Adoption of CRISPR-Cas for crop production: present status and future prospects.

Background: Global food systems in recent years have been impacted by some harsh environmental challenges and excessive anthropogenic activities. The increasing levels of both biotic and abiotic stressors have led to a decline in food production, safety, and quality. This has also contributed to a low crop production rate and difficulty in meeting the requirements of the ever-growing population. Several biotic stresses have developed above natural resistance in crops coupled with alarming contamination rates. In particular, the multiple antibiotic resistance in bacteria and some other plant pathogens has been a hot topic over recent years since the food system is often exposed to contamination at each of the farm-to-fork stages. Therefore, a system that prioritizes the safety, quality, and availability of foods is needed to meet the health and dietary preferences of everyone at every time. Methods: This review collected scattered information on food systems and proposes methods for plant disease management. Multiple databases were searched for relevant specialized literature in the field. Particular attention was placed on the genetic methods with special interest in the potentials of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and Cas (CRISPR associated) proteins technology in food systems and security. Results: The review reveals the approaches that have been developed to salvage the problem of food insecurity in an attempt to achieve sustainable agriculture. On crop plants, some systems tend towards either enhancing the systemic resistance or engineering resistant varieties against known pathogens. The CRISPR-Cas technology has become a popular tool for engineering desired genes in living organisms. This review discusses its impact and why it should be considered in the sustainable management, availability, and quality of food systems. Some important roles of CRISPR-Cas have been established concerning conventional and earlier genome editing methods for simultaneous modification of different agronomic traits in crops. Conclusion: Despite the controversies over the safety of the CRISPR-Cas system, its importance has been evident in the engineering of disease- and drought-resistant crop varieties, the improvement of crop yield, and enhancement of food quality.

Laccase is a multitasking protein for synthetic gene circuits in the yeast Saccharomycescerevisiae.

Laccase is a multicopper oxidase enzyme that oxidizes a variety of substrates, including polyphenols and polycyclic aromatic hydrocarbons (PAHs). It catalyzes the four-electron reduction of molecular oxygen that results in the production of water as a by-product. Thus, laccase can play an important role in environmental care. Previously, we have successfully expressed Trametes trogii laccase (TtLcc1) in the yeast Saccharomyces cerevisiae. In this work, we have expressed in yeast another laccase, LacA from Trametes sp. AH28-2, and tested its function on PAHs. Yeast cells engineered to produce the two laccases performed efficient PAH degradation. Both TtLcc1 and LacA led to the construction of spatiotemporal fluorescence-pulse generators when combined with a benzoate/salicylate yeast biosensor in a two-population system. Moreover, laccases returned a visual output signal in yeast synthetic circuits-upon reacting with ABTS (2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid)). Thus, in S. cerevisiae, laccases are a powerful alternative to fluorescent reporter proteins.

Allosteric-Regulation-Based DNA Circuits in Saccharomyces cerevisiae to Detect Organic Acids and Monitor Hydrocarbon Metabolism In Vitro.

We show the engineering of prokaryotic-transcription-factor-based biosensing devices in Saccharomyces cerevisiae cells for an in vitro detection of common hydrocarbon intermediates/metabolites and potentially, for monitoring of the metabolism of carbon compounds. We employed the bacterial receptor proteins MarR (multiple antibiotic-resistant receptor) and PdhR (pyruvate dehydrogenase-complex regulator) to detect benzoate/salicylate and pyruvate, respectively. The yeast-enhanced green fluorescence protein (yEGFP) was adopted as an output signal. Indeed, the engineered yeast strains showed a strong and dynamic fluorescent output signal in the presence of the input chemicals ranging from 2 fM up to 5 mM. In addition, we describe how to make use of these strains to assess over time the metabolism of complex hydrocarbon compounds due to the hydrocarbon-degrading fungus Trichoderma harzianum (KY488463).

Engineered yeasts and lignocellulosic biomaterials: shaping a new dimension for biorefinery and global bioeconomy.

