Secretome analysis of Trichoderma reesei and Aspergillus niger cultivated by submerged and sequential fermentation processes: Enzyme production for sugarcane bagasse hydrolysis

Cellulases and hemicellulases from Trichoderma reesei and Aspergillus niger have been shown to be powerful enzymes for biomass conversion to sugars, but the production costs are still relatively high for commercial application. The choice of an effective microbial cultivation process employed for enzyme production is important, since it may affect titers and the profile of protein secretion. We used proteomic analysis to characterize the secretome of T. reesei and A. niger cultivated in submerged and sequential fermentation processes. The information gained was key to understand differences in hydrolysis of steam exploded sugarcane bagasse for enzyme cocktails obtained from two different cultivation processes. The sequential process for cultivating A. niger gave xylanase and β-glucosidase activities 3- and 8-fold higher, respectively, than corresponding activities from the submerged process. A greater protein diversity of critical cellulolytic and hemicellulolytic enzymes were also observed through secretome analyses. These results helped to explain the 3-fold higher yield for hydrolysis of non-washed pretreated bagasse when combined T. reesei and A. niger enzyme extracts from sequential fermentation were used in place of enzymes obtained from submerged fermentation. An enzyme loading of 0.7 FPU cellulase activity/g glucan was surprisingly effective when compared to the 5–15 times more enzyme loadings commonly reported for other cellulose hydrolysis studies. Analyses showed that more than 80% consisted of proteins other than cellulases whose role is important to the hydrolysis of a lignocellulose substrate. Our work combined proteomic analyses and enzymology studies to show that sequential and submerged cultivation methods differently influence both titers and secretion profile of key enzymes required for the hydrolysis of sugarcane bagasse. The higher diversity of feruloyl esterases, xylanases and other auxiliary hemicellulolytic enzymes observed in the enzyme mixtures from the sequential fermentation could be one major reason for the more efficient enzyme hydrolysis that results when using the combined secretomes from A. niger and T. reesei.     

Synergistic effect of Aspergillus niger and Trichoderma reesei enzyme sets on the saccharification of wheat straw and sugarcane bagasse

Plant‐degrading enzymes can be produced by fungi on abundantly available low‐cost plant biomass. However, enzymes sets after growth on complex substrates need to be better understood, especially with emphasis on differences between fungal species and the influence of inhibitory compounds in plant substrates, such as monosaccharides. In this study, Aspergillus niger and Trichoderma reesei were evaluated for the production of enzyme sets after growth on two “second generation” substrates: wheat straw (WS) and sugarcane bagasse (SCB). A. niger and T. reesei produced different sets of (hemi‐)cellulolytic enzymes after growth on WS and SCB. This was reflected in an overall strong synergistic effect in releasing sugars during saccharification using A. niger and T. reesei enzyme sets. T. reesei produced less hydrolytic enzymes after growth on non‐washed SCB. The sensitivity to non‐washed plant substrates was not reduced by using CreA/Cre1 mutants of T. reesei and A. niger with a defective carbon catabolite repression. The importance of removing monosaccharides for producing enzymes was further underlined by the decrease in hydrolytic activities with increased glucose concentrations in WS media. This study showed the importance of removing monosaccharides from the enzyme production media and combining T. reesei and A. niger enzyme sets to improve plant biomass saccharification.     

Simplification of the Biomass to Ethanol Conversion Process by Using the Whole Medium of Filamentous Fungi Cultivated Under Solid-State Fermentation

A novel simplified configuration is proposed for the conversion of biomass to ethanol using whole medium enzymatic cocktails (WM) and enzymatic extracts (EE) from different filamentous fungi (Trichoderma reesei, Aspergillus niger, and Aspergillus oryzae) cultivated under solid-state fermentation (SSF) for the hydrolysis of steam-exploded sugarcane bagasse (SESB). The hydrolyzed material derived from the saccharification of SESB using the combinations A. niger WM + T. reesei EE, A. oryzae WM + A. niger EE, and A. niger EE + T. reesei WM resulted in the best biomass conversion yields (66, 65, and 64 % of the theoretical reducing sugar yields, respectively). The best ethanol production (84 % of the theoretical yield) was obtained using the material hydrolyzed by a combination of A. oryzae WM + A. niger EE. The enzymatic conversion of SESB using on-site produced enzymes from the whole SSF cultivation medium, followed by an ethanol production step, is a potential configuration for the biomass to ethanol conversion process. This novel simplified configuration would enable the use of a single reactor system, avoiding the need for additional separation steps.     

