Mycoprotein Production by Submerged Fermentation of the Edible Mushroom Pleurotus ostreatus in a Batch Stirred Tank Bioreactor Using Agro-Industrial Hydrolysate.

The demand for cheap, healthy, and sustainable alternative protein sources has turned research interest into microbial proteins. Mycoproteins prevail due to their quite balanced amino acid profile, low carbon footprint and high sustainability potential. The goal of this research was to investigate the capability of Pleurotus ostreatus to metabolize the main sugars of agro-industrial side streams, such as aspen wood chips hydrolysate, to produce high-value protein with low cost. Our results indicate that P. ostreatus LGAM 1123 could be cultivated both in a C-6 (glucose)- and C-5(xylose)-sugar-containing medium for mycoprotein production. A mixture of glucose and xylose was found to be ideal for biomass production with high protein content and rich amino acid profile. P. ostreatus LGAM 1123 cultivation in a 4 L stirred-tank bioreactor using aspen hydrolysate was achieved with 25.0 ± 3.4 g L-1 biomass production, 1.8 ± 0.4 d-1 specific growth rate and a protein yield of 54.5 ± 0.5% (g/100 g sugars). PCA analysis of the amino acids revealed a strong correlation between the amino acid composition of the protein produced and the ratios of glucose and xylose in the culture medium. The production of high-nutrient mycoprotein by submerged fermentation of the edible fungus P. ostreatus using agro-industrial hydrolysates is a promising bioprocess in the food and feed industry.

Bioprocess optimization for lactic and succinic acid production from a pulp and paper industry side stream.

The effective and cheap production of platform chemicals is a crucial step towards the transition to a bio-based economy. In this work, biotechnological methods using sustainable, cheap, and readily available raw materials bring bio-economy and industrial microbiology together: Microbial production of two platform chemicals is demonstrated [lactic (LA) and succinic acid (SA)] from a non-expensive side stream of pulp and paper industry (fibre sludge) proposing a sustainable way to valorize it towards economically important monomers for bioplastics formation. This work showed a promising new route for their microbial production which can pave the way for new market expectations within the circular economy principles. Fibre sludge was enzymatically hydrolysed for 72 h to generate a glucose rich hydrolysate (100 g·L-1 glucose content) to serve as fermentation medium for Bacillus coagulans A 541, A162 strains and Actinobacillus succinogenis B1, as well as Basfia succiniciproducens B2. All microorganisms were investigated in batch fermentations, showing the ability to produce either lactic or succinic acid, respectively. The highest yield and productivities for lactic production were 0.99 g·g-1 and 3.75 g·L-1·h-1 whereas the succinic acid production stabilized at 0.77 g·g-1 and 1.16 g·L-1·h-1.

A cellulolytic fungal biofilm enhances the consolidated bioconversion of cellulose to short chain fatty acids by the rumen microbiome.

The ability of the multispecies biofilm membrane reactors (MBM reactors) to provide distinguished niches for aerobic and anaerobic microbes at the same time was used for the investigation of the consolidated bioprocessing of cellulose to short chain fatty acids (SCFAs). A consortium based consolidated bioprocess (CBP) was designed. The rumen microbiome was used as the converting microbial consortium, co-cultivated with selected individual aerobic fungi which formed a biofilm on the tubular membrane flushed with oxygen. The beneficial effect of the fungal biofilm on the process yields and productivities was attributed to the enhanced cellulolytic activities compared with those achieved by the rumen microbiome alone. At 30 °C, the MBM system with Trichoderma reesei biofilm reached a concentration 39% higher (7.3 g/L SCFAs), than the rumen microbiome alone (5.1 g/L) using 15 g/L crystalline cellulose as the substrate. Fermentation temperature was crucial especially for the composition of the short chain fatty acids produced. The temperature increase resulted in shorter fatty acids produced. While a mixture of acetic, propionic, butyric, and caproic acids was produced at 30 °C with Trichoderma reesei biofilm, butyric and caproic acids were not detected during the fermentations at 37.5 °C carried out with Coprinopsis cinerea as the biofilm forming fungus. Apart from the presence of the fungal biofilm, no parameter studied had a significant impact on the total yield of organic acids produced, which reached 0.47 g of total SCFAs per g of cellulose (at 30 °C and at pH 6, with rumen inoculum to total volume ratio equal to 0.372).

