Penicillium Oxalicum Strain Research Articles

Developing endophytic Penicillium oxalicum as a source of lignocellulolytic enzymes for enhanced hydrolysis of biorefinery relevant pretreated rice straw.

Endophytic fungi, as plant symbionts, produce an elaborate array of enzymes for efficient disintegration of lignocellulosic biomass into constituent monomeric sugars, making them novel source of lignocellulolytic CAZymes with immense potential in future biorefineries. The present study reports lignocellulolytic enzymes production potential of an endophytic halotolerant Penicillium oxalicum strain isolated from Citrus limon, under submerged and solid-state fermentation (SmF & SSF, respectively), in the presence and absence of salt (1M NaCl). The comparative QTOF-LC/MS-based exoproteome analysis of the culture extracts unveiled differential expression of CAZymes, with the higher abundance of GH6 and GH7 family cellobiohydrolase in the presence of 1M salt. The strain improvement program, employing cyclic mutagenesis and diploidization, was utilized to develop hyper-cellulase producing mutant strains of P. oxalicum. The enzyme production of the developed strain (POx-M35) was further enhanced through statistical optimization of the culture conditions utilizing glucose mix disaccharides (GMDs) as an inducer. This optimization process resulted in the lignocellulolytic cocktail that contained high titers (U/mL) of endoglucanase (EG) (146.16), cellobiohydrolase (CBHI) (6.99), β-glucosidase (β-G) (26.21), xylanase (336.05) and FPase (2.02 U/mL), which were 5.47-, 5.54-, 8.55-, 4.96-, and 4.39-fold higher when compared to the enzyme titers obtained in wild HP1, respectively. Furthermore, the lignocellulolytic cocktails designed by blending secretome produced by mutant POx-M35 with xylanases (GH10 and GH11) derived from Malbranchea cinnamomea resulted in efficient hydrolysis of unwashed acid pretreated (UWAP) rice straw slurry and mild alkali deacetylated (MAD) rice straw. This study underscores the potential of bioprospecting novel fungus and developing an improved strain for optimized production and constitution of lignocellulolytic cocktails that can be an important determinant in advancing biomass conversion technologies.

Mechanisms of combined bioremediation by phosphate-solubilizing fungus and plants and its effects on cadmium contamination in phosphate-mining wastelands

Owing to uncontrolled mining activities and lack of ecological protection measures, phosphate-mining wastelands are contaminated with the heavy metal Cd. In this study, Penicillium oxalicum strain ZP6, a Cd-resistant phosphate-solubilizing fungus, was used in combination with the fast-growing, high-biomass plant Brassica juncea L. to enhance Cd remediation in phosphate-mining wastelands. Further, the bioremediation mechanisms were explored and elucidated. In pot experiments, strain ZP6 and Brassica juncea L. alone were significantly effective in removing Cd from phosphate-mining wastelands; however, their combination was more effective, exhibiting a high removal rate of 88.75%. The presence of phosphorite powder increases soil-enzyme activity, promotes plant growth, and reduces the bioaccumulation and translocation factors. However, Cd-inhibited plant growth and chlorophyll content increased malondialdehyde accumulation, which was alleviated by inoculation with strain ZP6. The results from the study indicate that bioremediation using a combination of strain ZP6 and plants is a restoration strategy with appreciable potential to resolve Cd contamination in phosphate-mining wastelands.

Metabolite Chemical Composition of the Bletilla striata (Thunb.) Reichb. f. Endophyte Penicillium oxalicum