Enzymatic hydrolyzing performance of Acremonium cellulolyticus and Trichoderma reesei against three lignocellulosic materials

Background  

Bioethanol isolated from lignocellulosic biomass represents one of the most promising renewable and carbon neutral alternative liquid fuel sources. Enzymatic saccharification using cellulase has proven to be a useful method in the production of bioethanol. The filamentous fungi Acremonium cellulolyticus and Trichoderma reesei are known to be potential cellulase producers. In this study, we aimed to reveal the advantages and disadvantages of the cellulase enzymes derived from these fungi.     

Fungal Bioconversion of Lignocellulosic Residues; Opportunities & Perspectives

The development of alternative energy technology is critically important because of the rising prices of crude oil, security issues regarding the oil supply, and environmental issues such as global warming and air pollution. Bioconversion of biomass has significant advantages over other alternative energy strategies because biomass is the most abundant and also the most renewable biomaterial on our planet. Bioconversion of lignocellulosic residues is initiated primarily by microorganisms such as fungi and bacteria which are capable of degrading lignocellulolytic materials. Fungi such as Trichoderma reesei and Aspergillus niger produce large amounts of extracellular cellulolytic enzymes, whereas bacterial and a few anaerobic fungal strains mostly produce cellulolytic enzymes in a complex called cellulosome, which is associated with the cell wall. In filamentous fungi, cellulolytic enzymes including endoglucanases, cellobiohydrolases (exoglucanases) and β-glucosidases work efficiently on cellulolytic residues in a synergistic manner. In addition to cellulolytic/hemicellulolytic activities, higher fungi such as basidiomycetes (e.g. Phanerochaete chrysosporium) have unique oxidative systems which together with ligninolytic enzymes are responsible for lignocellulose degradation. This review gives an overview of different fungal lignocellulolytic enzymatic systems including extracellular and cellulosome-associated in aerobic and anaerobic fungi, respectively. In addition, oxidative lignocellulose-degradation mechanisms of higher fungi are discussed. Moreover, this paper reviews the current status of the technology for bioconversion of biomass by fungi, with focus on mutagenesis, co-culturing and heterologous gene expression attempts to improve fungal lignocellulolytic activities to create robust fungal strains.     

Adipose Mesenchymal Stem Cell Secretome Modulated in Hypoxia for Remodeling of Radiation-Induced Salivary Gland Damage

Background and Purpose

This study was conducted to determine whether a secretome from mesenchymal stem cells (MSC) modulated by hypoxic conditions to contain therapeutic factors contributes to salivary gland (SG) tissue remodeling and has the potential to improve irradiation (IR)-induced salivary hypofunction in a mouse model.

Materials and Methods

Human adipose mesenchymal stem cells (hAdMSC) were isolated, expanded, and exposed to hypoxic conditions (O₂ < 5%). The hypoxia-conditioned medium was then filtered to a high molecular weight fraction and prepared as a hAdMSC secretome. The hAdMSC secretome was subsequently infused into the tail vein of C3H mice immediately after local IR once a day for seven consecutive days. The control group received equal volume (500 μL) of vehicle (PBS) only. SG function and structural tissue remodeling by the hAdMSC secretome were investigated. Human parotid epithelial cells (HPEC) were obtained, expanded in vitro, and then irradiated and treated with either the hypoxia-conditioned medium or a normoxic control medium. Cell proliferation and IR-induced cell death were examined to determine the mechanism by which the hAdMSC secretome exerted its effects.

Results

The conditioned hAdMSC secretome contained high levels of GM-CSF, VEGF, IL-6, and IGF-1. Repeated systemic infusion with the hAdMSC secretome resulted in improved salivation capacity and increased levels of salivary proteins, including amylase and EGF, relative to the PBS group. The microscopic structural integrity of SG was maintained and salivary epithelial (AQP-5), endothelial (CD31), myoepithelial (α-SMA) and SG progenitor cells (c-Kit) were successfully protected from radiation damage and remodeled. The hAdMSC secretome strongly induced proliferation of HPEC and led to a significant decrease in cell death in vivo and in vitro. Moreover, the anti-apoptotic effects of the hAdMSC secretome were found to be promoted after hypoxia-preconditioning relative to normoxia-cultured hAdMSC secretome.