A Multispecies Fungal Biofilm Approach to Enhance the Celluloyltic Efficiency of Membrane Reactors for Consolidated Bioprocessing of Plant Biomass.

The constraints and advantages in cellulolytic enzymes production by fungal biofilms for a consolidated bioconversion process were investigated during this study. The biofilm cultivations were carried out in reactors designed for consolidated bioprocessing Multispecies Biofilm Membrane reactors, (MBM) where an aerobic fungal biofilm produces the lignocellulolytic enzymes while a fermenting microorganism forms the fermentation product at anaerobic conditions. It was shown that although mycelial growth was limited in the MBM reactors compared to submerged cultivations, the secretion of cellulolytic enzymes per cell dry weight was higher. When Trichoderma reesei was used as the sole enzyme producer, cellobiose accumulated in the liquid medium as the result of the deficiency of ß-glucosidase in the fungal secretome. To enhance ß-glucosidase activity, T. reesei was co-cultivated with A. phoenicis which is a ß-glucosidase overproducer. The two fungi formed a multispecies biofilm which produced a balanced cellulolytic cocktail for the saccharification of plant biomass. The mixed biofilm reached a 2.5 fold increase in ß-glucosidase production, compared to the single T. reesei biofilm. The enzymatic systems of single and mixed biofilms were evaluated regarding their efficiency on cellulosic substrates degradation. Washed solids from steam pretreated beechwood, as well as microcrystalline cellulose were used as the substrates. The enzymatic system of the multispecies biofilm released four times more glucose than the enzymatic system of T. reesei alone from both substrates and hydrolyzed 78 and 60% of the cellulose content of washed solids from beechwood and microcrystalline cellulose, respectively.

Life cycle impacts of ethanol production from spruce wood chips under high-gravity conditions.

BACKGROUND: Development of more sustainable biofuel production processes is ongoing, and technology to run these processes at a high dry matter content, also called high-gravity conditions, is one option. This paper presents the results of a life cycle assessment (LCA) of such a technology currently in development for the production of bio-ethanol from spruce wood chips. RESULTS: The cradle-to-gate LCA used lab results from a set of 30 experiments (or process configurations) in which the main process variable was the detoxification strategy applied to the pretreated feedstock material. The results of the assessment show that a process configuration, in which washing of the pretreated slurry is the detoxification strategy, leads to the lowest environmental impact of the process. Enzyme production and use are the main contributors to the environmental impact in all process configurations, and strategies to significantly reduce this contribution are enzyme recycling and on-site enzyme production. Furthermore, a strong linear correlation between the ethanol yield of a configuration and its environmental impact is demonstrated, and the selected environmental impacts show a very strong cross-correlation ([Formula: see text] in all cases) which may be used to reduce the number of impact categories considered from four to one (in this case, global warming potential). Lastly, a comparison with results of an LCA of ethanol production under high-gravity conditions using wheat straw shows that the environmental performance does not significantly differ when using spruce wood chips. For this comparison, it is shown that eutrophication potential also needs to be considered due to the fertilizer use in wheat cultivation. CONCLUSIONS: The LCA points out the environmental hotspots in the ethanol production process, and thus provides input to the further development of the high-gravity technology. Reducing the number of impact categories based only on cross-correlations should be done with caution. Knowledge of the analyzed system provides further input to the choice of impact categories.

Ethanol effect on metabolic activity of the ethalogenic fungus Fusarium oxysporum.

BACKGROUND: Fusarium oxysporum is a filamentous fungus which has attracted a lot of scientific interest not only due to its ability to produce a variety of lignocellulolytic enzymes, but also because it is able to ferment both hexoses and pentoses to ethanol. Although this fungus has been studied a lot as a cell factory, regarding applications for the production of bioethanol and other high added value products, no systematic study has been performed concerning its ethanol tolerance levels. RESULTS: In aerobic conditions it was shown that both the biomass production and the specific growth rate were affected by the presence of ethanol. The maximum allowable ethanol concentration, above which cells could not grow, was predicted to be 72 g/L. Under limited aeration conditions the ethanol-producing capability of the cells was completely inhibited at 50 g/L ethanol. The lignocellulolytic enzymatic activities were affected to a lesser extent by the presence of ethanol, while the ethanol inhibitory effect appears to be more severe at elevated temperatures. Moreover, when the produced ethanol was partially removed from the broth, it led to an increase in fermenting ability of the fungus up to 22.5%. The addition of F. oxysporum's system was shown to increase the fermentation of pretreated wheat straw by 11%, in co-fermentation with Saccharomyces cerevisiae. CONCLUSIONS: The assessment of ethanol tolerance levels of F. oxysporum on aerobic growth, on lignocellulolytic activities and on fermentative performance confirmed its biotechnological potential for the production of bioethanol. The cellulolytic and xylanolytic enzymes of this fungus could be exploited within the biorefinery concept as their ethanol resistance is similar to that of the commercial enzymes broadly used in large scale fermentations and therefore, may substantially contribute to a rational design of a bioconversion process involving F. oxysporum. The SSCF experiments on liquefied wheat straw rich in hemicellulose indicated that the contribution of the metabolic system of F. oxysporum in a co-fermentation with S. cerevisiae may play a secondary role.