Penicillium oxalicum strain can be isolated from the Bletilla striata (Thunb.) Reichb. f. tubers. Its solid-state fermentation products are concentrated by percolation extraction. Separation and purification have been conducted to the ethyl acetate extracts by preparative HPLC. Based on the use of spectrometry, we have determined 17 known compounds, 12,13-dihydroxy-fumitremorgin C (1), pseurotin A (2), tyrosol (3), cyclo-(L-Pro-L-Val) (4), cis-4-hydroxy-8-O-methylmellein (5), uracil (6), cyclo-(L-Pro-L-Ala) (7), 1,2,3,4-tetrahydro-4-hydroxy-4-quinolin carboxylic acid (8), cyclo-(Gly-L-Pro) (9), 2'-deoxyuridine (10), 1-(β-D-ribofuranosyl)thymine (11), cyclo-(L-Val-Gly) (12), 2'-deoxythymidine (13), cyclo-(Gly-D-Phe) (14), cyclo-L-(4-hydroxyprolinyl)-D-leucine (15), cyclo-(L)-4-hydroxy-Pro-(L)-Phe (16), uridine (17). Here, we report compounds 1–3, 5, 7–8, 11–12, 14–17 are first found and isolated from this endophyte.

Environmentally-friendly biorecovery of manganese from electrolytic manganese residue using a novel Penicillium oxalicum strain Z6-5-1: Kinetics and mechanism

Bioleaching is a promising route for electrolytic manganese (Mn) residue (EMR) reutilization due to being eco-friendly and cost-effective. However, microbes with high bioleaching efficiency are scarce. This work aimed to isolate, screen, and characterize a novel fungal strain with high Mn-bioleaching efficiency from EMR, and study the kinetics and mechanism. The novel Penicillium oxalicum strain Z6-5-1 was found to selectively bioleach Mn from EMR. A maximum Mn2+ recovery of 93.3 % was achieved after 7 days and was mainly dependent upon acidolysis of the bio-organic acids, specifically gluconic acid and oxalic acid, as well as mycelial biosorption. This efficiency was the highest reported in the literature for a fungus over such a short time. EMR strongly induced P. oxalicum to produce gluconic acid and oxalic acid. The novel transcription factor PoxCxrE of P. oxalicum controlled the production of bio-organic acids by regulating the expression of rate-limiting enzyme genes involved in the biosynthesis of bio-organic acids. Scanning electron microscopy, laser particle size analysis, X-ray diffraction, X-ray photoelectron spectroscopy, and Fourier transform infrared spectroscopy were employed to analyze EMR changes after bioleaching. This study provides an alternative fungal resource for Mn-bioleaching of EMR, and a novel target for metabiotic engineering to improve bio-organic acid production.

Construction of a novel filamentous fungal protein expression system based on redesigning of regulatory elements.

Filamentous fungi are extensively used as an important expression host for the production of a variety of essential industrial proteins. They have significant promise as an expression system for protein synthesis due to their inherent superior secretory capabilities. The purpose of this study was to develop a novel expression system by utilizing a Penicillium oxalicum strain that possesses a high capacity for protein secretion. The expression of glycoside hydrolases in P. oxalicum was evaluated in a cleaner extracellular background where the formation of two major amylases was inhibited. Four glycoside hydrolases (CBHI, Amy15B, BGL1, and Cel12A) were expressed under the highly constitutive promoter PubiD. It was found that the proteins exhibited high purity in the culture supernatant after cultivation with starch. Two inducible promoters, Pamy15A and PempA, under the activation of the transcription factor AmyR were used as elements in the construction of versatile vectors. When using the cellobiohydrolase CBHI as the extracellular quantitative reporter, the empA promoter screened from the AmyR-overexpressing strain was shown to be superior to the amy15A promoter based on RNA-sequencing data. Therefore, we designed an expression system consisting of a cleaner background host strain and an adjustable promoter. This system enables rapid and high-throughput evaluation of glycoside hydrolases from filamentous fungi.Key points• A new protein expression system derived from Penicillium oxalicum has been developed.• The expression platform is capable of secreting recombinant proteins with high purity.• The adjustable promoter may allow for further optimization of recombinant protein synthesis.

Biologically formed silver nanoparticles and in vitro study of their antimicrobial activities on resistant pathogens.