Conclusion

These results show that the hAdMSC secretome from hypoxic-conditioned medium may provide radioprotection and tissue remodeling via release of paracrine mediators.     

Study of a High-Yield Cellulase System Created by Heavy-Ion Irradiation-Induced Mutagenesis of Aspergillus niger and Mixed Fermentation with Trichoderma reesei

The aim of this study was to evaluate and validate the efficiency of ¹²C⁶⁺ irradiation of Aspergillus niger (A. niger) or mutagenesis via mixed Trichoderma viride (T. viride) culturing as well as a liquid cultivation method for cellulase production via mixed Trichoderma reesei (T. reesei) and A. niger culture fermentation. The first mutagenesis approach was employed to optimize yield from a cellulase-producing strain via heavy-ion mutagenesis and high-throughput screening, and the second was to effectively achieve enzymatic hydrolysis of cellulase from a mixed culture of mutant T. viride and A. niger. We found that ¹²C⁶⁺-ion irradiation induced changes in cellulase biosynthesis in A. niger but had no effect on the time course of the synthesis. It is notable that the exoglucanases (CBH) activities of A. niger strains H11-1 and H differed (6.71 U/mL vs. 6.01 U/mL) and were significantly higher than that of A. niger mutant H3-1. Compared with strain H, the filter paper assay (FPA), endoglucanase (EG) and β-glucosidase (BGL) activities of mutant strain H11-1 were increased by 250.26%, 30.26% and 34.91%, respectively. A mixed culture system was successfully optimized, and the best ratio of T. reesei to A. niger was 5:1 for 96 h with simultaneous inoculation. The BGL activity of the mixed culture increased after 72 h. At 96 h, the FPA and BGL activities of the mixed culture were 689.00 and 797.15 U/mL, respectively, significantly higher than those of monocultures, which were 408.70 and 646.98 U/mL for T. reesei and 447.29 and 658.89 U/mL for A. niger, respectively. The EG activity of the mixed culture was 2342.81 U/mL, a value that was significantly higher than that of monocultures at 2206.57 U/mL for T. reesei and 1727.62 U/mL for A. niger. In summary, cellulose production and hydrolysis yields were significantly enhanced by the proposed combination scheme.     

The Penicillium echinulatum Secretome on Sugar Cane Bagasse

Plant feedstocks are at the leading front of the biofuel industry based on the potential to promote economical, social and environmental development worldwide through sustainable scenarios related to energy production. Penicillium echinulatum is a promising strain for the bioethanol industry based on its capacity to produce large amounts of cellulases at low cost. The secretome profile of P. echinulatum after grown on integral sugarcane bagasse, microcrystalline cellulose and three types of pretreated sugarcane bagasse was evaluated using shotgun proteomics. The comprehensive chemical characterization of the biomass used as the source of fungal nutrition, as well as biochemical activity assays using a collection of natural polysaccharides, were also performed. Our study revealed that the enzymatic repertoire of P. echinulatum is geared mainly toward producing enzymes from the cellulose complex (endogluganases, cellobiohydrolases and β-glucosidases). Glycoside hydrolase (GH) family members, important to biomass-to-biofuels conversion strategies, were identified, including endoglucanases GH5, 7, 6, 12, 17 and 61, β-glycosidase GH3, xylanases GH10 and GH11, as well as debranching hemicellulases from GH43, GH62 and CE2 and pectinanes from GH28. Collectively, the approach conducted in this study gave new insights on the better comprehension of the composition and degradation capability of an industrial cellulolytic strain, from which a number of applied technologies, such as biofuel production, can be generated.     

Arsenal of plant cell wall degrading enzymes reflects host preference among plant pathogenic fungi

Background  

The discovery and development of novel plant cell wall degrading enzymes is a key step towards more efficient depolymerization of polysaccharides to fermentable sugars for the production of liquid transportation biofuels and other bioproducts. The industrial fungus Trichoderma reesei is known to be highly cellulolytic and is a major industrial microbial source for commercial cellulases, xylanases and other cell wall degrading enzymes. However, enzyme-prospecting research continues to identify opportunities to enhance the activity of T. reesei enzyme preparations by supplementing with enzymatic diversity from other microbes. The goal of this study was to evaluate the enzymatic potential of a broad range of plant pathogenic and non-pathogenic fungi for their ability to degrade plant biomass and isolated polysaccharides.     

