Tailoring a cellulolytic enzyme cocktail for efficient hydrolysis of mildly pretreated lignocellulosic biomass.

Commercially available cellulase cocktails frequently demonstrate high efficiency in hydrolyzing easily digestible pretreated biomass, which often lacks hemicellulose and/or lignin fractions. However, the challenge arises with enzymatic hydrolysis of mildly pretreated lignocellulosic biomasses, which contain cellulose, hemicellulose and lignin in high proportions. This study aimed to address this question by evaluating the supplementation of a commercial cellulolytic cocktail with accessory hemicellulases and two additives (H2O2 and Tween® 80). Statistical optimization methods were employed to enhance the release of glucose and xylose from mildly pretreated sugarcane bagasse. The optimized supplement composition resulted in the production of 304 and 124 mg g-1 DM of glucose and xylose, respectively, significantly increasing glucose release by 84% and xylose release by 94% compared to using only the cellulolytic cocktail. This enhancement might be attributed to a coordinated hemicellulases action degrading hemicellulose, creating more space for cellulase activity, potentially boosted by the presence of H2O2 and Tween® 80. However, the addition of different concentrations of H2O2 in combination with hemicellulase and Tween® 80 did not result a significant difference on sugar release, which could be attributed to the limited range of concentrations studied (5 to 65 µM). The results obtained in this study using the mix of three supplements were also compared to the addition of only hemicellulase and only Tween® 80 to the cellulolytic cocktail. A significant increase in glucose release of 39% and 41%, respectively, was observed when using the optimized combination. For xylose, the increase was 38% and 41%, respectively. This study underscores the substantial potential in optimizing enzyme cocktails for the hydrolysis of mildly pretreated lignocellulosic biomass by using enzymes and additive combinations tailored to the specific biomass composition.

Enzyme-constrained metabolic model and in silico metabolic engineering of Clostridium ljungdahlii for the development of sustainable production processes.

Constraint-based genome-scale models (GEMs) of microorganisms provide a powerful tool for predicting and analyzing microbial phenotypes as well as for understanding how these are affected by genetic and environmental perturbations. Recently, MATLAB and Python-based tools have been developed to incorporate enzymatic constraints into GEMs. These constraints enhance phenotype predictions by accounting for the enzyme cost of catalyzed model´s reactions, thereby reducing the space of possible metabolic flux distributions. In this study, enzymatic constraints were added to an existing GEM of Clostridium ljungdahlii, a model acetogenic bacterium, by including its enzyme turnover numbers (kcats) and molecular masses, using the Python-based AutoPACMEN approach. When compared to the metabolic model iHN637, the enzyme cost-constrained model (ec_iHN637) obtained in our study showed an improved predictive ability of growth rate and product profile. The model ec_iHN637 was then employed to perform in silico metabolic engineering of C. ljungdahlii, by using the OptKnock computational framework to identify knockouts to enhance the production of desired fermentation products. The in silico metabolic engineering was geared towards increasing the production of fermentation products by C. ljungdahlii, with a focus on the utilization of synthesis gas and CO2. This resulted in different engineering strategies for overproduction of valuable metabolites under different feeding conditions, without redundant knockouts for different products. Importantly, the results of the in silico engineering results indicated that the mixotrophic growth of C. ljungdahlii is a promising approach to coupling improved cell growth and acetate and ethanol productivity with net CO2 fixation.

Adaptive laboratory evolution of Bacillus subtilis to overcome toxicity of lignocellulosic hydrolysate derived from Distiller's dried grains with solubles (DDGS).

Microbial tolerance to toxic compounds formed during biomass pretreatment is a significant challenge to produce bio-based products from lignocellulose cost effectively. Rational engineering can be problematic due to insufficient prerequisite knowledge of tolerance mechanisms. Therefore, adaptive laboratory evolution was applied to obtain 20 tolerant lineages of Bacillus subtilis strains able to utilize Distiller's Dried Grains with Solubles-derived (DDGS) hydrolysate. Evolved strains showed both improved growth performance and retained heterologous enzyme production using 100% hydrolysate-based medium, whereas growth of the starting strains was essentially absent. Whole-genome resequencing revealed that evolved isolates acquired mutations in the global regulator codY in 15 of the 19 sequenced isolates. Furthermore, mutations in genes related to oxidative stress (katA, perR) and flagella function appeared in both tolerance and control evolution experiments without toxic compounds. Overall, tolerance adaptive laboratory evolution yielded strains able to utilize DDGS-hydrolysate to produce enzymes and hence proved to be a valuable tool for the valorization of lignocellulose.