Background and Objectives:Silver nanoparticles (AgNPs) have been found to have multiple uses as antibacterial, antifungal and anti-biofilm agents because of their biological activities and safety. The present study was aimed to analyze the antimicrobial and anti-biofilm activities as well as the cytotoxic effect of AgNPs against different human pathogens.Materials and Methods:AgNPs were synthesized using cell free supernatants of Escherichia coli (ATCC 25922), Enterococcus faecalis (ATCC 19433), Pseudomonas aeruginosa (ATCC 27856), Enterobacter cloacae (ATCC 13047) and Penicillium oxalicum strain, then were analyzed using UV/Vis Spectral Analysis, Transmission electron microscopy (TEM). Scanning Electron Microscope (SEM) and Energy Dispersive-X-ray Spectroscopy (EDX) analysis. Antimicrobial activities of biosynthesized AgNPs were assessed with selected antimicrobial agents against multidrug resistant bacteria and candida. Anti-biofilm and cytotoxicity assays of these biosynthesized AgNPs were also done.Results:The synthesis of AgNPs were confirmed through observed color change and monitoring UV-Vis spectrum which showed homogeneous (little agglomeration) distribution of silver nanoparticles. TEM and SEM have shown that the particle size ranged from 13 to 34 (nm) with spherical shape and a high signal with EDX analysis. Antibacterial and antifungal efficacy of antibiotics and fluconazole were increased in combination with biosynthesized AgNPs against resistant bacteria and candida. Significant reduction in biofilm formation was found better with Penicillium oxalicum AgNPs against biofilm forming bacteria.Conclusion:Penicillium oxalicum has the best effect towards synthesizing AgNPs, for antimicrobial activities against resistant bacteria and candida, in addition to anti-biofilm activities against biofilm forming Staphylococcus aureus and E. coli and the safest cytotoxicity effect on (MRC-5) cell line.

Functional Attributes of Myco-Synthesized Silver Nanoparticles from Endophytic Fungi: A New Implication in Biomedical Applications.

Simple SummaryGreener fabrication of silver nanoparticles has received extensive attention owing to their low cytotoxicity, tunable surface features, and stability. Fabrication of AgNPs using natural entities has received substantial consideration as an alternative to toxic chemicals. In this study, we have screened the potential of an endophytic fungi Penicillium oxalicum strain LA-1 for the synthesis of AgNPs and analyzed their potential multipurpose values in biology and medicine.To develop a benign nanomaterial from biogenic sources, we have attempted to formulate and fabricate silver nanoparticles synthesized from the culture filtrate of an endophytic fungus Penicillium oxalicum strain LA-1 (PoAgNPs). The synthesized PoAgNPs were exclusively characterized through UV–vis absorption spectroscopy, Fourier Transform Infra-Red spectroscopy (FT-IR), X-ray powder diffraction (XRD), and Transmission Electron Microscopy (TEM) with energy dispersive X-ray spectroscopy (EDX). The synthesized nanoparticles showed strong absorbance around 430 nm with surface plasmon resonance (SPR) and exhibited a face-centered cubic crystalline nature in XRD analysis. Proteins presented in the culture filtrate acted as reducing, capping, and stabilization agents to form PoAgNPs. TEM analysis revealed the generation of polydispersed spherical PoAgNPs with an average size of 52.26 nm. The PoAgNPs showed excellent antibacterial activity against bacterial pathogens. The PoAgNPs induced a dose-dependent cytotoxic activity against human adenocarcinoma breast cancer cell lines (MDA-MB-231), and apoptotic morphological changes were observed by dual staining. Additionally, PoAgNPs demonstrated better larvicidal activity against the larvae of Culex quinquefasciatus. Moreover, the hemolytic test indicated that the as-synthesized PoAgNPs are a safe and biocompatible nanomaterial with versatile bio-applications.

Complete Genome Sequence of Penicillium oxalicum Strain SGAir0226 Isolated from Outdoor Tropical Air in Singapore.

Penicillium oxalicum strain SGAir0226 was isolated from a tropical air sample collected in Singapore. The complete genome was assembled from long reads obtained from single-molecule real-time sequencing and was further polished and error corrected using short read sequencing data. The assembly comprises 20 contigs with a total length of 30.7Mb. The genome was predicted to contain 8310 protein-coding genes, 237 tRNAs and 83 rRNAs.