Lignocellulolytic characterization and comparative secretome analysis of a Trichoderma erinaceum strain isolated from decaying sugarcane straw

The fungus Trichoderma reesei is employed in the production of most enzyme cocktails used by the lignocellulosic biofuels industry today. Despite significant improvements, the cost of the required enzyme preparations remains high, representing a major obstacle for the industrial production of these alternative fuels. In this study, a new Trichoderma erinaceum strain was isolated from decaying sugarcane straw. The enzyme cocktail secreted by the new isolate during growth in pretreated sugarcane straw-containing medium presented higher specific activities of β-glucosidase, endoxylanase, β-xylosidase and α-galactosidase than the cocktail of a wild T. reesei strain and yielded more glucose in the hydrolysis of pretreated sugarcane straw. A proteomic analysis of the two strains' secretomes identified a total of 86 proteins, of which 48 were exclusive to T. erinaceum, 35 were exclusive to T. reesei and only 3 were common to both strains. The secretome of T. erinaceum also displayed a higher number of carbohydrate-active enzymes than that of T. reesei (37 and 27 enzymes, respectively). Altogether, these results reveal the significant potential of the T. erinaceum species for the production of lignocellulases, both as a possible source of enzymes for the supplementation of industrial cocktails and as a candidate chassis for enzyme production.     

Comparative secretome analysis of <Emphasis Type="Italic">Trichoderma asperellum</Emphasis> S4F8 and <Emphasis Type="Italic">Trichoderma reesei</Emphasis> Rut C30 during solid-state fermentation on sugarcane bagasse

Background

The lignocellulosic enzymes of Trichoderma species have received particular attention with regard to biomass conversion to biofuels, but the production cost of these enzymes remains a significant hurdle for their commercial application. In this study, we quantitatively compared the lignocellulolytic enzyme profile of a newly isolated Trichoderma asperellum S4F8 strain with that of Trichoderma reesei Rut C30, cultured on sugarcane bagasse (SCB) using solid-state fermentation (SSF).

Results

Comparison of the lignocellulolytic enzyme profiles of S4F8 and Rut C30 showed that S4F8 had significantly higher hemicellulase and β-glucosidase enzyme activities. Liquid chromatography tandem mass spectrometry analysis of the two fungal secretomes enabled the detection of 815 proteins in total, with 418 and 397 proteins being specific for S4F8 and Rut C30, respectively, and 174 proteins being common to both strains. In-depth analysis of the associated biological functions and the representation of glycoside hydrolase family members within the two secretomes indicated that the S4F8 secretome contained a higher diversity of main and side chain hemicellulases and β-glucosidases, and an increased abundance of some of these proteins compared with the Rut C30 secretome.

Conclusions

In SCB SSF, T. asperellum S4F8 produced a more complex lignocellulolytic cocktail, with enhanced hemicellulose and cellobiose hydrolysis potential, compared with T. reesei Rut C30. This bodes well for the development of a more cost-effective and efficient lignocellulolytic enzyme cocktail from T. asperellum for lignocellulosic feedstock hydrolysis.

     

Production of recombinant lytic polysaccharide monooxygenases and evaluation effect of its addition into Aspergillus fumigatus var. niveus cocktail for sugarcane bagasse saccharification

Lignocellulosic biomass is a promising alternative for producing biofuels, despite its recalcitrant nature. There are microorganisms in nature capable of efficiently degrade biomass, such as the filamentous fungi. Among them, Aspergillus fumigatus var. niveus (AFUMN) has a wide variety of carbohydrate-active enzymes (CAZymes), especially hydrolases, but a low number of oxidative enzymes in its genome. To confirm the enzymatic profile of this fungus, this study analyzed the secretome of AFUMN cultured in sugarcane bagasse as the sole carbon source. As expected, the secretome showed a predominance of hydrolytic enzymes compared to oxidative activity. However, it is known that hydrolytic enzymes act in synergy with oxidative proteins to efficiently degrade cellulose polymer, such as the Lytic Polysaccharide Monooxygenases (LPMOs). Thus, three LPMOs from the fungus Thermothelomyces thermophilus (TtLPMO9D, TtLPMO9H, and TtLPMO9O) were selected, heterologous expressed in Aspergillus nidulans, purified, and used to supplement the AFUMN secretome to evaluate their effect on the saccharification of sugarcane bagasse. The saccharification assay was carried out using different concentrations of AFUMN secretome supplemented with recombinant T. thermophilus LPMOs, as well as ascorbic acid as reducing agent for oxidative enzymes. Through a statistic design created by Design-Expert software, we were able to analyze a possible cooperative effect between these components. The results indicated that, in general, the addition of TtLPMO9D and ascorbic acid did not favor the conversion process in this study, while TtLPMO9O had a highly significant cooperative effect in bagasse saccharification compared to the control using only AFUMN secretome.     

