The characteristics of insoluble softwood substrates affect fungal morphology, secretome composition, and hydrolytic efficiency of enzymes produced by Trichoderma reesei

Background On-site enzyme production using Trichoderma reesei can improve yields and lower the overall cost of lignocellulose saccharification by exploiting the fungal gene regulatory mechanism that enables it to continuously adapt enzyme secretion to the substrate used for cultivation. To harness this, the interrelation between substrate characteristics and fungal response must be understood. However, fungal morphology or gene expression studies often lack structural and chemical substrate characterization. Here, T. reesei QM6a was cultivated on three softwood substrates: northern bleached softwood Kraft pulp (NBSK) and lodgepole pine pretreated either by dilute-acid-catalyzed steam pretreatment (LP-STEX) or mild alkaline oxidation (LP-ALKOX). With different pretreatments of similar starting materials, we presented the fungus with systematically modified substrates. This allowed the elucidation of substrate-induced changes in the fungal response and the testing of the secreted enzymes’ hydrolytic strength towards the same substrates. Results Enzyme activity time courses correlated with hemicellulose content and cellulose accessibility. Specifically, increased amounts of side-chain-cleaving hemicellulolytic enzymes in the protein produced on the complex substrates (LP-STEX; LP-ALKOX) was observed by secretome analysis. Confocal laser scanning micrographs showed that fungal micromorphology responded to changes in cellulose accessibility and initial culture viscosity. The latter was caused by surface charge and fiber dimensions, and likely restricted mass transfer, resulting in morphologies of fungi in stress. Supplementing a basic cellulolytic enzyme mixture with concentrated T. reesei supernatant improved saccharification efficiencies of the three substrates, where cellulose, xylan, and mannan conversion was increased by up to 27, 45, and 2800%, respectively. The improvement was most pronounced for proteins produced on LP-STEX and LP-ALKOX on those same substrates, and in the best case, efficiencies reached those of a state-of-the-art commercial enzyme preparation. Conclusion Cultivation of T. reesei on LP-STEX and LP-ALKOX produced a protein mixture that increased the hydrolytic strength of a basic cellulase mixture to state-of-the-art performance on softwood substrates. This suggests that the fungal adaptation mechanism can be exploited to achieve enhanced performance in enzymatic hydrolysis without a priori knowledge of specific substrate requirements.

Sequence analysis of 18SrDNA gene from sagoplast degrading fungi

The bioplastic can be made from sago flour and known as sagoplast. It was widely known that for making bioplastic, the addition of acetic acid and glycerol are needed. Products that are air-dried are easy to grow fungi within a few weeks. This makes the basis for researchers to undestand more about the character and identity of the sagoplast degrading fungi. Characterization and identification were carried out by observed morphology and analyzing the 18SrDNA gene sequence of fungal isolates that had grown on the sagoplast. Fungal isolates morphology showed yellowish-orange color with white thread-like mycelia and a blackish brown mace with white thread-shaped mycelia. These characters of fungal morphology that similar with Aspergillus. The gene sequences of the fungal isolates were aligned with reference gene sequences of the fungi obtained from the Gen Bank of the National Center for Biotechnology Information (NCBI). Sequence data analysis was performed by using the Clustal X program to determine the kinship and taxonomy of the fungal isolates that able to degrade sagoplast. The result showed that two fungal isolates, DFSP.J1 and DFSP.J4, were found and demonstrated their ability for degrading sagoplast. Isolate DFSP.J1 is related to Aspergillus flavus strain PSU2 LC127086.1, while isolate DFSP.J4 is related to Aspergillus niger IFO4033 D63697.1.

Sequence analysis of 18SrDNA gene from sagoplast degrading fungi

The bioplastic can be made from sago flour and known as sagoplast. It was widely known that for making bioplastic, the addition of acetic acid and glycerol are needed. Products that are air-dried are easy to grow fungi within a few weeks. This makes the basis for researchers to undestand more about the character and identity of the sagoplast degrading fungi. Characterization and identification were carried out by observed morphology and analyzing the 18SrDNA gene sequence of fungal isolates that had grown on the sagoplast. Fungal isolates morphology showed yellowish-orange color with white thread-like mycelia and a blackish brown mace with white thread-shaped mycelia. These characters of fungal morphology that similar with Aspergillus. The gene sequences of the fungal isolates were aligned with reference gene sequences of the fungi obtained from the Gen Bank of the National Center for Biotechnology Information (NCBI). Sequence data analysis was performed by using the Clustal X program to determine the kinship and taxonomy of the fungal isolates that able to degrade sagoplast. The result showed that two fungal isolates, DFSP.J1 and DFSP.J4, were found and demonstrated their ability for degrading sagoplast. Isolate DFSP.J1 is related to Aspergillus flavus strain PSU2 LC127086.1, while isolate DFSP.J4 is related to Aspergillus niger IFO4033 D63697.1.