The next milestone of synthetic biology research relies on the development of customized microbes for specific industrial purposes. Metabolic pathways of an organism, for example, depict its chemical repertoire and its genetic makeup. If genes controlling such pathways can be identified, scientists can decide to enhance or rewrite them for different purposes depending on the organism and the desired metabolites. The lignocellulosic biorefinery has achieved good progress over the past few years with potential impact on global bioeconomy. This principle aims to produce different bio-based products like biochemical(s) or biofuel(s) from plant biomass under microbial actions. Meanwhile, yeasts have proven very useful for different biotechnological applications. Hence, their potentials in genetic/metabolic engineering can be fully explored for lignocellulosic biorefineries. For instance, the secretion of enzymes above the natural limit (aided by genetic engineering) would speed-up the down-line processes in lignocellulosic biorefineries and the cost. Thus, the next milestone would greatly require the development of synthetic yeasts with much more efficient metabolic capacities to achieve basic requirements for particular biorefinery. This review gave comprehensive overview of lignocellulosic biomaterials and their importance in bioeconomy. Many researchers have demonstrated the engineering of several ligninolytic enzymes in heterologous yeast hosts. However, there are still many factors needing to be well understood like the secretion time, titter value, thermal stability, pH tolerance, and reactivity of the recombinant enzymes. Here, we give a detailed account of the potentials of engineered yeasts being discussed, as well as the constraints associated with their development and applications.

Metabolic pathways of an organism depict its chemical repertoire and its genetic makeup.Autonomous synthetic microbes can be developed for lignocellulose biorefinery (LCB).LCBs can be harnessed with synthetic microbes to boost global bioeconomy.Yeasts can be engineered to enhance downstream process of LCB.

Synthetic metabolic transducers in Saccharomyces cerevisiae as sensors for aromatic permeant acids and bioreporters of hydrocarbon metabolism.

Yeast-based biosensors have great potential for various applications, although the present range of detectable chemicals is still very minimal. This work provides an enlargement of the knowledge on detectable chemicals and creates an additional basis for engineering modular yeast biosensors. Bacterial allosteric transcription factors, such as MarR and PdhR, were recruited to build transducer circuits in Saccharomyces cerevisiae. MarR-based biosensors were designed for the detection of aromatic permeant acids (benzoate and salicylate), whereas the PdhR-expressing yeast cells were engineered for responding to pyruvate. In general, all our engineered strains showed a fast response time and a strong fluorescent output signal to chemical concentrations ranging from 5 mM down to 2 fM. They exhibited versatile dynamic range and were capable of operating in a variety of complex media that might contain any of these compounds. A new milestone in biosensor design is the engineering of inter/intracellular metabolic biosensors that would allow real-time monitoring of either the metabolism of particular compounds, or the detection of their intermediate/end products. Our synthetic cells are applicable to different areas, from adequate real-time detection of aromatic permeant acids to regulation/monitoring of different hydrocarbon metabolisms. The new strains engineered in this study could be of great importance because of the ecological significance of aromatic permeant acids from their formations during either hydrocarbon degradation or metabolism of different chemicals to their involvement in different biological and non-biological systems.

Biotreatment of Selected Agrowaste Products with Some Culinary-Medicinal Mushrooms Enhances Their Nutrient Composition for Use as Animal Feed in Nigeria.

Biological treatment of agrowaste products using white-rot fungi can enhance their physicochemical and nutritional values for further use as animal feed. In this study, sweet orange and ripe plantain peels were biotreated during their use as substrates to grow three mushroom species (Lentinus squarrosulus, Pleurotus pulmonarius, and P. ostreatus), with the aim to reuse these wastes for animal feed. The effects of mushroom biotreatment on these wastes were analyzed at 0, 30, and 60 days of incubation with respect to physical and chemical properties of the substrates. Results from the mineral composition revealed the presence of appreciable quantities of sodium, magnesium, phosphorus, nitrogen, calcium, potassium, and iron for both peels after treatment with all of the mushrooms. The amino acid content of orange peels treated with L. squarrosulus and P. pulmonarius increased after treatment compared with the control and plantain peel. The carbohydrate level in both wastes also increased after treatment with L. squarrosulus, P. pulmonarius, and P. ostreatus, respectively, while the moisture and ash contents of treated wastes decreased as the incubation periods progressed. However, there was no significant difference (P ≤ 0.05) in the protein, fiber, and fat content of the control compared to those of treated wastes. This study affirms that biotreatment of these agrowaste products using the selected mushrooms can enhanced their value for further use.