Transcription Factor Atf1 Regulates Expression of Cellulase and Xylanase Genes during Solid-State Fermentation of Ascomycetes.

Transcriptional regulation of cellulolytic and xylolytic genes in ascomycete fungi is controlled by specific carbon sources in different external environments. Here, comparative transcriptomic analyses of Penicillium oxalicum grown on wheat bran (WB), WB plus rice straw (WR), or WB plus Avicel (WA) as the sole carbon source under solid-state fermentation (SSF) revealed that most of the differentially expressed genes (DEGs) were involved in metabolism, specifically, carbohydrate metabolism. Of the DEGs, the basic core carbohydrate-active enzyme-encoding genes which responded to the plant biomass resources were identified in P. oxalicum, and their transcriptional levels changed to various extents depending on the different carbon sources. Moreover, this study found that three deletion mutants of genes encoding putative transcription factors showed significant alterations in filter paper cellulase production compared with that of a parental P. oxalicum strain with a deletion of Ku70 (ΔPoxKu70 strain) when grown on WR under SSF. Importantly, the ΔPoxAtf1 mutant (with a deletion of P. oxalicumAtf1, also called POX03016) displayed 46.1 to 183.2% more cellulase and xylanase production than a ΔPoxKu70 mutant after 2 days of growth on WR. RNA sequencing and quantitative reverse transcription-PCR revealed that PoxAtf1 dynamically regulated the expression of major cellulase and xylanase genes under SSF. PoxAtf1 bound to the promoter regions of the key cellulase and xylanase genes in vitro This study provides novel insights into the regulatory mechanism of fungal cellulase and xylanase gene expression under SSF.IMPORTANCE The transition to a more environmentally friendly economy encourages studies involving the high-value-added utilization of lignocellulosic biomass. Solid-state fermentation (SSF), that simulates the natural habitat of soil microorganisms, is used for a variety of applications such as biomass biorefinery. Prior to the current study, our understanding of genome-wide gene expression and of the regulation of gene expression of lignocellulose-degrading enzymes in ascomycete fungi during SSF was limited. Here, we employed RNA sequencing and genetic analyses to investigate transcriptomes of Penicillium oxalicum strain EU2101 cultured on medium containing different carbon sources and to identify and characterize transcription factors for regulating the expression of cellulase and xylanase genes during SSF. The results generated will provide novel insights into genetic engineering of filamentous fungi to further increase enzyme production.

Differential transcriptomic profiling of filamentous fungus during solid-state and submerged fermentation and identification of an essential regulatory gene PoxMBF1 that directly regulated cellulase and xylanase gene expression

BackgroundSolid-state fermentation (SSF) mimics the natural decay environment of soil fungi and can be employed to investigate the production of plant biomass-degrading enzymes. However, knowledge on the transcriptional regulation of fungal genes during SSF remains limited. Herein, transcriptional profiling was performed on the filamentous fungus Penicillium oxalicum strain HP7-1 cultivated in medium containing wheat bran plus rice straw (WR) under SSF (WR_SSF) and submerged fermentation (WR_SmF; control) conditions. Novel key transcription factors (TFs) regulating fungal cellulase and xylanase gene expression during SSF were identified via comparative transcriptomic and genetic analyses.ResultsExpression of major cellulase genes was higher under WR_SSF condition than that under WR_SmF, but the expression of genes involved in the citric acid cycle was repressed under WR_SSF condition. Fifty-six candidate regulatory genes for cellulase production were screened out from transcriptomic profiling of P. oxalicum HP7-1 for knockout experiments in the parental strain ∆PoxKu70, resulting in 43 deletion mutants including 18 constructed in the previous studies. Enzyme activity assays revealed 14 novel regulatory genes involved in cellulase production in P. oxalicum during SSF. Remarkably, deletion of the essential regulatory gene PoxMBF1, encoding Multiprotein Bridging Factor 1, resulted in doubled cellulase and xylanase production at 2 days after induction during both SSF and SmF. PoxMBF1 dynamically and differentially regulated transcription of a subset of cellulase and xylanase genes during SSF and SmF, and conferred stress resistance. Importantly, PoxMBF1 bound specifically to the putative promoters of major cellulase and xylanase genes in vitro.ConclusionsWe revealed differential transcriptional regulation of P. oxalicum during SSF and SmF, and identified PoxMBF1, a novel TF that directly regulates cellulase and xylanase gene expression during SSF and SmF. These findings expand our understanding of regulatory mechanisms of cellulase and xylanase gene expression during fungal fermentation.

