Culturable Bacterial Isolates from Arctic Soil shows High Biotechnological Potential

Polar microbiology remains as the most fascinating area of research which mainly focuses on exploration of psychrophilic organisms for having their cold-active enzymes of biotechnological potential. In this study, we have explored a culturable bacterial community and isolated 27 bacterial isolates with a different morphology from an unexplored site of Arctic region, for the possibility of identifying various active biomolecules. Screening of various isolates in a culture dependent manner helped us to identify strains capable of producing extracellular enzymes. The optimal growth parameters of most of the isolates are ranges between 18-22°C temperature, 3-5 days of incubation, 6-9 pH, and 3-5% (w/v) NaCl in LB media. It has also been found that among these isolates, 63% are able to produce lipase, 17% amylase, 7% xylanase and 7% isolates have responded for phosphatase activity but there are no isolates found for gelatinase and cellulase production ability. In addition, few isolates can also produce secretory protease, urease, β-galactosidase, etc. 16SrRNA gene sequence-based phylogeny revealed that the isolates belong to the genera of Psychrobacter, Planococcus, Halomonas, Arthrobacter, Oceanisphaera, Marinbacter, Pseudomonas, Algoriphagus. Strikingly, none of the Arctic isolates showed resistance towards commonly used antibiotics which indicates that the unexplored habitat is devoid of antibiotic exposure and so does the rise of antimicrobial resistance. The structure-function relationship of the isolated bioactive compounds from these isolates are the major focus of future research.

Intron Retention Coupled with Nonsense-Mediated Decay is Involved in Cellulase Biosynthesis in Cellulolytic Fungi

Background Knowledge on regulatory networks associated with cellulase biosynthesis is prerequisite for exploitation of such regulatory systems in ehancing cellulase production with low cost. The biological functions of intron retention (IR) and nonsense-mediated mRNA decay (NMD) in filamentous fungi is lack of study, let alone their roles in cellulase biosynthesis. Result We found that major cellulase genes (cel7a, cel7b, and cel3a) exhibited concomitant decrease in IR rates and increase in their gene expression in T. reesei under cellulase-producing condition (cellulose and lactose) that was accompanied with a more active NMD pathway, as compared to non cellulase-producing condition (glucose). In the presence of the NMD pathway inhibitor that successfully repressed the NMD pathway, the mRNA levels of cellulase genes were sharply down-regulated, but the rates of IR in these genes were significantly up-regulated. Consistently, the cellulase activities were severely inhibited. In addition, the NMD pathway inhibitor caused the downregulated mRNA levels of two important genes of the target of rapamycin (TOR) pathway, trfkbp12 and trTOR1. The absence of gene trfkbp12 made the cellulase production in T. reesei more sensitive to the NMD pathway inhibitor. Conclusion All these findings suggest that the IR of cellulase genes regulates their own gene expression by coupling with the NMD pathway, which might involve the TOR pathway. Our results provide better understanding on intron retention, the NMD pathway, and cellulase production mechanism in filamentous fungi.

Inducer-Free Cellulase Production System Based on the Constitutive Expression of Mutated Xyr1 and Ace3 in the Industrial Fungus Trichoderma Reesei

Background: Trichoderma reesei (Hypocrea jecorina) is a filamentous fungus that can produce extremely high levels of protein; consequently, it is utilized as a host for the production of cellulase and hemicellulase cocktails for lignocellulosic biomass degradation. Several hyper-producer strains of T. reesei have been bred for use in industrial production, but they generally require inducers to achieve high production capacities. The most commonly used inducers are soluble sugars produced by the degradation of cellulose; however, the dependence on cellulose degradation is problematic because cellulose is insoluble and has poor handling properties as a carbon source. Furthermore, once cellulose is decomposed, little cellulase is produced, making it difficult to produce the enzyme continuously and efficiently. The aim of this study was to establish a simple, inducer-free, cellulase production system using glucose as the sole carbon source.Results: Here, we focused on transcription factors that regulate both cellulase and hemicellulase genes. First, we verified that the previously reported Xylanase regulator 1 (Xyr1) mutation had a glucose-blind phenotype in T. reesei, and confirmed that constitutive expression of the V821F mutation in Xyr1 produced high levels of proteins, especially hemicellulase and cellulase, even in inducer-free conditions. However, the majority of proteins were hemicellulases. To reproduce cellulase/hemicellulase production similar to those observed under induced conditions, an activator of cellulase expression 3 (Ace3) was expressed in Xyr1V821F expressed strain additionally. As a result, the T. reesei strain constitutively expressing Xyr1V821F and Ace3 exhibited a 1.5-fold increase than Xyr1V821F expressed only in protein productivity under inducer-free conditions. Notably, the enzyme composition significantly improved for cellulases ratio and similar to that induced by cellulose. Furthermore, the enzymes exhibited a high saccharification efficiency when compared to that of produced by the strain expressing only the mutated Xyr1.Conclusions: This work shows that the constitutive expression of mutated Xyr1 and Ace3 can increase cellulase and hemicellulase production in T. reesei without inducers. This inducer-free enzyme production method could provide an effective system to reduce costs and simplify production processes, and is expected to be applied in the production of various proteins.