Orchestration an extracellular lipase production from Aspergillus niger MYA 135: biomass morphology and fungal physiology

The impact of biomass morphology and culture conditions on fungal fermentation was widely reviewed in the literature. In this work, we presented three independent experiments in order to evaluate the influence of some of those input factors on a lipase production separately by using the Aspergillus niger MYA 135 and the two-stage fermentation technique. Regarding the culture modality, the biomass was pre-grown in a first reactor. Then, the washed mycelium was transferred to a second reactor to continue the study. Firstly, linear effects of fungal morphology and several physiological parameters on a lipase production were explored using the Plackett–Burman design. The dispersed fungal morphology was confirmed as a proper quality characteristic for producing an extracellular lipase activity. Concerning the impact of the carbon source on the biomass pre-growth, the sucrose (E = 9.923, p < 0.001) and the l-arabinose (E = 4.198, p = 0.009) presented positive and significant effects on the enzyme production. On the contrary, the supplementation of 0.05 g/L CaCl2 displayed a highly negative and significant effect on this process (E = − 7.390, p < 0.001). Secondly, the relationship between the enzyme production and the input variables N:C ratio, FeCl3 and olive oil was explored applying the central composite design. Among the model terms, the N:C ratio of the production medium had the most negative and significant influence on the enzyme synthesis. Thus, it was concluded that a low N:C ratio was preferable to increase its production. In addition, the bifunctional role of FeCl3 on this fungus was presented. Thirdly, a prove of concept assay was also discussed.

The Characteristics of Insoluble Softwood Substrates Affect Fungal Morphology, Secretome Composition, and Hydrolytic Efficiency of Enzymes Produced by Trichoderma Reesei

BackgroundOn-site enzyme production using Trichoderma reesei can improve yields and lower the overall cost of lignocellulose saccharification by exploiting the fungal gene regulatory mechanism that enables it to continuously adapt enzyme secretion to the substrate used for cultivation. To harness this, the interrelation between substrate characteristics and fungal response must be understood. However, fungal morphology or gene expression studies often lack structural and chemical substrate characterization. Here, T. reesei QM6a was cultivated on three softwood substrates: northern bleached softwood Kraft pulp (NBSK) and lodgepole pine pretreated by dilute-acid-catalyzed steam pretreatment (LP-STEX) and mild alkaline oxidation (LP-ALKOX). With different pretreatments of similar starting materials, we presented the fungus with systematically modified substrates. This allowed the elucidation of substrate-induced changes in the fungal response and the testing of the secreted enzymes’ hydrolytic strength towards the same substrates.ResultsEnzyme activity time courses correlated with hemicellulose content and cellulose accessibility. Specifically, increased amounts of side chain-cleaving hemicellulolytic enzymes in the protein produced on the complex substrates (LP-STEX; LP-ALKOX) was observed by secretome analysis. Confocal laser scanning micrographs showed that fungal micromorphology responded to changes in cellulose accessibility and initial culture viscosity. The latter was caused by surface charge and fiber dimensions, and likely restricted mass transfer, resulting in morphologies of fungi in stress. Supplementing a basic cellulolytic enzyme mixture with concentrated T. reesei supernatant improved saccharification efficiencies of the three substrates, where cellulose, xylan, and mannan conversion was increased by up to 27%, 45%, and 2800%, respectively. The improvement was most pronounced for proteins produced on LP-STEX and LP-ALKOX on those same substrates, and in the best case, efficiencies reached those of a state-of-the-art enzyme preparation.ConclusionCultivation of T. reesei on LP-STEX and LP-ALKOX produced a protein mixture that increased the hydrolytic strength of a basic cellulase mixture to state-of-the-art performance on softwood substrates. This suggests that the fungal adaptation mechanism can be exploited to achieve enhanced performance in enzymatic hydrolysis without a priori knowledge of specific substrate requirements.

First Report and New Hosts of Pseudofabraea citricarpa Causing Citrus Target Spot in China

Citrus target spot, caused by Pseudofabraea citricarpa (Zhu et al.) Chen, Verkley & Crous, was a recently reported disease on satsuma mandarin and kumquat in Chenggu (Shaanxi province, China). In January 2019, target spot-like disease was also observed on ‘Eureka’ lemon and ‘Beijing’ lemon in Wanzhou (Chongqing province), satsuma in Yichang (Hubei province), and ‘Ponkan’ in Jishou (Hunan province). The identity of the causative agent was conducted and confirmed as P. citricarpa based on symptoms, fungal morphology, and multigene phylogenetic analysis, as well as pathogenicity tests. Investigations revealed that P. citricarpa can also infect ‘Tarocco’ blood orange and ‘Newhall’ navel orange. It can not only infect leaves and shoots but also can infect fruits. These results suggest that P. citricarpa could potentially spread to other citrus-growing regions in China.

Misidentification of Scedosporium boydii Infection as Aspergillosis in a Patient with Chronic Renal Failure

Aspergillosis is a commonly diagnosed fungal infection. Histopathologic examination alone can have diagnostic pitfalls due to the overlapping of fungal morphology. We report a case of Scedosporium boydii infection initially misdiagnosed as aspergillosis. The patient presented to the hospital with shortness of breath and chest and abdominal pain. Laboratory tests revealed leukocytosis and elevated serum liver enzymes, myoglobin and lipase. He died of hypotensive shock and brain abscesses despite antibiotic treatment. Autopsy revealed invasive fungal infection in the heart, thyroid, and brain with presence of 45-degree angled, branching hyphae. The initial diagnosis of aspergillosis was made; however, further molecular studies identified the organism as S. boydii. This report reveals the potential pitfalls of morphologic diagnosis alone; and the necessity of other testing modalities to render an accurate diagnosis which is crucial for appropriate.