Compatibility of Inherent Fungal Endophytes of Withania somnifera with Trichoderma viride and its Impact on Plant Growth and Withanolide Content

Withania somnifera (Ashwagandha), also known as Indian ginseng, is an important ancient medicinal plant, used in the Indian traditional systems of medicine. In view of increasing demand for roots of Ashwagandha, the present study was undertaken to investigate the compatibility of inherent fungal endophytes along with the biocontrol agent, Trichoderma viride, for enhancing W. somnifera plant growth and root secondary metabolites (withaferin A). It has frequently been emphasized by the World Health Organization the use of healthy roots of Ashwagandha for therapeutic applications. To maintain quality of W. somnifera roots, an option could be eco-friendly management of root-knot diseases and co-inoculation of native endophytes along with T. viride. The in vitro antagonistic activity of T. viride (TV) against the W. somnifera pathogens, Alternaria alternata and Sclerotium rolfsii, showed 64.3% and 69.5% growth inhibition, respectively. Here, we investigated the compatibility of TV along with the native endophytic fungi Aspergillus terreus strain 2aWF (2aWF), Penicillium oxalicum strain 5aWF (5aWF), and Sarocladium kiliense strain 10aWF (10aWF) for the cultivation of W. somnifera. The co-inoculation of TV and native endophytic fungi resulted in increased shoot, root weight, and plant height to 65–150%, 35–74.5%, and 15–35%, respectively, compared to untreated plants. Withanolide A content in leaves of TV-treated plants increased significantly by 260%, whereas in co-inoculation treatments, it was enhanced up to 109–242%. However, no considerable change was noticed with withaferin A content in leaves, except the 2aWF + TV treatment significantly increased by 27%. In contrast, withanolide A content in roots was not affected by TV alone but co-inoculation with endophyte treatments significantly increased its content (19–73%). TV alone had increased chlorophyll a by 23%; however, in combination treatments, it increased up to 115–164% compared to control. Besides secondary metabolites in roots and leaves, co-inoculation of TV and native endophytes modulated the expression of the withanolide biosynthetic pathway genes HMGR, DXR, FPPS, SQS, SQE, CAS, SMT1, STE1, and CYP710A1 compared to control treatments. Apart from withanolide biosynthetic pathway genes, co-inoculation of TV also ameliorated the host-resistant-related gene NPR1 which was upregulated by ninefold in the TV treatment and 3- to 7-fold in the combination treatment. Overall, our results show that co-inoculation of TV along with inherent endophytes of W. somnifera enhanced plant growth and withanolides accumulation.

Fungal endophytes attune withanolide biosynthesis in Withania somnifera, prime to enhanced withanolide A content in leaves and roots.