Synthetic Saccharomyces cerevisiae tolerate and degrade highly pollutant complex hydrocarbon mixture.

Fungal laccase (Lac) has become a very useful biocatalyst in different industries, bio-refineries and, most importantly, bioremediation. Many reports have also linked hydrocarbon tolerance and degradation by various microorganisms with Lac secretion. In this study, Trametes trogii Lac (Ttlcc1) was engineered into Saccharomyces cerevisiae strain CEN.PK2-1 C under the constitutive GPD promoter (pGPD) for multi-fold synthesis with efficient hydrocarbon tolerance and degradation. Protein expression in heterologous hosts is strictly strain-specific, it can also be influenced by the synthetic design and culture conditions. We compared synthetic designs with different shuttle vectors for the yeast strains and investigated the best culture conditions by varying the pH, temperature, carbon, nitrogen sources, and CuSO4 amount. Two S. cerevisiae strains were built in this study: byMM935 and byMM938. They carry the transcription unit pGPD-Ttlcc1-CYC1t either inside the pRSII406 integrative plasmid (byMM935) or the pRSII426 multicopy plasmid (byMM938). The performance of these two synthetic strains were studied by comparing them to the wild-type strain (byMM584). Both byMM935 and byMM938 showed significant response to different carbon sources (glucose, galactose, lactose, maltose, and sucrose), nitrogen sources (NH4Cl, NH4NO3, KNO3, malt extract, peptone, and yeast extract), and solid state fermentation of different plant biomasses (bagasse, banana peels, corn cob, mandarin peels, and peanut shells). They performed best in optimized growth conditions with specific carbon and nitrogen sources, and a preferred pH in the range 3.5-4.5, temperature between 30 and 40 0C, and 1 mM CuSO4. In optimized yeast-growth medium, strain byMM935 showed the highest laccase activities of 1.621 ±â€¯0.063 U/mL at 64 h, whereas byMM938 gave its highest activity (1.417 ±â€¯0.055 U/mL) at 48 h. In this work, we established, by using Bushnell Hass synthetic medium, that the new Ttlcc1-yeast strains tolerated extreme pH and complex hydrocarbon mixture (CHM) toxicity. They degraded 60-90% of the key components in CHM within 48 h, including poly-cyclic aromatic hydrocarbons, alkyl indenes, alkyl tetralines, alkyl benzenes, alkyl biphenyls, and BTEX (Benzene, Toluene, Ethylbenzene, and Xylenes). This is the first report on the hydrocarbon degradation potential of a Ttlcc1-yeast. Compared to the native organism, such synthetic strains are better suited for meeting growing demands and have potentials for application in large-scale in situ bioremediation of hydrocarbon-polluted sites.

Trichoderma: Potential bio-resource for the management of tomato root rot diseases in Africa.

Trichoderma spp. are among the front-line microorganisms commonly employed in novel biotechnology applications. They have been well-proven as biopesticides, biofertilizers, and biostimulants for managing plants against biotic and abiotic stresses. They are instrumental in managing plant diseases of economic importance, such as tomato root rot. However, this group of fungi has not been well-exploited en-mass in developing countries, while the use of bioagents in-lieu of chemical pesticides is still not a common practice in many African countries. Africa contributes 11.8% to global tomato production. Unfortunately, more than half of the actual product is lost due to diseases. The root rot of tomatoes predominantly caused by soil-borne fungal pathogens are among significant problems of tomato cultivation in Africa. Here, we review the constraints of tomato root rot in Africa and the roles of Trichoderma in repositioning the crop for optimum productivity. We gave a comprehensive overview of the economic importance, root rot epidemiology, and how to circumvent it through gene pool to resistant tomato and employ Trichoderma's biological control potentials. Furthermore, this review gives an overview of the mechanisms of action of Trichoderma, gaps in the advocacy, adoption, commercialization, and regulation of Trichoderma as biocontrol agents of tomato rot diseases in Africa.