Enzymatic hydrolysis cocktail optimization for the intensification of sugar extraction from sugarcane bagasse.

Lignocellulosic biomasses have a very important role as a raw material to produce biofuels and biochemicals. However, a sustainable, efficient, and economically competitive process for the release of sugars from such materials has still not been achieved. In this work, the optimization of the enzymatic hydrolysis cocktail was evaluated as an approach to maximize sugar extraction from mildly pretreated sugarcane bagasse. Different additives and enzymes, including hydrogen peroxide (H2O2), laccase, hemicellulase and the surfactants Tween 80 and PEG4000 were added to a cellulolytic cocktail with the aim of improving biomass hydrolysis. An increase of 39 % and 46 % of glucose and xylose concentrations, respectively, compared to the control (when only the cellulolytic cocktail (20 or 35 FPU g-1 dry mass), was obtained when H2O2 (0.24 mM) was added at the beginning of the hydrolysis. On the other hand, the addition of hemicellulase (81-162 µL g-1 DM) increased the production of glucose up to 38 % and xylose up to 50 %. The findings of this study reveal that it is possible to increase the extraction of sugars from mildly pretreated lignocellulosic biomass by using an appropriate enzymatic cocktail supplemented with additives. This opens up new opportunities for the development of a more sustainable, efficient, and economically competitive process for biomass fractionation.

Improving Saccharomyces cerevisiae acid and oxidative stress resistance using a prokaryotic gene identified by functional metagenomics.

Innovations in obtaining products from lignocellulosic biomass have been largely based on the improvement of microorganisms and enzymes capable of degrading these materials. To complete the whole process, microorganisms must be able to ferment the resulting sugars and tolerate high concentrations of product, osmotic pressure, ion toxicity, temperature, toxic compounds from lignocellulose pretreatment, low pH, and oxidative stress. In this work, we engineered laboratory and industrial Saccharomyces cerevisiae strains by combining a gene (hu) recovered from a metagenomic approach with different native and synthetic promoters to obtain improved acid and oxidative stress resistance. Laboratorial strains harboring hu gene under the control of the synthetic stress responsive PCCW14v5 showed increased survival rates after 2 h exposure to pH 1.5. The hu gene was also able to significantly enhance the tolerance of the industrial strain to high concentrations of H2O2 when combined with PTEF1, PYGP1 or PYGP1v7 after 3 h exposure.

Microbial meat: A sustainable vegan protein source produced from agri-waste to feed the world.

In the modern world, animal and plant protein may not meet the sustainability criteria due to their high need for arable land and potable water consumption, among other practices. Considering the growing population and food shortage, finding alternative protein sources for human consumption is an urgent issue that needs to be solved, especially in developing countries. In this context, microbial bioconversion of valuable materials in nutritious microbial cells represent a sustainable alternative to the food chain. Microbial protein, also known as single-cell protein (SCP), consist of algae biomass, fungi or bacteria that are currently used as food source for both humans and animals. Besides contributing as a sustainable source of protein to feed the world, producing SCP, is important to reduce waste disposal problems and production costs meeting the sustainable development goals. However, for microbial protein as feed or food to become an important and sustainable alternative, addressing the challenges of raising awareness and achieving wider public regulatory acceptance is real and must be addressed with care and convenience. In this work, we critically reviewed the potential technologies for microbial protein production, its benefits, safety, and limitations associated with its uses, and perspectives for broader large-scale implementation. We argue that the information documented in this manuscript will assist in developing microbial meat as a major protein source for the vegan world.

Extrusion of process flavorings from methionine and dextrose using modified starch as a carrier.