Fermentation performance and physiology of two strains of Saccharomyces cerevisiae during growth in high gravity spruce hydrolysate and spent sulphite liquor.

BACKGROUND: Lignocellulosic materials are a diverse group of substrates that are generally scarce in nutrients, which compromises the tolerance and fermentation performance of the fermenting organism. The problem is exacerbated by harsh pre-treatment, which introduces sugars and substances inhibitory to yeast metabolism. This study compares the fermentation behaviours of two yeast strains using different types of lignocellulosic substrates; high gravity dilute acid spruce hydrolysate (SH) and spent sulphite liquor (SSL), in the absence and presence of yeast extract. To this end, the fermentation performance, energy status and fermentation capacity of the strains were measured under different growth conditions. RESULTS: Nutrient supplementation with yeast extract increased sugar uptake, cell growth and ethanol production in all tested fermentation conditions, but had little or no effect on the energy status, irrespective of media. Nutrient-supplemented medium enhanced the fermentation capacity of harvested cells, indicating that cell viability and reusability was increased by nutrient addition. CONCLUSIONS: Although both substrates belong to the lignocellulosic spruce hydrolysates, their differences offer specific challenges and the overall yields and productivities largely depend on choice of fermenting strain.

Lignocellulosic ethanol production at high-gravity: challenges and perspectives.

In brewing and ethanol-based biofuel industries, high-gravity fermentation produces 10-15% (v/v) ethanol, resulting in improved overall productivity, reduced capital cost, and reduced energy input compared to processing at normal gravity. High-gravity technology ensures a successful implementation of cellulose to ethanol conversion as a cost-competitive process. Implementation of such technologies is possible if all process steps can be performed at high biomass concentrations. This review focuses on challenges and technological efforts in processing at high-gravity conditions and how these conditions influence the physiology and metabolism of fermenting microorganisms, the action of enzymes, and other process-related factors. Lignocellulosic materials add challenges compared to implemented processes due to high inhibitors content and the physical properties of these materials at high gravity.

Factors affecting cellulose and hemicellulose hydrolysis of alkali treated brewers spent grain by Fusarium oxysporum enzyme extract.

The enzymatic degradation of polysaccharides to monosaccharides is an essential step in bioconversion processes of lignocellulosic materials. Alkali treated brewers spent grain was used as a model substrate for the study of cellulose and hemicellulose hydrolysis by Fusarium oxysporum enzyme extract. The results obtained showed that cellulose and hemicellulose conversions are not affected by the same factors, implementing different strategies for a successful bioconversion. Satisfactory cellulose conversion could be achieved by increasing the enzyme dosage in order to overcome the end-product inhibition, while the complexity of hemicellulose structure imposes the presence of specific enzyme activities in the enzyme mixture used. All the factors investigated were combined in a mathematical model describing and predicting alkali treated brewers spent grain conversion by F. oxysporum enzyme extract.

Factors affecting ferulic acid release from Brewer's spent grain by Fusarium oxysporum enzymatic system.

In this study, the factors affecting ferulic acid (FA) release from Brewer's spent grain (BSG), by the crude enzyme extract of Fusarium oxysporum were investigated. In order to evaluate the importance of the multienzyme preparation on FA release, the synergistic action of feruloyl esterase (FAE, FoFaeC-12213) and xylanase (Trichoderma longibrachiatum M3) monoenzymes was studied. More than double amount of FA release (1 mg g(-1) dry BSG) was observed during hydrolytic reactions by the crude enzyme extract compared to hydrolysis by the monoenzymes (0.37 mg g(-1) dry BSG). The protease content of the crude extract and the inhibitory effect of FA as an end-product were also evaluated concerning their effect on FA release. The protease treatment prior to hydrolysis by monoenzymes enhanced FA release about 100%, while, for the first time in literature, FA in solution found to have a significant inhibitory effect on FAE activity and on total FA release.