Modified filters with Penicillium chrysogenum culture enhance removal of copper and iron contaminants in water.

Development of a cost-effective and broad-based practical approach to wastewater treatment is of utmost importance, especially in developing countries. In this study, we investigated the efficacy of using Penicillium chrysogenum cells with sand filters for removal of heavy metals from contaminated water. This was done by designing three fungal-based sand filters (FSF) containing 30, 40, 50 spores/mL and sand filter (SF) without the fungus. These preparations were used to treat deionized water simulated with two concentrations of copper and iron (5 and 10 mg L-1 respectively). These simulations were prepared to create commonly observed contamination levels in many water sources. Effluent reductions relative to treatments effects were analysed using the standard protocol for eight days under aseptic conditions. On the eighth day, it was observed that the copper concentration (10 mg L-1) was reduced to 0.106 mg L-1 in the 30 spores/mL treatment as compared to 0.198 mg L-1 observed in SF. It was also observed that copper concentrations were significantly reduced (p ≤ 0.05) between FSF and the SF. There was also a significant reduction while comparing the metal removal in treatments after the second and eighth days. The general affinity range for iron in the four treatments was 30 > 40 > SF > 50, in that order has 94.26, 91.66, 87.98 and 85.48 as removal efficiency for iron (5 mg L-1) on the eighth day. P. chrysogenum is therefore a valuable biosorbent that can help improve the quality of wastewater by biosand filter treatment.

Phytoremediation technology and food security impacts of heavy metal contaminated soils: A review of literature.

Heavy metal accumulation in soil and water is one of major problems caused by inorganic contaminants. Their presence in agricultural soils in high quantities have impacted the food security significantly and, by extension, the human health. Amongst various physico-chemical methods available for remediation of heavy-metals-polluted-sites, phytoremediation approaches have been found to be safe and environment friendly. This review gathered scattered information on heavy metal phytoremediation studies published in both review and research articles. It described the impact of heavy metals on food security and comprehensively discussed the application of different phytoremediation approaches for treatment of heavy metal-polluted soils, the basic principles underlining them, their strengths and weaknesses. Our findings indicated that, while hundreds of hyper-accumulator plants are being reported yearly, only few describe limitations inherent in them, such as low growth rate, low biomass production, and low metal tolerance. Hence, this review also gave a detailed overview of research gaps in phytotechnology and advocates consideration of the 'omics' studies; genomics, proteomics, metabolomics and likes in selecting and enhancing potential plants for phytoremediation. For a sustainable large-scale phytoremediation application, we established a multi-technology repair strategy via the combination of different methods like application of biological composts, plant-growth promoting microorganisms, and phytohormones for stimulation of the plant-growth during phytoremediation. We also gave comprehensive insights to proper disposal of plants used for phytoremediation, this subject is often not well considered/planned while deciding the application of plants for removal of heavy metals from polluted environments.

Antimicrobial Activities of Laetiporus conifericola (Agaricomycetes) from the United States.

In this study, samples of polypore mushroom Laetiporus conifericola were collected from Pennsylvania, USA. The antimicrobial activity (AMA) of ethanolic, methanolic, and water extracts of this fungus were tested in vitro by the agar diffusion test against some selected clinically important microorganisms. These microorganisms included three Gram-positive bacteria (Staphylococcus aureus 5W1941, S. epidermidis 85W1940, and Bacillus cereus 85W1815), three Gram-negative bacteria (Escherichia coli 85W1860, Salmonella typhimurium 85W1956, and Pseudomonas aeruginosa 85W1903), and one fungus (Candida albicans). These extracts demonstrated varying degrees of inhibition against all of the test pathogenic microorganisms except C. albicans. Methanolic and ethanolic extracts of L. conifericola were very effective against S. aureus, while the aqueous extract was the least effective. All tested extracts were effective against S. epidermidis, methanolic extract produced the best zone of inhibition followed by the aqueous extract while ethanolic extract had the least zone of inhibition. B. cereus and P. aeruginosa were highly susceptible to ethanol extract. In addition, the growth of E. coli was best inhibited by the aqueous extract, followed by the methanolic and ethanolic extracts, respectively. The aqueous and methanolic extracts were most effective against S. typhimurium; however, this bacterium was not susceptible to ethanolic extract. The significance of these findings is discussed.

