The effects of monoculture and intercropping on photosynthesis performance correlated with growth of garlic and perennial ryegrass response to different heavy metals.

BACKGROUND: The potential of phytoremediation using garlic monoculture (MC) and intercropping (IC) system with perennial ryegrass to enhance the uptake of cadmium (Cd), chromium (Cr), and lead (Pb) were investigated. RESULTS: Positive correlations were found between MC and IC systems, with varying biomass. Production of perennial ryegrass was affected differently depending on the type of toxic metal present in the soil. Root growth inhibition was more affected than shoot growth inhibition. The total biomass of shoot and root in IC was higher than MC, increasing approximately 3.7 and 2.9 fold compared to MC, attributed to advantages in root IC crop systems. Photosystem II efficiency showed less sensitivity to metal toxicity compared to the control, with a decrease between 10.07-12.03%. Among gas exchange parameters, only Cr significantly affected physiological responses by reducing transpiration by 69.24%, likely due to leaf chlorosis and necrosis. CONCLUSION: This study exhibited the potential of garlic MC and IC with perennial ryegrass in phytoremediation. Although the different metals affect plant growth differently, IC showed advantages over MC in term biomass production.

Purification and Characterization of a recombinant ß-Xylosidase from Bacillus licheniformis ATCC 14580 into E. coli Bl21.

Present research work is aimed to purify and characterize a recombinant ß-xylosidase enzyme which was previously cloned from Bacillus licheniformis ATCC 14580 in to Escherichia coli BL21. Purification of recombinant enzyme was carried out by using ammonium sulphate precipitation method followed by single step immobilized metal ion affinity chromatography. Specific activity of purified recombinant ß-xylosidase enzyme was 20.78 Umg-1 with 2.58 purification fold and 33.75% recovery. SDS-PAGE was used to determine the molecular weight of recombinant purified ß-xylosidase and it was recorded as 52 kDa. Purified enzyme showed stability upto 90°C within a pH range of 3-8 with and optimal temperature and pH, 55ºC and 7.0, respectively. The enzyme activity was not considerably affected in the presence of EDTA. An increase in the enzyme activity was found in the manifestation of Mg+2. Enzyme activity was also increased by 6%, 18% and 22% in the presence of 1% Tween 80, ß-mercaptoethanol and DTT, respectively. Higher concentrations (10 - 40%) of organic solvents did not show any effect upon activity of enzyme. All these characteristics of the recombinant enzyme endorsed it as a potential candidate for biofuel industry.

Purification and characterization of cloned alkaline protease gene of Geobacillus stearothermophilus.

Thermostable alkaline serine protease gene of Geobacillus stearothermophilus B-1172 was cloned and expressed in Escherichia coli BL21 (DE3) using pET-22b(+), as an expression vector. The growth conditions were optimized for maximal production of the protease using variable fermentation parameters, i.e., pH, temperature, and addition of an inducer. Protease, thus produced, was purified by ammonium sulfate precipitation followed by ion exchange chromatography with 13.7-fold purification, with specific activity of 97.5 U mg(-1) , and a recovery of 23.6%. Molecular weight of the purified protease, 39 kDa, was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was stable at 90 °C at pH 9. The enzyme activity was steady in the presence of EDTA indicating that the protease was not a metalloprotease. No significant change in the activity of protease after addition of various metal ions further strengthened this fact. However, an addition of 1% Triton X-100 or SDS surfactants constrained the enzyme specific activity to 34 and 19%, respectively. Among organic solvents, an addition of 1-butanol (20%) augmented the enzyme activity by 29% of the original activity. With casein as a substrate, the enzyme activity under optimized conditions was found to be 73.8 U mg(-1) . The effect of protease expression on the host cells growth was also studied and found to negatively affect E. coli cells to certain extent. Catalytic domains of serine proteases from eight important thermostable organisms were analyzed through WebLogo and found to be conserved in all serine protease sequences suggesting that protease of G. stearothermophilus could be beneficially used as a biocontrol agent and in many industries including detergent industry.

Prospective Roles of Extremophilic Fungi in Climate Change Mitigation Strategies.