Endophytes have been reported from all plant species from different parts of tissue including root, stem and leaves. Here we report, three fungal endophytes, Aspergillus terreus strain 2aWF (2aWF), Penicillium oxalicum strain 5aWF (5aWF), and Sarocladium kiliense strain 10aWF (10aWF) from Withania somnifera, which could enhance withanolides content in leaf and root. Upon treatment with the above endophytes to 4weeks old plants in field conditions, W. somnifera elicited withanolide A content (97 to 100%) in leaves without considerable changes in withaferin A content. Furthermore, withanolide A content in roots of 5aWF and 10aWF endophyte treated W. somnifera plants increased up to 52% and 65% respectively. Incidentally, expression profile of withanolide and sterol biosynthetic pathway genes HMGR, DXR, FPPS, SQS, SQE, CAS, SMT1, STE1 and CYP710A1 were significantly upregulated in2aWF, 5aWF and 10aWF fungal endophyte treated plants. Besides, modulation of withanolide biosynthetic pathway genes, fungal endophytes also induce a host resistant related gene, NPR1 resulting in 2, 4 and 16 fold expression levels in 2aWF, 10aWF and 5aWF endophyte treatments respectively, compared to control plants. Overall, our results illustrate that application of native-fungal endophytes 2aWF (96.60%), 5aWF (95%) and 10aWF (147%) enhances plant biomass in addition to withanolide content.

Hydrolysis of nicosulfuron under acidic environment caused by oxalate secretion of a novel Penicillium oxalicum strain YC-WM1

A novel Penicillium oxalicum strain YC-WM1, isolated from activated sludge, was found to be capable of completely degrading 100 mg/L of nicosulfuron within six days when incubated in GSM at 33 °C. Nicosulfuron degradation rates were affected by GSM initial pH, nicosulfuron initial concentration, glucose initial concentration, and carbon source. After inoculation, the medium pH was decreased from 7.0 to 4.5 within one day and remained at around 3.5 during the next few days, in which nicosulfuron degraded quickly. Besides, 100 mg/L of nicosulfuron were completely degraded in GSM medium at pH of 3.5 without incubation after 4 days. So, nicosulfuron degradation by YC-WM1 may be acidolysis. Based on HPLC analysis, GSM medium acidification was due to oxalate accumulation instead of lactic acid and oxalate, which was influenced by different carbon sources and had no relationship to nicosulfuron initial concentration. Furthermore, nicosulfuron broke into aminopyrimidine and pyridylsulfonamide as final products and could not be used as nitrogen source and mycelium didn’t increase in GSM medium. Metabolomics results further showed that nicosulfuron degradation was not detected in intracellular. Therefore, oxalate secretion in GSM medium by strain YC-WM1 led to nicosulfuron acidolysis.

Production and characterization of a neutral phytase of Penicillium oxalicum EUFR-3 isolated from Himalayan region

Kaur R, Saxena A, Sangwan P, Yadav AN, Kumar V, Dhaliwal HC. 2017. Production and characterization of a neutral phytase of Penicillium oxalicum EUFR-3 isolated from Himalayan region. Nusantara Bioscience 9: 68-76. Micronutrient bioavailability from cereals is poor to monogastric animals due to presence of phytate as a chelating agent in seeds and an exogenous application of phytases is required to overcome antinutritional effect of phytate. In the present investigation, 40 fungal isolates including Aspergillus, Penicillium, Trichoderma, Toliposporium, Cladosporium, Talaromyces etc. from different habitats were screened positive for phytase activity on specific media. On the basis of phytase production potential in solid state fermentation conditions, plant epiphytic fungi Penicillium oxalicum strain EUFR-3 has been observed with maximum phytase activity of 12.8 U/g. The biochemical properties i.e. activity and stability under optimum pH and temperature, and effect of modulators on phytase activity from this isolate were studied for its prospective application in human food. The P. oxalicum EUFR-3 phytase (PhyP-EUFR3) had maximum activity at pH 7.0 and temperature 40 °C. It was stable in pH range of 3-8 with more than 60 % activity throughout investigation. The thermostability of PhyP-EUFR3 was about 30 % residual activities after 10 min at 80 °C. Further purification, characterization and study of catalytic mechanism will be useful in better utilization of this phytase in human food for increased bioavailability of important metal ions.

The development of genetic and molecular markers to register and commercialize Penicillium rubens (formerly Penicillium oxalicum) strain 212 as a biocontrol agent.