Evaluation of Fusarium oxysporum cellulolytic system for an efficient hydrolysis of hydrothermally treated wheat straw.

The crude multienzyme extract produced by Fusarium oxysporum cultivated under submerged conditions in 20 L bioreactor using brewers spent grain and corn cobs in a ratio 2:1 as the carbon source was evaluated with regard to an efficient saccharification of hydrothermally treated wheat straw. Several factors concerning the obtained hydrolysis yield and reaction rate were investigated. The takeout of product sugars (in situ) was effective at reducing end-product inhibition and lead to a bioconversion about 80% of the theoretical. A kinetic model incorporating dynamic adsorption, enzymatic hydrolysis, and product inhibition was developed. The model predicted very satisfactorily the experimental data.

Enhanced ethanol production from brewer's spent grain by a Fusarium oxysporum consolidated system.

BACKGROUND: Brewer's spent grain (BG), a by-product of the brewing process, is attracting increasing scientific interest as a low-cost feedstock for many biotechnological applications. BG in the present study is evaluated as a substrate for lignocellulolytic enzyme production and for the production of ethanol by the mesophilic fungus Fusarium oxysporum under submerged conditions, implementing a consolidated bioconversion process. Fermentation experiments were performed with sugar mixtures simulating the carbohydrate content of BG in order to determine the utilization pattern that could be expected during the fermentation of the cellulose and hemicellulose hydrolysate of BG. The sugar mixture fermentation study focused on the effect of the initial total sugar concentration and on the effect of the aeration rate on fermenting performance of F. oxysporum. The alkali pretreatment of BG and different aeration levels during the ethanol production stage were studied for the optimization of the ethanol production by F. oxysporum. RESULTS: Enzyme yields as high as 550, 22.5, 6.5, 3225, 0.3, 1.25 and 3 U per g of carbon source of endoglucanase, cellobiohydrolase, beta-D-glucosidase, xylanase, feruloyl esterase, beta-D-xylosidase and alpha-L-arabinofuranosidase respectively, were obtained during the growth stage under optimized submerged conditions. An ethanol yield of 109 g ethanol per kg of dry BG was obtained with alkali-pretreated BG under microaerobic conditions (0.01 vvm), corresponding to 60% of the theoretical yield based on total glucose and xylose content of BG. CONCLUSION: The enzymatic profile of the extracellular extract from F. oxysporum submerged cultures using BG and corn cob as the carbon source was proved efficient for a successful hydrolysis of BG. The fermentation study carried out using sugar mixtures simulating BG's carbohydrates content and consecutively alkali-pretreated and untreated BG, indicates that BG hydrolysis is the bottleneck of the bioconversion process. However, a considerable bioconversion yield was achieved (60% of the theoretical) making this bioprocess worthy of further investigation for a potential commercial application.

Hydrolysis and fermentation of brewer's spent grain by Neurospora crassa.

In this study, the ethanol production by the mesophilic fungus Neurospora crassa from BG was studied and optimized concerning the induction of lignocellulose degrading enzymes and the production phase as well. The production of cellulolytic and hemicellulolytic enzymes was studied under solid-state cultivation (SSC). SSC in a laboratory horizontal bioreactor using the optimized medium, WS and BG in the ratio 1:1 and initial moisture level 61.5%, allowed the large scale production of the multienzymatic system. Similar yields with those from flasks experiments, as high as 1073,56,4.2,1.6,3.1,5.7 and 0.52 U g(-1) carbon source of xylanase, endoglucanase, cellobiohydrolase, beta-glucosidase, alpha-l-arabinofuranosidase, acetyl esterase and feruloyl esterase, respectively, were obtained. Chromogenic (fluorogenic) 4-methylumbelliferyl substrates were used to characterize the major activities of the multienzyme component, after the separation by isoelectric focusing (IEF) electrophoresis. Alkali pre-treated BG was used for ethanol production. A yield of about 74 g of ethanol kg(-1) dry BG (5,6 g L(-1)) was obtained under optimum conditions (aeration 0.1 vvm, pre-treatment with 1g NaOH 10 g(-1)dry BG).