This study aimed to produce process flavorings from methionine and glucose via Maillard reaction by extrusion method. Modified starch was used as a carrier to reduce the torque and facilitate the production process. Five formulations of process flavorings with different ratios of methionine: dextrose: modified starch: water as MS5 (72:18:5:5), MS15 (64:16:15:5), MS25 (56:14:25:5), MS35 (42:12:35:5), and MS45 (40:10:45:5) were prepared and feded into the extruder. The temperatures of the extruder barrel in zones 1 and 2 were controlled at 100, and 120°C, with a screw speed of 30 rpm. The appearance of the obtained products, torque, pH before and after extrusion, color, volatile compounds, and sensory evaluation were determined. The extrudate from the formulation containing the highest amount of modified starch (MS45) gave the highest L* (lightness) of 88.00, which increased to 93.00 (very light) after grinding into a powder. The process flavorings from all formulations exhibited similar sensory scores in terms of aroma, taste, and water solubility, with a very slight difference in color. However, MS25, MS35 and MS45 indicated the torque at 10 Nm/cm3, while MS5 and MS 15 exhibited higher torque at 18, and 25 Nm/cm3, respectively. Extruded process flavorings from MS25 were analyzed for their flavor profiles by gas chromatography-mass spectrometry. Twelve volatile compounds including the key volatile compounds for sulfurous and vegetable odor type, dimethyl disulfide, methional, and methanethiol, were found. Four pyrazine compounds presented nutty, musty and caramelly odor; and 3-hydroxybutan-2-one and heptane-2,3-dione, which gave buttery odor type, were also detected. The results demonstrated a successful production of process flavorings using modified starch as carrier to facilitate and reduce the torque during the extrusion process.

Subcritical water pretreatment enhanced methane-rich biogas production from the anaerobic digestion of brewer's spent grains.

ABSTRACTThis study evaluated the effectiveness of a semi-continuous flow-through subcritical water hydrolysis (SWH) pretreatment of brewer's spent grains (BSG) for subsequent application in the anaerobic digestion (AD) process. BSG pretreatment was conducted at 160 °C and 15 MPa with a flow rate of 10 mL water min-1 and 15 g water g-1 BSG. The results revealed that SWH attacked the hemicellulose structure, releasing arabinose (46.54 mg g-1) and xylose (39.90 mg g-1) sugars, and proteins (34.89 mg g-1). The start-up of anaerobic reactors using pretreated BSG (747.71 L CH4 kg-1 TVS) increased the methane yield compared with the reactor without pretreatment (53.21 L CH4 kg-1 TVS). For the process with pretreatment, the generation of electricity (134 kWh t-1 BSG) and heat (604 MJ t-1) are responsible for the mitigation of 43.90 kg CO2 eq t-1 BSG. The adoption of SWH as an eco-friendly pretreatment of biomass for AD could be a technological route to increase methane-rich biogas and bioenergy production, supporting the circular economy transition by reducing the carbon footprint of the beer industry.

Recovery of sugars and amino acids from brewers' spent grains using subcritical water hydrolysis in a single and two sequential semi-continuous flow-through reactors.

This study evaluated the subcritical water hydrolysis (SWH) of brewer's spent grains (BSG) to obtain sugars and amino acids. The experimental conditions investigated the hydrolysis of BSG in a single flow-through reactor and in two sequential reactors operated in semi-continuous mode. The hydrolysis experiments were carried out for 120 min at 15 MPa, 5 mL water min-1, at different temperatures (80 - 180 °C) and using an S/F of 20 and 10 g solvent g-1 BSG, for the single and two sequential reactors, respectively. The highest monosaccharide yields were obtained at 180 °C in a single reactor (47.76 mg g-1 carbohydrates). With these operational conditions, the hydrolysate presented xylose (0.477 mg mL-1) and arabinose (1.039 mg mL-1) as main sugars, while low contents of furfural (310.7 µg mL-1), 5-hydroxymethylfurfural (<1 mg L-1), and organic acids (0.343 mg mL-1) were obtained. The yield of proteins at 180 °C in a process with a single reactor was 43.62 mg amino acids g-1 proteins, where tryptophan (215.55 µg mL-1), aspartic acid (123.35 µg mL-1), valine (64.35 µg mL-1), lysine (16.55 µg mL-1), and glycine (16.1 µg mL-1) were the main amino acids recovered in the hydrolysate. In conclusion, SWH pretreatment is a promising technology to recover bio-based compounds from BSG; however, further studies are still needed to increase the yield of bioproducts from lignocellulosic biomass to explore two sequential reactors.