Analysis of culturable airborne fungi in outdoor environments in Tianjin, China.

BACKGROUND: Fungal spores dispersed in the atmosphere may become cause of different pathological conditions and allergies for human beings. A number of studies have been performed to analyze the diversity of airborne fungi in different environments worldwide, and in particular in many urban areas in China. We investigated, for the first time, the diversity, concentration and distribution of airborne fungi in Tianjin city. We sampled 8 outdoor environments, using open plate method, during a whole winter season. Isolated fungi were identified by morphological and molecular analysis. Environmental factors which could influence the airborne fungi concentration (temperature, humidity, wind speed, and air pressure) were monitored and analyzed. The effect of different urban site functions (busy areas with high traffic flow and commercial activities vs. green areas) on airborne fungal diversity was also analyzed. RESULTS: A total of 560 fungal strains, belonging to 110 species and 49 genera of Ascomycota (80 %), Basidiomycota (18 %), and Mucoromycota (2 %) were isolated in this study. The dominant fungal genus was Alternaria (22 %), followed by Cladosporium (18.4 %), Naganishia (14.1 %), Fusarium (5.9 %), Phoma (4.11 %), and Didymella (4.8 %). A fungal concentration ranging from 0 to 3224.13 CFU m- 3 was recorded during the whole study. Permutational multivariate analysis showed that the month was the most influential factor for airborne fungal community structure, probably because it can be regarded as a proxy of environmental variables, followed by wind speed. The two analyzed environments (busy vs. green) had no detectable effect on the air fungal community, which could be related to the relatively small size of parks in Tianjin and/or to the study season. CONCLUSIONS: Our study shed light on the highly diverse community of airborne fungi characterizing the outdoor environments of Tianjin, and clarified the role that different environmental factors played in shaping the analyzed fungal community. The dominant presence of fungi with potential hazardous effect on human health, such as Alternaria, Cladosporium and Naganishia, deserves further attention. Our results may represent a valuable source of information for air quality monitoring, microbial pollution control, and airborne diseases prevention.

Genome-based engineering of ligninolytic enzymes in fungi.

BACKGROUND: Many fungi grow as saprobic organisms and obtain nutrients from a wide range of dead organic materials. Among saprobes, fungal species that grow on wood or in polluted environments have evolved prolific mechanisms for the production of degrading compounds, such as ligninolytic enzymes. These enzymes include arrays of intense redox-potential oxidoreductase, such as laccase, catalase, and peroxidases. The ability to produce ligninolytic enzymes makes a variety of fungal species suitable for application in many industries, including the production of biofuels and antibiotics, bioremediation, and biomedical application as biosensors. However, fungal ligninolytic enzymes are produced naturally in small quantities that may not meet the industrial or market demands. Over the last decade, combined synthetic biology and computational designs have yielded significant results in enhancing the synthesis of natural compounds in fungi. In this review, we gave insights into different protein engineering methods, including rational, semi-rational, and directed evolution approaches that have been employed to enhance the production of some important ligninolytic enzymes in fungi. We described the role of metabolic pathway engineering to optimize the synthesis of chemical compounds of interest in various fields. We highlighted synthetic biology novel techniques for biosynthetic gene cluster (BGC) activation in fungo and heterologous reconstruction of BGC in microbial cells. We also discussed in detail some recombinant ligninolytic enzymes that have been successfully enhanced and expressed in different heterologous hosts. Finally, we described recent advance in CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR associated) protein systems as the most promising biotechnology for large-scale production of ligninolytic enzymes. SHORT CONCLUSION: Aggregation, expression, and regulation of ligninolytic enzymes in fungi require very complex procedures with many interfering factors. Synthetic and computational biology strategies, as explained in this review, are powerful tools that can be combined to solve these puzzles. These integrated strategies can lead to the production of enzymes with special abilities, such as wide substrate specifications, thermo-stability, tolerance to long time storage, and stability in different substrate conditions, such as pH and nutrients.

Hydrocarbon Degradation and Enzyme Activities of Aspergillus oryzae and Mucor irregularis Isolated from Nigerian Crude Oil-Polluted Sites.