Penicillium oxalicum strain 212 (PO212) is an effective biocontrol agent (BCA) against a large number of economically important fungal plant pathogens. For successful registration as a BCA in Europe, PO212 must be accurately identified. In this report, we describe the use of classical genetic and molecular markers to characterize and identify PO212 in order to understand its ecological role in the environment or host. We successfully generated pyrimidine (pyr-) auxotrophic mutants. In addition we also designed specific oligonucleotides for the pyrF gene at their untranslated regions for rapid and reliable identification and classification of strains of P. oxalicum and P. rubens, formerly P. chrysogenum. Using these DNA-based technologies, we found that PO212 is a strain of P. rubens, and is not a strain of P. oxalicum. This work presents PO212 as the unique P. rubens strain to be described as a BCA and the information contained here serves for its registration and commercialization in Europe.

Isolation and characterization of a novel Penicillium oxalicum strain Z1-3 with enhanced cellobiohydrolase production using cellulase-hydrolyzed sugarcane bagasse as carbon source

Biofuels from lignocellulosic biomass are acknowledged to be a promising alternative to fossil fuels. During biomass saccharification, nearly complete conversion is rarely achieved due to the recalcitrance of the residual biomass to enzymatic hydrolysis. Few studies have examined schemes for searching for novel fungal strains that could improve the conversion. In the current study, for the first time, we searched for efficient fungal producers of cellulases in a natural decay system, using a screening process containing cellulase-hydrolyzed sugarcane bagasse as carbon source. Three hundred and seventy-three fungal strains were isolated from 69 samples collected in Guangxi Zhuang Autonomous Region, China. Twelve of the 373 strains produced more than 0.5 filter paper units (FPU)/mL and were identified as Penicillium oxalicum strains. Among them, P. oxalicum Z1-3 yielded the highest cellulase production at 2.74FPU/ml, especially cellobiohydrolase, leading to about 0.5–2.0-fold higher synergistic enzyme activities including sugarcane bagasse residue cellulase, potassium hydroxide (KOH)-pretreated sugarcane bagasse cellulase, filter paper cellulase (FPase) and Avicelase, than the four isolates which yielded higher or similar FPase activities in comparison with the P. oxalicum reference strain HP7-1. Furthermore, a crude enzyme solution of Z1-3 was able to hydrolyze 92.5% of KOH-pretreated sugarcane bagasse to glucose within 96h. It was also shown that the cellobiohydrolase genes of Z1-3 and their promoter regions were the same as those of the reference strain HP7-1. Altogether, this scheme, which used recalcitrant biomass residues after cellulase hydrolysis, is feasible for searching for efficient cellulase producers. This work demonstrates the great potential of P. oxalicum Z1-3 in future biotechnological applications of lignocellulosic biomass collected from industry and agriculture.

Ecophysiological requirements on growth and survival of the biocontrol agent Penicillium oxalicum 212 in different sterile soils

In order to optimize the practical application of Penicillium oxalicum strain 212 (PO212) as a biological soil treatment, and to achieve optimal biocontrol efficacy, it is essential to understand how the physical environment influences on its survival and growth. The combined effects of temperature, water potential and soil type on the survival and growth of PO212 were monitored for 30 days in microcosms. Three levels of water potential (dry, intermediate, and wet), two temperatures (15 and 25°C), and three soils with different physical–chemical properties were used to examine the temporal survival and growth of two PO212 conidial formulations (F1, dried conidia without additives and F2, dried conidia with additives). Traditional serial dilution (CFU) on potato dextrose agar (PDA) and quantitative real-time PCR (qPCR) were used to track the PO212 treatments in the different soils. The survival (CFU) of the F1 was greater than that of the F2. PO212 was able to grow (qPCR) and survive (CFU) at all temperatures and water potentials in the three soils tested. The highest growth and survival rate was observed under some drought stress and in more acidic soils. Regression models were developed for the combined effect of temperature, water stress and soil organic matter content related to the survival and growth of PO212 in soil microcosms. Finally, the growth rate was positively correlated with the survival rate. The results could be successfully employed as an empirical approach in the development and prediction of risk of PO212 failure to grow and survive in different environments.