Production of process flavorings from methionine, thiamine with d-xylose or dextrose by direct extrusion: Physical properties and volatile profiles.

The conventional method to produce process flavoring is non-continuous, time consuming, and generates a high volume of effluent. This research aimed to evaluate the use of methionine, thiamine, and reducing sugars to develop process flavorings by direct extrusion, as a potential alternative to the conventional method. The mixed substrates consisted of methionine: d-xylose (MX), methionine: dextrose (MD), thiamine: d-xylose (TX), and thiamine: dextrose (TD) at 80:20 w/w. Three barrel temperatures of the extruder were controlled at 65, 80, and 50°C, respectively, a screw speed of 30 rpm and feed rate at 3 kg/hr. Appearance, pH, odor, and taste description of the product from each mixture were determined. Volatile compounds, possibly occurred from the Maillard reaction during the extrusion were analyzed by gas chromatography-mass spectrometry. The products exhibited different levels of meaty odor and bitter taste. Those obtained from MD showed the highest L* (lightness, 85.37) and frequency for just-about-right in terms of taste (33.33%) and odor (60.00%). Products from MX and MD presented the highest frequency for intense taste, and higher frequency for color compared to TX and TD. More volatile compounds were detected from the use of methionine than from thiamine. The key meaty odor compounds such as dimethyl disulfide, dimethyl trisulfide, methional, and methanethiol were found in the samples from MX and MD, while only dimethyl disulfide was detected in the mixture of TX and TD. Finally, the results demonstrated that direct extrusion reaction from methionine and d-xylose or dextrose is a highly efficient method to produce meaty process flavorings. PRACTICAL APPLICATION: The manuscript describes the production of process flavorings that exhibited meaty flavors by extrusion process. Physical properties, volatile profiles, and sensory evaluation of the products from methionine, thiamine, d-xylose, and glucose were evaluated. The extruded products from methionine and dextrose exhibited acceptable color, taste, and odor and presented many volatiles compounds contributing to meaty flavors. The results revealed the high potential to use a direct extrusion process with very low effluent, compared to the conventional method, to produce meaty flavors for industrial application.

Bacteriocin-like inhibitory substances production by Enterococcus faecium 135 in co-culture with Ligilactobacillus salivarius and Limosilactobacillus reuteri.

The use of lactic acid bacteria (LAB) and probiotic cultures in the breeding of animals such as poultry and swine are quite common. It is known that those strains can produce bacteriocins when grown in pure culture. However, the production of bacteriocin using co-culture of microorganisms has not been much studied so far. The present study contributes with innovation in this area by embracing the production of bacteriocin-like inhibitory substances (BLIS) by a newly isolated strain of Enterococcus faecium 135. Additionally, the co-cultivation of this strain with Ligilactobacillus salivarius and Limosilactobacillus reuteri was also investigated. The antimicrobial activity of the produced BLIS was evaluated against Listeria monocytogenes, Listeria innocua, Salmonella enterica, and Salmonella enterica serovar Typhimurium using two methods: turbidimetric and agar diffusion. In addition, the presence of enterocin genes was also evaluated. The BLIS produced showed a bacteriostatic effect against the bio-indicator strains, and the highest antimicrobial activities expressed by arbitrary units per mL (AU/mL) were obtained against L. monocytogenes in monoculture (12,800 AU/mL), followed by the co-culture of E. faecium with Limosilactobacillus reuteri (400 AU/mL). After concentration with ammonium sulfate, the antimicrobial activity raised to 25,600 AU/mL. Assays to determine the proteinaceous nature of the BLIS showed susceptibility to trypsin and antimicrobial activity until 90 °C. Finally, analysis of the presence of structural genes of enterocins revealed that four enterocin genes were present in E. faecium 135. These results suggest that BLIS produced by E. faecium 135 has potential to be a bacteriocin and, after purification, could potentially be used as an antimicrobial agent in animal breeding.