Engineered Penicillium funiculosum produces potent lignocellulolytic enzymes for saccharification of various pretreated biomasses

PfMig1⁸⁸, a catabolically derepressed engineered strain of the hyper-cellulolytic fungus Penicillium funiculosum NCIM1228, was investigated for the efficacy of its secretome for biomass saccharification. An inexpensive version of media containing microcrystalline cellulose, wheat bran and soya protein was optimized for producing a high-quality secretome from the PfMig1⁸⁸ strain in both shake flasks and in a 20-L bioreactor. The activities of four classes of core cellulolytic enzymes required for saccharification in the PfMig1⁸⁸ secretome, namely, cellobiohydrolase (Avicelase activity), endoglucanase (CMCase activity), β-glucosidase (PNPGase activity) and xylanase (xylanase activity), were found to be 2.29 U/mL, 28.24 U/mL, 150 U/mL and 76 U/mL, respectively. The saccharification potential of the PfMig1⁸⁸ secretome was evaluated on rice straw and sugarcane bagasse pretreated with nitric acid and/or ammonium hydroxide. Saccharification performed using a 15 % (w/v) biomass load and a 3% (w/w) enzyme load released >100 g/L sugar in the hydrolysate, irrespective of the type of biomass and pre-treatment, with >80 % hydrolysis. Furthermore, the presence of lignin in nitric acid-pretreated biomass only marginally affected saccharification. Overall, the results demonstrated that the PfMig1⁸⁸ secretome, having relatively broad substrate specificity, is a viable and efficient substitute for T. reesei-based secretomes for diverse biomass saccharification.     

Enhancement of Synthetic Trichoderma-Based Enzyme Mixtures for Biomass Conversion with an Alternative Family 5 Glycosyl Hydrolase from Sporotrichum thermophile

Enzymatic conversion of lignocellulosic materials to fermentable sugars is a limiting step in the production of biofuels from biomass. We show here that combining enzymes from different microbial sources is one way to identify superior enzymes. Extracts of the thermophilic fungus Sporotrichum thermophile (synonym Myceliophthora thermophila) gave synergistic release of glucose (Glc) and xylose (Xyl) from pretreated corn stover when combined with an 8-component synthetic cocktail of enzymes from Trichoderma reesei. The S. thermophile extracts were fractionated and an enhancing factor identified as endo-β1,4- glucanase (StCel5A or EG2) of subfamily 5 of Glycosyl Hydrolase family 5 (GH5_5). In multi-component optimization experiments using a standard set of enzymes and either StCel5A or the ortholog from T. reesei (TrCel5A), reactions containing StCel5A yielded more Glc and Xyl. In a five-component optimization experiment (i.e., varying four core enzymes and the source of Cel5A), the optimal proportions for TrCel5A vs. StCel5A were similar for Glc yields, but markedly different for Xyl yields. Both enzymes were active on lichenan, glucomannan, and oat β-glucan; however, StCel5A but not TrCel5A was also active on β1,4-mannan, two types of galactomannan, and β1,4-xylan. Phylogenetically, fungal enzymes in GH5_5 sorted into two clades, with StCel5A and TrCel5A belonging to different clades. Structural differences with the potential to account for the differences in performance were deduced based on the known structure of TrCel5A and a homology-based model of StCel5A, including a loop near the active site of TrCel5A and the presence of four additional Trp residues in the active cleft of StCel5A. The results indicate that superior biomass-degrading enzymes can be identified by exploring taxonomic diversity combined with assays in the context of realistic enzyme combinations and realistic substrates. Substrate range may be a key factor contributing to superior performance within GH5_5.     