Persistence, survival, vertical dispersion, and horizontal spread of the biocontrol agent, Penicillium oxalicum strain 212, in different soil types

The potential adverse effects of a biological control agent can be assessed from the knowledge on its environmental fate and behaviour. This study focuses on environmental fate and behaviour of Penicillium oxalicum Currie and Thom strain 212 (PO212), a promising biocontrol agent, after its application to three different soil substrates under different crop conditions in terms of the persistence, survival, vertical dispersion, and horizontal spread. Two different PO212 conidial formulations, one without any additives (F1) and one with additives (F2), were applied to the seedbeds of tomato plants seven days before their transplanting into three soil types: sterilized peat in an experimental glasshouse and the sandy loam and the loamy sand soils in two commercial orchards. The size of the Penicillium spp. population in the substrate samples was quantified using the classical microbiological method of counting of number of colony-forming units on a semi-selective culture medium. The size of PO212 biomass in the samples was quantified by a specific quantitative real-time polymerase chain reaction. We found that PO212 had a very limited vertical dispersion and horizontal spread in the three soil substrates that were tested in the study. PO212 persisted in the natural soil substrates for at least one year at very low levels (0.45–61.2ng dried PO212 biomass/g dry soil or 2.71–367.2ng fresh PO212 biomass/g dry soil). Treatment influenced the horizontal spread of the PO212 biomass in the sterilized peat and sandy loam soils. We did not find significant increases in the size of indigenous Penicillium spp. population (102–103CFU/g dry soil) in the three soil substrates following application of the F1 or F2 conidial formulations. In the sandy loam soil of the orchard in the VO2009 field trial the Penicillium spp. population decreased at increasing depths. Although PO212 persists in the soil after its application, it does not proliferate. We conclude that PO212 is an effective and safe biocontrol agent to control soil-borne pathogens.

Effects of Different Carbon Sources on Pectinase Production by Penicillium oxalicum

Currently, obtaining enzymatic preparations with low production costs is the ultimate challenge for researchers worldwide. This study compares the productivity of pectinolytic enzymes using Penicillium oxalicum strain in submerged and solid state fermentation. Agro-industrial residues used as carbon sources were beet pulp, wheat bran and rapeseed cake. Enzyme production was higher in both fermentation types using wheat bran. Comparing the two types of fermentation, it was observed that enzyme activity is higher in solid state fermentation. The maximal activity was reached after seven days by solid state fermentation. Pectinase activity decreased progressively after 96 days, in solid state fermentation when was used beet pulp and rapeseed cake. Optimum pH and temperature for the crude enzyme activity was obtained by wheat bran in solid state fermentation at 5 and 60°C, respectively. The crude enzyme lost 50% of its activity after 40 minutes, when was heated at 60°C.Â

Biodegradation of poly(ε-caprolactone) (PCL) by a new Penicillium oxalicum strain DSYD05-1

In this study, fungi isolated from soil were screened for their ability to form clear zones on agar plates with emulsified poly(ε-caprolactone) (PCL). The most active strain, designated as DSYD05, was identified as Penicillium oxalicum on the basis of morphological characteristics and phylogenetic analysis. Mutant DSYD05-1, obtained by ultraviolet-light mutagenesis from strain DSYD05, was more effective in PCL degradation. In liquid cultures of the mutant strain with PCL emulsion, DSYD05-1 showed the highest PCL-degrading activity after 4days of cultivation. The products of PCL degradation were analysed by mass spectrometry; the results indicated that 6-hydroxyhexanoic acid was produced and assimilated during cultivation. The degradation of PCL film by DSYD05-1 was observed by scanning electron microscopy, and was indicative of a three-stage degradation process. The degradation of amorphous parts of the film preceded that of the crystalline center and then the peripheral crystalline regions. In addition, DSYD05-1 showed a wide range of substrate specificity, with capability to degrade PCL, poly(β-hydroxybutyrate), and poly(butylene succinate), but not poly(lactic acid), indicating that the strain could have potential for application in the treatment or recycling of bio-plastic wastes.