Maximizing the simultaneous production of lipids and carotenoids by Rhodosporidium toruloides from wheat straw hydrolysate and perspectives for large-scale implementation.

This study aimed to select fermentation conditions able to simultaneously maximize the production of lipids and carotenoids by oleaginous yeast cultivated in wheat straw hydrolysate. An evolved strain of Rhodosporidium toruloides with improved tolerance to toxic compounds present in hydrolysate medium was used. Experiments were performed in order to investigate the effect of the temperature and inoculum load on the production of lipids and carotenoids by R. toruloides. Results revealed that the accumulation of both products can be simultaneously maximized when performing the fermentation at 17 °C and using 3.5 g/L of inoculum. This maximum simultaneous production opens up new perspectives for the establishment of a feasible and more sustainable large-scale process for the production of lipids and carotenoids. Even corresponding to only 1% of the cell mass, due to the high market value, carotenoids would account for more than 90% of the total income of the industrial plant.

Adaptive laboratory evolution of Rhodosporidium toruloides to inhibitors derived from lignocellulosic biomass and genetic variations behind evolution.

Using lignocellulosic biomass hydrolysate for the production of microbial lipids and carotenoids is still a challenge due to the poor tolerance of oleaginous yeasts to the inhibitors generated during biomass pretreatment. In this study, a strategy of adaptive laboratory evolution in hydrolysate-based medium was developed to improve the tolerance of Rhodosporidium toruloides to inhibitors present in biomass hydrolysate. The evolved strains presented better performance to grow in hydrolysate medium, with a significant reduction in their lag phases, and improved ability to accumulate lipids and produce carotenoids when compared to the wild-type starting strain. In the best cases, the lag phase was reduced by 72 h and resulted in lipid accumulation of 27.89 ± 0.80% (dry cell weight) and carotenoid production of 14.09 ± 0.12 mg/g (dry cell weight). Whole genome sequencing analysis indicated that the wild-type strain naturally contained tolerance-related genes, which provided a background that allowed the strain to evolve in biomass-derived inhibitors.

Bioprocess intensification: Cases that (don't) work.

Development of affordable and low carbon biobased manufacturing depends critically on strategies that reduce cost and emission profiles. This paper indicates that efforts around the reduction of capital costs by intensification of process equipment need to be carefully weighed against the inherently fast increasing financial and climate costs of driving forces used for the intensification. The fundamental relation between capital expenditures (CAPEX) and operational expenditures (OPEX) of intensified and non-intensified biobased processes and their financial and climatic impacts are emphasized and provisionally explored for a few industrial processes. General learnings flag the importance in particular of OPEX minimisation for sustainable bio-economic development.

A spatially explicit assessment of sugarcane vinasse as a sustainable by-product.

This study evaluates the benefits of mineral fertilizers replacement for biodigested vinasse. Data from experimental anaerobic digestion (AD) of vinasse were applied to support the analysis. Based on previous experiments, this assessment assumed that vinasse production could reach 2.38 × 107 m3/year generating around 66,585 MWh/year of electric energy from biogas burning in the Administrative Region of Campinas (ARC). This amount of energy could supply more than 103,000 inhabitants and avoid 35,892 tCO2eq/year (from electric energy replacement). The biodigested vinasse might also reduce the total N, P, and K mineral fertilizers demand per hectare of sugarcane crop in 30%, 1%, and 46%, respectively, avoiding additional greenhouse gas emissions of 111,877 tCO2eq/year. There is no biodigested vinasse surplus for a moderate fertigation rate of 100 m3/ha, complying with local environmental laws related to nutrients excess side effects in areas destined to sugarcane crop. Notwithstanding, a Geographic Information System analysis for a small adjacent area to ARC indicated nine different fertigation rates, ranging from 50 to 100 m3/ha. Even though the general analysis for ARC shows high NPK replacement levels, the fertigation practices should be subsidized for robust soil analysis and adequate to safe environmental levels. A management tool can be designed using the results here presented to subsidize investments for AD widespread adoption by the sugarcane industry to catch a reasonable practice from the economic and environmental perspectives.