Many free-living saprobic fungi are nature recruited organisms for the degradation of wastes, ranging from lignocellulose biomass to organic/inorganic chemicals, aided by their production of enzymes. In this study, fungal strains were isolated from contaminated crude-oil fields in Nigeria. The dominant fungi were selected from each site and identified as Aspergillus oryzae and Mucor irregularis based on morphological and molecular characterization, with site percentage incidences of 56.67% and 66.70%, respectively. Selected strains response/tolerance to complex hydrocarbon (used engine oil) was studied by growing them on Bushnell Haas (BH) mineral agar supplemented with the hydrocarbon at different concentrations, i.e., 5%, 10%, 15%, and 20%, with a control having dextrose. Hydrocarbon degradation potentials of these fungi were confirmed in BH broth culture filtrates pre-supplemented with 1% engine oil after 15 days of incubation using GC/MS. In addition, the presence of putative enzymes, laccase (Lac), manganese peroxidase (MnP), and lignin peroxidase (LiP) was confirmed in culture filtrates using appropriate substrates. The analyzed fungi grew in hydrocarbon supplemented medium with no other carbon source and exhibited 39.40% and 45.85% dose inhibition response (DIR) respectively at 20% hydrocarbon concentration. An enzyme activity test revealed that these two fungi produced more Lac than MnP and LiP. It was also observed through the GC/MS analyses that while A. oryzae acted on all hydrocarbon components in the used engine oil, M. irregularis only degraded the long-chain hydrocarbons and BTEX. This study confirms that A. oryzae and M. irregularis have the potential to be exploited in the bio-treatment and removal of hydrocarbons from polluted soils.

Synergistic plant-microbes interactions in the rhizosphere: a potential headway for the remediation of hydrocarbon polluted soils.

Soil pollution is an unavoidable evil; many crude-oil exploring communities have been identified to be the most ecologically impacted regions around the world due to hydrocarbon pollution and their concurrent health risks. Several clean-up technologies have been reported on the removal of hydrocarbons in polluted soils but most of them are either very expensive, require the integration of advanced mechanization and/or cannot be implemented in small scale. However, "Bioremediation" has been reported as an efficient, cost-effective and environment-friendly technology for clean-up of hydrocarbon"s contaminated soils. Here, we suggest the implementation of synergistic mechanism of bioremediation such as the use of rhizosphere mechanism which involves the actions of plant and microorganisms, which involves the exploitation of plant and microorganisms for effective and speedy remediation of hydrocarbon"s contaminated soils. In this mechanism, plant"s action is synergized with the soil microorganisms through the root rhizosphere to promote soil remediation. The microorganisms benefit from the root metabolites (exudates) and the plant in turn benefits from the microbial recycling/solubilizing of mineral nutrients. Harnessing the abilities of plants and microorganisms is a potential headway for cost-effective clean-up of hydrocarbon"s polluted sites; such technology could be very important in countries with great oil producing activities/records over many years but still developing.

Degradation of 2, 2-Dichlorovinyl dimethyl phosphate (dichlorvos) through the rhizosphere interaction between Panicum maximum Jacq and some selected fungi.

Many fungi have been reported to enhance the plant responses and degradation of several persistent pollutants in soils. In this study, five dominant fungi strains were identified from a pesticides polluted soil in Nigeria and screened for the expression of phosphoesterase (opd and mpd) and catechol 1, 2-dioxygenase (afk2 and afk4) genes using Reverse Transcriptase-PCR technique. Their rhizosphere interaction with plant (Panicum maximum) was further studied for the degradation of 2, 2 Dichlorovinyl dimethyl phosphate (dichlorvos). Fungal strains were mixed with Spent Mushroom Compost (SMC) of Pleurotus ostreatus in 1:100 w/w and then applied to a sterilized pesticide polluted soil (5 kg) at increasing concentrations of 10, 20, 30 and 40% with two controls (plant only and fungi-SMC mixture only). Degradation efficiency (DE), degradation rate (K1) and half-life (t1/2) of dichlorvos was calculated in each treatment after 90-day of planting. All the strains were registered at NCBI gene-bank with accession numbers KY693969, KY488464, KY488465, KY693971 and KY693972: they all possess the tested genes although mpd and opd were over-expressed in all the strains while afk2 and afk4 were moderately expressed. The plant-fungi-SMC interaction synergistically sped-up dichlorvos degradation rate in less time period, appreciable loss of dichlorvos at 72.23 and 82.70% DE were observed in 30 and 40% treatments respectively as compared to controls 1 and 2 having 62.20 ±â€¯3.07 and 62.33 ±â€¯4.69% DE respectively. In the same way, the 40% treatment gave the best k1 and t1/2 of 1.755 and 0.40 ±â€¯0.02/day respectively.

