whole algae-based biorefinery sector with additional biochemical and thermochemical processes and integration with ecological constraints. This review finds that a biorefinery approach with integrated biology, ecology, and engineering could lead to a feasible algal-based technology for variety of biofuels and bioproducts.
Biosurfactants are natural surface-active compounds produced by a variety of microorganisms. The high cost of culture media limits the large-scale production and use of biosurfactants. It is therefore necessary to develop an efficient and cost-effective bioprocess to improve the yield of biosurfactants from microorganisms. In this study, the response surface method was used to optimize the production of biosurfactants by a Lactobacillus strain and the antimicrobial activity of the biosurfactants was assessed.
The biosurfactant-producing strain was identified as Lactobacillus paracasei subsp. tolerans N2 after 16S rRNA gene analysis. Among the different variables studied using a Plackett–Burman statistical design, temperature and peptone and sugar cane molasses concentrations were found to be the main factors that had significant (p < 0.05) influence on biosurfactant production. The results of this study showed that molasses concentration at 59.5 g/L, peptone at 6.20 g/L and temperature of 33 °C were optimal conditions for biosurfactant production, with a maximum yield of 2.70 g/L. The biosurfactant exhibited surface tension reduction of 37.85 mN/m and antimicrobial activity expressed as inhibition diameter of 63 mm. Partial characterizations by elemental, biochemical and Fourier transmission infrared spectroscopy analysis of the biosurfactant produced revealed that it was glycolipoprotein in nature. The biosurfactant exhibited bactericidal activity against Pseudomonas aeruginosa PSB2, Pseudomonas putida PSJ1, Salmonella sp. SL2, Escherichia coli MTCC 118, Bacillus sp. BC1 and Staphylococcus aureus STP1 at concentrations ranging from 6.4 to 50 mg/mL.
The yield of biosurfactant was four-fold higher after optimization of media components and culture conditions using response surface methodology. The results of this study suggested that sugar cane molasses can be used as a low-cost substrate to enhance the yield of biosurfactants with antimicrobial activity.
Enzymatic biodegradation of organophosphate pesticides (OPs) is a promising technology to remove these toxic compounds. However, its application in industrial washing was restricted by the lack of efficient immobilized enzymes that can work at high temperatures and high pHs in the presence of various detergents. Therefore, it is necessary to develop a simple method to prepare a robust immobilized enzyme for efficient degradation of OPs.
An organophosphate hydrolase (OPH), PoOPHM9, was conjugated and immobilized with a commercially available polymer, Pluronic F127. The prepared cross-linked enzyme-polymer conjugate (CLEPC) displayed higher pH stability in the range from 7.0 to 11.0 and a higher optimal temperature (50 °C) than that of free PoOPHM9 (30 °C). Its half-life and apparent kcat/KM reached 12.8 h at 50 °C and 390.3 ± 7.8 mM−1 s−1, respectively, which were even better than that of the traditional cross-linked enzyme aggregates (CLEA, 7.2 h and 10.9 ± 1.7 mM−1 s−1). The activity of PoOPHM9 CLEPC was further enhanced up to 2.5-fold by the anionic, nonionic and biocompatible detergents, which was first observed. 0.15 mM Malathion was degraded completely by PoOPHM9 CLEPC after activation within 10 min in the presence of 0.1% (w/w) detergents of all types at pH 9.0 and 25 °C, demonstrating its capability in degrading OPs at practically relevant conditions.
The conjugation of Pluronic F127 in enzyme immobilization could effectively reduce the activity loss of immobilized enzymes and enhance their stability and activity at high temperatures and high pHs. In addition, the activity of CLEPC can be even enhanced in the presence of various detergents. This technology can be extended easily to produce other immobilized polymer-enzyme conjugates due to its simplicity.