Transformation of sugarcane molasses into fructooligosaccharides with enhanced prebiotic activity using whole-cell biocatalysts from Aureobasidium pullulans FRR 5284 and an invertase-deficient Saccharomyces cerevisiae 1403-7A.

Fructooligosaccharides (FOS) can be used as feed prebiotics, but are limited by high production costs. In this study, low-cost sugarcane molasses was used to produce whole-cell biocatalysts containing transfructosylating enzymes by Aureobasidium pullulans FRR 5284, followed by FOS production from molasses using the whole-cells of A. pullulans. A. pullulans in molasses-based medium produced cells and broth with a total transfructosylating activity of 123.6 U/mL compared to 61.0 and 85.8 U/mL in synthetic molasses-based and sucrose-based media, respectively. It was found that inclusion of glucose in sucrose medium reduced both transfructosylating and hydrolytic activities of the produced cells and broth. With the use of pure glucose medium, cells and broth had very low levels of transfructosylating activities and hydrolytic activities were not detected. These results indicated that A. pullulans FRR 5284 produced both constitutive and inducible enzymes in sucrose-rich media, such as molasses while it only produced constitutive enzymes in the glucose media. Furthermore, treatment of FOS solutions generated from sucrose-rich solutions using an invertase-deficient Saccharomyces yeast converted glucose to ethanol and acetic acid and improved FOS content in total sugars by 20-30%. Treated FOS derived from molasses improved the in vitro growth of nine probiotic strains by 9-63% compared to a commercial FOS in 12 h incubation. This study demonstrated the potential of using molasses to produce FOS for feed application.

Enzymatic Hydrolysis of Sugarcane Bagasse in Aqueous Two-Phase Systems (ATPS): Exploration and Conceptual Process Design.

The enzymatic conversion of lignocellulosic material to sugars can provide a carbon source for the production of energy (fuels) and a wide range of renewable products. However, the efficiency of this conversion is impaired due to product (sugar) inhibition. Even though several studies investigate how to overcome this challenge, concepts on the process to conduct the hydrolysis are still scarce in literature. Aqueous two-phase systems (ATPS) can be applied to design an extractive reaction due to their capacity to partition solutes to different phases in such a system. This work presents strategies on how to conduct extractive enzymatic hydrolysis in ATPS and how to explore the experimental results in order to design a feasible process. While only a limited number of ATPS was explored, the methods and strategies described could easily be applied to any further ATPS to be explored. We studied two promising ATPS as a subset of a previously high throughput screened large set of ATPS, providing two configurations of processes having the reaction in either the top phase or in the bottom phase. Enzymatic hydrolysis in these ATPS was performed to evaluate the partitioning of the substrate and the influence of solute partitioning on conversion. Because ATPS are able to partition inhibitors (sugar) between the phases, the conversion rate can be maintained. However, phase forming components should be selected to preserve the enzymatic activity. The experimental results presented here contribute to a feasible ATPS-based conceptual process design for the enzymatic conversion of lignocellulosic material.

Exploiting new biorefinery models using non-conventional yeasts and their implications for sustainability.

Feasible bioprocessing of lignocellulosic biomass requires the use of microbial strains with tolerance to inhibitor compounds and osmotic pressure, able to provide high product yield and productivity. In this sense, this study evaluated the potential of two non-conventional yeasts, Hansenula polymorpha CBS 4732 and Debaryomyces hansenii CBS 767, for use on biomass conversion in a biorefinery perspective. The ability of the strains to consume pentose and hexose sugars, to resist the toxic compounds present in hydrolysates, as well as to produce sugar alcohols and ethanol, was investigated. H. polymorpha showed highlighted resistance to toxic compounds and relevant ability to consume xylose and produce xylitol and ethanol under these conditions, at 37 °C. D. hansenii was a great producer of arabitol from glucose. The implications for sustainability due to the use of these yeasts in biorefineries was discussed. These results open up new perspectives for the development of the biorefinery sector.