Transcriptomic responses of catalase, peroxidase and laccase encoding genes and enzymatic activities of oil spill inhabiting rhizospheric fungal strains.

Fungi are well associated with the degradation of hydrocarbons by the production of different enzymes, among which catalases (CBH), laccases (LCC) and peroxidases (LiP and MnP) are of immense importance. In this study, crude oil tolerance and enzyme secretions were demonstrated by rhizospheric fungal strains. Four most abundant strains were isolated from the rhizosphere of grasses growing in aged oil spill sites and identified through morphological characterization and molecular PCR-amplification of 5.8-28S ribosomal rRNA using ITS1 and ITS4 primers. These strains were subjected to crude oil tolerance test at 0-20% concentrations. Presence and transcriptase responses of putative genes lig (1-6), mnp, cbh (1.1, 1.1 and 11), and lcc encoding lignin peroxidase, manganese peroxidase, catalase, and laccase enzymes respectively were also studied in these strains using RT-PCR. In addition, activities of secreted enzymes by each strain were studied in aliquots. The strains were identified as Aspergillus niger asemoA (KY473958), Talaromyces purpurogenus asemoF (KY488463), Trichoderma harzianum asemoJ (KY488466), and Aspergillus flavus asemoM (KY488467) through sequencing and comparing the sequences' data at NCBI BLAST search software. All the isolated strains showed tolerance to crude oil at 20% concentration, but the growth rate reduced with increasing in oil concentrations. All the isolated strains possess the tested genes and lig 1-6 gene was overexpressed in A. niger and T. harzianum while lcc and mnp genes were moderately expressed in all the four strains. Almost 145 U.mL-1 of lignin and manganese peroxidase, 87 U.mL-1 of catalase, and 180 U.mL-1 of laccase enzymes were produced by these strains and it was also observed that these strain mostly produced studied enzymes in response to increasing crude oil concentrations. Considering the robust nature and diverse production of these catalytic enzymes by these strains, they can be exploited for various bioremediation technologies as well as other biotechnological applications.

Synergistic rhizosphere degradation of γ-hexachlorocyclohexane (lindane) through the combinatorial plant-fungal action.

Fungi are usually involved in degradation/deterioration of many anthropogenic wastes due to their verse enzyme secretions and adaptive capabilities. In this study, five dominant fungal strains were isolated from an aged lindane polluted site, they were all mixed (100 mg each) together with pent mushroom compost (SMC) and applied to lindane polluted soil (5 kg) at 10, 20, 30, 40% and control 0% (soil with no treatment), these were used to grow M. maximus Jacq for 3 months. To establish lindane degradation, deductions such as Degradation rate (K1), Half-life (t1/2) and Degradation efficiency (DE) were made based on the analyzed lindane concentrations before and after the experiment. We also tested the presence and expressions of phosphoesterases (mpd and opd-A) and catechol 1,2-dioxygenases (efk2 and efk4) genes in the strains. The stains were identified as Aspergillus niger (KY693970); Talaromyces atroroseus (KY488464), Talaromyces purpurogenus (KY488468), Yarrowia lipolytica (KY488469) and Aspergillus flavus (KY693973) through morphological and molecular methods. Combined rhizospheric action of M. maximus and fungi speed up lindane degradation rate, initially detected lindane concentration of 45 mg/kg was reduced to 11.26, 9.34 and 11.23 mg/kg in 20, 30 and 40% treatments respectively making 79.76, 85.93 and 88.67% degradation efficiencies. K1 of 1.29 was recorded in control while higher K1 of 1.60, 1.96 and 2.18 /day were recorded in 20, 30 and 40% treatments respectively. The best t1/2 of 0.32 and 0.35 /day were recorded in 40 and 30% compared to control (0.54 /day). All the strains were also affirmed to possess the tested genes; opd was overexpressed in all the strains except KY693973 while mpd was overexpressed in KY693970, KY488464 but moderately expressed in KY488468, KY488469 and KY693973. However, efk genes were under-expressed in most of the strains except KY488469 and KY693973 which showed moderate expression of efk4. This work suggests that the synergistic association of the identified rhizospheric fungi and M. maximus roots could be used to remove lindane in soil at a limited time period and this combination could be used at large scale.