Climate change and the resultant environmental deterioration signify one of the most challenging problems facing humankind in the 21st century. The origins of climate change are multifaceted and rooted in anthropogenic activities, resulting in increasing greenhouse gases in the environment and leading to global warming and weather drifts. Extremophilic fungi, characterized by their exceptional properties to survive extreme habitats, harbor great potential in mitigating climate change effects. This review provides insight into the potential applications of extremophilic fungi in climate change mitigation strategies. They are able to metabolize organic biomass and degrade carbon compounds, thereby safely sequestering carbon and extenuating its release into the environment as noxious greenhouse gases. Furthermore, they possess extremozymes, which break down recalcitrant organic species, including lignocellulosic biomass and hydrocarbons. Enzymatic machinery equips these extremophilic fungi to perform the bioremediation of polluted environments. Extremophilic fungi can also be exploited for various biological interventions, such as biofuels, bioplastics, and other bioprocessing applications. However, these fungi characterize a valued but underexplored resource in the arsenal of climate change mitigation strategies.

Efficient biomass saccharification using a novel cellobiohydrolase from Clostridium clariflavum for utilization in biofuel industry.

The present study describes the cloning of the cellobiohydrolase gene from a thermophilic bacterium Clostridium clariflavum and its expression in Escherichia coli BL21(DE3) utilizing the expression vector pET-21a(+). The optimization of various parameters (pH, temperature, isopropyl ß-d-1-thiogalactopyranoside (IPTG) concentration, time of induction) was carried out to obtain the maximum enzyme activity (2.78 ± 0.145 U ml-1) of recombinant enzyme. The maximum expression of recombinant cellobiohydrolase was obtained at pH 6.0 and 70 °C respectively. Enzyme purification was performed by heat treatment and immobilized metal anionic chromatography. The specific activity of the purified enzyme was 57.4 U mg-1 with 35.17% recovery and 3.90 purification fold. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the molecular weight of cellobiohydrolase was 78 kDa. Among metal ions, Ca2+ showed a positive impact on the cellobiohydrolase enzyme with increased activity by 115%. Recombinant purified cellobiohydrolase enzyme remained stable and exhibited 77% and 63% residual activity in comparison to control in the presence of n-butanol and after incubation at 80 °C for 1 h, respectively. Our results indicate that our purified recombinant cellobiohydrolase can be used in the biofuel industry.

Correction: Efficient biomass saccharification using a novel cellobiohydrolase from Clostridium clariflavum for utilization in biofuel industry.

[This corrects the article DOI: 10.1039/D1RA00545F.].

Metabolic Engineering of Microalgae for Biofuel Production.

Microalgae are considered as promising cell factories for the production of various types of biofuels, including bioethanol, biodiesel, and biohydrogen by using carbon dioxide and sunlight. In spite of unique advantages of these microorganisms, the commercialization of microalgal biofuels has been hindered by poor economic features. Metabolic engineering is among the most promising strategies put forth to overcome this challenge. In this chapter, metabolic pathways involved in lipid and hydrogen production by microalgae are reviewed and discussed. Moreover, metabolic and genetic engineering approaches investigated for improving the rate of lipid (as a feedstock for biodiesel production) and biohydrogen synthesis are presented. Finally, genetic engineering tools and approaches employed for engineering microalgal metabolic pathways are elaborated. A thorough step-by-step protocol for reconstructing the metabolic pathway of various microorganisms including microalgae is also presented.

Pilot-scale production of a highly thermostable α-amylase enzyme from Thermotoga petrophila cloned into E. coli and its application as a desizer in textile industry.

In this study, the industrial applications of a highly thermostable α-amylase as a desizer in the textile industry was evaluated. The cloned gene was expressed in different media (ZBM, LB, ZYBM9, and ZB) with IPTG (isopropyl ß-d-1-thiogalactopyranoside) used as an inducer. Lactose was also used as an alternate inducer for the T7 promoter system in E. coli. For the large-scale production of the enzyme, different parameters were optimized. The maximum enzyme production was achieved when the volume of medium was 70% of the total volume of fermenter with a 2.0 vvm air supply and 20% dissolved oxygen at a 200 rpm agitation rate. Under all the optimized conditions, the maximum enzyme production was 22.08 U ml-1 min-1 with lactose (200 mM) as an inducer in ZBM medium. The desizing potential of the purified α-amylase enzyme was calculated with different enzyme concentrations (50-300 U ml-1) at different temperatures (50-100 °C), and pHs (4-9) with varying time intervals (30-120 min). The highest desizing activity was found when 150 U ml-1 enzyme units were utilized at 85 °C and at 6.5 pH for 1 h.