Mutagenesis of Trichoderma reesei endoglucanase I: impact of expression host on activity and stability at elevated temperatures

Background

Trichoderma reesei is a key cellulase source for economically saccharifying cellulosic biomass for the production of biofuels. Lignocellulose hydrolysis at temperatures above the optimum temperature of T. reesei cellulases (~50°C) could provide many significant advantages, including reduced viscosity at high-solids loadings, lower risk of microbial contamination during saccharification, greater compatibility with high-temperature biomass pretreatment, and faster rates of hydrolysis. These potential advantages motivate efforts to engineer T. reesei cellulases that can hydrolyze lignocellulose at temperatures ranging from 60–70°C.

Results

A B-factor guided approach for improving thermostability was used to engineer variants of endoglucanase I (Cel7B) from T. reesei (TrEGI) that are able to hydrolyze cellulosic substrates more rapidly than the recombinant wild-type TrEGI at temperatures ranging from 50–70°C. When expressed in T. reesei, TrEGI variant G230A/D113S/D115T (G230A/D113S/D115T Tr_TrEGI) had a higher apparent melting temperature (3°C increase in T_m) and improved half-life at 60°C (t_1/2 = 161 hr) than the recombinant (T. reesei host) wild-type TrEGI (t_1/2 = 74 hr at 60°C, Tr_TrEGI). Furthermore, G230A/D113S/D115T Tr_TrEGI showed 2-fold improved activity compared to Tr_TrEGI at 65°C on solid cellulosic substrates, and was as efficient in hydrolyzing cellulose at 60°C as Tr_TrEGI was at 50°C. The activities and stabilities of the recombinant TrEGI enzymes followed similar trends but differed significantly in magnitude depending on the expression host (Escherichia coli cell-free, Saccharomyces cerevisiae, Neurospora crassa, or T. reesei). Compared to N.crassa-expressed TrEGI, S. cerevisiae-expressed TrEGI showed inferior activity and stability, which was attributed to the lack of cyclization of the N-terminal glutamine in Sc_TrEGI and not to differences in glycosylation. N-terminal pyroglutamate formation in TrEGI expressed in S. cerevisiae was found to be essential in elevating its activity and stability to levels similar to the T. reesei or N. crassa-expressed enzyme, highlighting the importance of this ubiquitous modification in GH7 enzymes.

Conclusion

Structure-guided evolution of T. reesei EGI was used to engineer enzymes with increased thermal stability and activity on solid cellulosic substrates. Production of TrEGI enzymes in four hosts highlighted the impact of the expression host and the role of N-terminal pyroglutamate formation on the activity and stability of TrEGI enzymes.

Electronic supplementary material

The online version of this article (doi:10.1186/s12896-015-0118-z) contains supplementary material, which is available to authorized users.     

Reduced genomic potential for secreted plant cell-wall-degrading enzymes in the ectomycorrhizal fungus Amanita bisporigera,based on the secretome of Trichoderma reesei

Based on the analysis of its genome sequence, the ectomycorrhizal (ECM) basidiomycetous fungus Laccaria bicolor was shown to be lacking many of the major classes of secreted enzymes that depolymerize plant cell wall polysaccharides. To test whether this is also a feature of other ECM fungi, we searched a survey genome database of Amanita bisporigera with the proteins found in the secretome of Trichoderma reesei (syn. Hypocrea jecorina), a biochemically well-characterized industrial fungus. Additional proteins were also used as queries to compensate for major groups of cell-wall-degrading enzymes lacking in the secretome of T. reesei and to substantiate conclusions drawn from the T. reesei collection. By MS/MS-based “shotgun” proteomics, 80 proteins were identified in culture filtrates of T. reesei strain RUTC30 grown on corn cell walls and in a commercial “cellulase” preparation, Spezyme CP. The two T. reesei enzyme preparations were qualitatively and quantitatively similar, the most striking difference being the lack of at least five major peptidases from the commercial enzyme mixture. Based on our analysis of A. bisporigera, this ECM fungus is deficient in many major classes of cell-wall-degrading enzymes, including both glycosyl hydrolases and carbohydrate esterases. By comparison, the genomes of the saprophytic basidiomycetes Coprinopsis cinerea and Galerina marginata (using a genome survey sequence approximately equivalent in depth to that of A. bisporigera) have, like T. reesei, a much more complete complement of cell-wall-degrading enzymes.     