Synergistic action of rhizospheric fungi with Megathyrsus maximus root speeds up hydrocarbon degradation kinetics in oil polluted soil.

This study was aimed at combining the potentials of plant and some rhizospheric fungal strains in remediation of crude-oil polluted soil. Four new rhizospheric fungi were identified from an aged crude-oil polluted site and used with Megathyrsus maximus (guinea grass) for a 90 day synergistic remediation experiment. Cultures of these strains were first mixed with spent mushroom compost (SMC), the mixture was then applied to a sterilized crude oil polluted soil at concentrations of 10%, 20%, 30% and 40% potted in three replicates. Soil with plant alone (0%1) and soil with fungi-SMC alone (0%2) served as controls. The soil's initial and final pH, nutrient, 16 EPA PAHs and heavy metal contents were determined, degradation rate, half-life and percentage loss of the total polyaromatic hydrocarbon (TPAH) were also calculated. Finally, the remediated soils were further screened for seed germination supporting index. The fungal strains were identified and registered at NCBI as Aspergillus niger asemoA (KY473958.1), Talaromyces purpurogenus asemoF (KY488463.1), Trichoderma harzianum asemoJ (KY488466.1) and Aspergillus flavus asemoM (KY488467.1). We observed for the first time that the synergistic mechanism improved the soil nutrient, reduced the heavy metal concentration and sped up hydrocarbon degradation rate. Using the initial and final concentrations of the TPAH, we recorded highest biodegradation rates (K1) and half-life (t1/2) in 30 and 40% treatments over controls, these treatments also had highest seed germination supporting index. This work suggests that the set-up synergistic remediation could be used to remediate crude oil polluted soil and this could be used in large scale.

Mediational influence of spent mushroom compost on phytoremediation of black-oil hydrocarbon polluted soil and response of Megathyrsus maximus Jacq.

Ability of a plant to develop different adaptive strategies can also determine its capability for effective soil remediation. In this study, influence of spent mushroom compost (SMC) was tested on the phytoremediation of black oil hydrocarbon polluted soil and the response of Megathyrsus maximus (guinea grass). Studies were carried out in microcosm conditions by mixing different concentration of SMC viz., 10, 20, 30 and 40% in a 5 kg of contaminated soil along with control. Seeds of M. maximus was sown in tray for two weeks and allowed to grow for height of 10 cm and transplanted in to the different experimental pots. Soil nutrient, heavy metal and PAH contents were analyzed before and after the experiment. Ecophysiological and anatomical responses due to the contaminants in the soil by M. Maximus were analyzed after 120 days. Phytomass efficiency, potential photosynthesis (Amax) and contents of chlorophylls (a and b) as well as the total chlorophyll along with anatomical evaluations were recorded. Plant alone (control) reduced the soil heavy metal and PAH contents but further improvements were observed in SMC treatments, similar results were also observed as regards to the plant's phytoremediation efficiency (PE), phytomass and potential photosynthetic rates (m mol O2 M-2S-1). The plant's root and shoot anatomical responses were enhanced in treatments compared to control, study infers that the treatment enhances the biostimulation and development of adaptive characteristics for M. maximus survival in contaminated soils and promotes its co-degradation of hydrocarbon. SMC supports remediation and as well enhances the anatomical evaluations, we therefore recommend the use of SMC on response of Megathyrsus maximus Jacq for remediation of petrochemical based phytoremediation.