Cellulase Production by Penicillium Oxalicum Ti-11 with Traditional Chinese Medicine Residue as Substrate

PurposeThe study aims to search for efficient cellulase producer and explore the possibility of traditional Chinese medicine residue as a substrate for cellulase production, so as to realize the waste utilization of traditional Chinese medicine residue.MethodsThe cellulase-producing strain was identified through morphological and molecular biological methods. Scanning electron microscope (SEM) and Fourier transform infrared spectroscopy (FTIR) were used to characterize the structure of traditional Chinese medicine residues before and after fermentation. The enzyme activity was determined by DNS method, and the enzyme production conditions were optimized by single factor and response surface methodology.ResultThe strain grew well in forsythia leaf residue, and the highest FPA could reach 2.06 IU/mL. In addition, the structural characteristics of traditional Chinese medicine residue that before and after enzymatic hydrolysis were characterized by SEM and FTIR. The results showed that the structure of the residue was destroyed after enzymatic hydrolysis, the damage of forsythia leaf residue was the most serious, and enzymatic hydrolysis promoted the dissolution of cellulose, lignin and hemicellulose. The enzyme production conditions of the strain were optimized by Plackett-Burman design and response surface analysis. The FPA could reach 2.79 IU/mL under the optimal conditions of FLR concentration 24.84 g/L, (NH4)2SO4 concentration 2 g/L, temperature 34.44℃, pH 6.20, rotational speed 200rpm, inoculum 6%, which was 35.44% higher than that before optimization.ConclusionsThe results showed that traditional Chinese medicine residue could be used as the induced substrate for fungal cellulase production. This study provides an idea for the low-cost production of fungal cellulase and the waste utilization of traditional Chinese medicine residue.

Evaluation of Lactobacillus spp. Based on Phenotypical Profile as Direct-Fed Microbial Candidate for Poultry Nutrition

The present study was conducted to isolate, identify and characterize a lactic acid bacteria strain from turkey ileum content (46-day-old). The new strain was phenotypical confirmed as Lactobacillus acidophilus (L. acidophilus) and conserved under the code IBNA 09. Bacterial profile of L. acidophilus was compared with other strains known as L. paracasei CCM 1837 and L. plantarum ATCC 8014, based on cultural, morphological, biochemical and enzymatic activity (amylase and cellulase). The strains appear as Gram positive bacilli, thin, non-spore-forming, isolated, diplo form, in short chains or in small irregular piles on Man Rogosa and Sharp (MRS) broth and agar medium. The identification and biochemical traits were performed by catalase assay, API 50 CHL V 5.1 soft (L. acidophilus biotype 2, 99.9% ID; good identification to the genus L. paracasei spp. paracasei 1 or 3, 48-51% ID; L. plantarum 1, 99.9% ID) and ABIS online (L. acidophilus ~ 88%; L. paracasei spp. paracasei, ~ 90%; L. plantarum, ~91%). The highest total score of extracellular amylase activity was recorded by L. acidophilus IBNA 09 at 24-48 h (5.10 ± 0.176 U/mL, 4.99 ± 0.409 U/mL), follow by L. paracasei CCM 1837(0.12 ± 0.002 U/mL, 0.15 ± 0.001 U/mL). During entire period, cellulase production was observed only for L. acidophilus (0.28 ± 0.019 U/mL), comparative with L. paracasei where the activity was observed in the first 24 h, respectively at 72 h for L. plantarum. These results suggest that L. acidophilus IBNA 09 possesses potential probiotic traits as a suitable candidate for amylase and cellulase production, and starter culture can improve cereal fermentation and the process of digestion in poultry nutrition.