Ocular Dominance Is Associated with the Ganglion Cell-Inner Plexiform Layer Thickness Profile in the Macula

Purpose

To investigate the characteristics of macular ganglion cell-inner plexiform layer (GCIPL) thickness profiles associated with ocular dominance.

Setting

Private practice, Seoul, Republic of Korea.

Design

Comparative case-control study.

Methods

Both eyes of 199 participants with no ophthalmic abnormalities were included. Participants were imaged by spectral-domain optical coherence tomography, and underwent dominant eye testing using a hole-in-a-card test (sighting dominance) at the same visit. Macular GCIPL, as well as circumpapillary retinal nerve fiber layer (RNFL) thickness were compared for individual patients, according to ocular dominance.

Results

Ocular dominance occurred predominantly in the right eye (right vs. left: 72.36 vs. 27.60%; P < 0.001). In the comparison of macular GCIPL thickness, the average (81.27±5.01 μm vs. 80.66±6.31 μm in dominant vs. non-dominant eyes), inferonasal (81.39±5.47μm vs. 80.33±6.82μm, and inferior sectors (77.95±6.05μm vs. 76.97±8.15μm) were significantly different between dominant and non-dominant eyes (P = 0.040, 0.005, and 0.032, respectively). Significant predictors of average GCIPL thickness were spherical equivalent (β = 1.37, P<0.001), astigmatic power (β = 1.44, P = 0.009), disc area (β = 3.90, P < 0.001), average RNFL thickness (β = 0.22, P<0.001), average cup-to-disc ratio (β = 5.74, P = 0.002), difference between the inferior and superior quadrant RNFL thicknesses (β = 0.08, P = 0.024), and ocular dominance (β = 2.10, P = 0.020). On multivariate regression analysis, ocular dominance was correlated with average GCIPL thickness after adjusting for potential confounders (β = 1.63, P = 0.048).

Conclusions

Dominant eyes accompanied significantly thicker average macular GCIPL. This information suggests that macular GCIPL thickness may provide an indicator of the relative dominance of an eye.     

A time course analysis of the extracellular proteome of Aspergillus nidulans growing on sorghum stover

Background

Fungi are important players in the turnover of plant biomass because they produce a broad range of degradative enzymes. Aspergillus nidulans, a well-studied saprophyte and close homologue to industrially important species such as A. niger and A. oryzae, was selected for this study.

Results

A. nidulans was grown on sorghum stover under solid-state culture conditions for 1, 2, 3, 5, 7 and 14?days. Based on analysis of chitin content, A. nidulans grew to be 4-5% of the total biomass in the culture after 2?days and then maintained a steady state of 4% of the total biomass for the next 12?days. A hyphal mat developed on the surface of the sorghum by day one and as seen by scanning electron microscopy the hyphae enmeshed the sorghum particles by day 5. After 14?days hyphae had penetrated the entire sorghum slurry. Analysis (1-D PAGE LC-MS/MS) of the secretome of A. nidulans, and analysis of the breakdown products from the sorghum stover showed a wide range of enzymes secreted. A total of 294 extracellular proteins were identified with hemicellulases, cellulases, polygalacturonases, chitinases, esterases and lipases predominating the secretome. Time course analysis revealed a total of 196, 166, 172 and 182 proteins on day 1, 3, 7 and 14 respectively. The fungus used 20% of the xylan and cellulose by day 7 and 30% by day 14. Cellobiose dehydrogenase, feruloyl esterases, and CAZy family 61 endoglucanases, all of which are thought to reduce the recalcitrance of biomass to hydrolysis, were found in high abundance.

Conclusions

Our results show that A. nidulans secretes a wide array of enzymes to degrade the major polysaccharides and lipids (but probably not lignin) by 1?day of growth on sorghum. The data suggests simultaneous breakdown of hemicellulose, cellulose and pectin. Despite secretion of most of the enzymes on day 1, changes in the relative abundances of enzymes over the time course indicates that the set of enzymes secreted is tailored to the specific substrates available. Our findings reveal that A. nidulans is capable of degrading the major polysaccharides in sorghum without any chemical pre-treatment.