Optimization of saccharification potential of recombinant xylanase from Bacillus licheniformis.

Saccharification potential of xylanase enzyme cloned from Bacillus licheniformis into E. coli BL21 (DE3) was evaluated against plant biomass for the production of bioethanol. The expression of cloned gene was studied and conditions were optimized for its large scale production. The parameters effecting enzyme production were examined in a fermenter. Recombinant xylanase has the ability to breakdown birchwood xylan to release xylose as well as the potential to treat plant biomass, such as wheat straw, rice straw, and sugarcane bagass. The saccharification ability of this enzyme was optimized by studying various parameters. The maximum saccharification percentage (84%) was achieved when 20 units of recombinant xylanase were used with 8% sugarcane bagass at 50°C and 120 rpm after 6 hours of incubation. The results indicated that the bioconversion of natural biomass by recombinant xylanase into simple sugars can be used for biofuel (bioethanol) production. This process can replace the use of fossil fuels, and the use of bioethanol can significantly reduce the emission of toxic gases. Future directions regarding pre-treatment of cellulosic and hemicellulosic biomass and other processes that can reduce the cost and enhance the yield of biofuels are briefly discussed.

Cloning, Purification and Characterization of a Highly Thermostable Amylase Gene of Thermotoga petrophila into Escherichia coli.

A putative α-amylase gene of Thermotoga petrophila was cloned and expressed in Escherichia coli BL21 (DE3) using pET-21a (+), as an expression vector. The growth conditions were optimized for maximal expression of the α-amylase using various parameters, such as pH, temperature, time of induction and addition of an inducer. The optimum temperature and pH for the maximum expression of α-amylase were 22 °C and 7.0 pH units, respectively. Purification of the recombinant enzyme was carried out by heat treatment method, followed by ion exchange chromatography with 34.6-fold purification having specific activity of 126.31 U mg(-1) and a recovery of 56.25%. Molecular weight of the purified α-amylase, 70 kDa, was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was stable at 100 °C temperature and at pH of 7.0. The enzyme activity was increased in the presence of metal ions especially Ca(+2) and decreased in the presence of EDTA indicating that the α-amylase was a metalloenzyme. However, addition of 1% Tween 20, Tween 80 and ß-mercaptoethanol constrained the enzyme activity to 87, 96 and 89%, respectively. No considerable effect of organic solvents (ethanol, methanol, isopropanol, acetone and n-butanol) was observed on enzyme activity. With soluble starch as a substrate, the enzyme activity under optimized conditions was 73.8 U mg(-1). The α-amylase enzyme was active to hydrolyse starch forming maltose.

Cloning, Expression, and Purification of Xylanase Gene from Bacillus licheniformis for Use in Saccharification of Plant Biomass.

The xylanase gene (xynA) of Bacillus licheniformis 9945A was cloned and expressed in Escherichia coli BL21(DE3) using pET-22b(+) as an expression vector. The recombinant xylanase enzyme was purified by ammonium sulfate precipitation, followed by single-step immobilized metal ion affinity chromatography with a 57.58-fold purification having 138.2 U/mg specific activity and recovery of 70.08 %. Molecular weight of the purified xylanase, 23 kDa, was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was stable for up to 70 °C with a broad pH range of 4-9 pH units. The enzyme activity was increased in the presence of metal ions especially Ca(+2) and decreased in the presence of EDTA, indicating that the xylanase was a metalloenzyme. However, an addition of 1-4 % Tween 80, ß-mercaptoethanol, and DTT resulted in the increase of enzyme activity by 51, 52, and 5 %, respectively. Organic solvents with a concentration of 10-40 % slightly decreased the enzyme activity. The xylanase enzyme possesses the ability of bioconversion of plant biomasses like wheat straw, rice straw, and sugarcane bagasse. Among the different tested biomasses, the highest saccharification percentage was observed with 1 % sugarcane bagasse after 72 h of incubation at 50 °C with 20 units of enzyme. The results suggest that recombinant xylanase can be used in the bioconversion of natural biomasses into simple sugars which could be further used for the production of biofuel.