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.

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.

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.

Innovation and strategic orientations for the development of advanced biorefineries.

Advanced biorefineries, which aim at valorizing biomass (from agriculture, forestry, aquaculture, among others) into a wide spectrum of products and bioenergy, are seen today as key to implement a sustainable biobased economy. Although different concepts of biorefinery are currently under development, further research and improvement are still required to obtain environmentally friendly and economically feasible commercial scale biorefineries. Valorization of all biomass components and integration of different disciplines are some of the strategies that have been considered to improve the economic and environmental performance. This paper summarizes and discusses the most recent innovations and strategic orientations for the development of advanced biorefineries. Focus is given on the valorization of non-carbohydrate components of biomass (protein, acetic acid and lignin), on-site and tailor-made production of enzymes, big data analytics, and interdisciplinary efforts. The idea is to provide new insights and directions to support the development and large-scale implementation of biorefineries.

Oleaginous yeasts for sustainable lipid production-from biodiesel to surf boards, a wide range of "green" applications.

A growing world population and a growing number of applications for vegetable oils are generating an increasing demand for these oils, causing serious environmental problems. A sustainable lipid production is then fundamental to address these problems. Oleaginous yeasts are a promising solution for sustainable lipid production, but, with the current knowledge and technology, they are still not a serious alternative in the market. In this review, the potential of these yeasts is highlighted and a discussion is made mainly focused on the economics of the oleaginous yeast oil production and identification of the key points to be improved to achieve lower production costs and higher income. Three main stages of the production process, where costs are higher, were identified. To render economically feasible the production of oils using oleaginous yeasts, a reduction in production costs must occur in all stages, lipid yields and productivities must be improved, and production must be targeted to high-value product applications.

Optimization of lipid extraction from the oleaginous yeasts Rhodotorula glutinis and Lipomyces kononenkoae.

The constant growing demand for vegetable oil for biodiesel and food is raising many environmental concerns about the sustainability of its production based on crops. Oleaginous yeasts show great potential to end with those concerns due to their high lipid productivity in small areas. To evaluate their productivity in lipids, an efficient and reproducible extraction process should be used. As no standard extraction process is available for the extraction of yeast lipids, an optimized extraction process is presented. In this work, the lipids extraction process for the yeasts Rhodotorula glutinis and Lipomyces kononenkoae is optimized using bead beating for cell rupture and introducing adaptations of the two most used extraction methods (Bligh and Dyer and Folch). For Rhodotorula g. the optimum extraction conditions are obtained by the Bligh and Dyer method applying 4.8 cycles of 47 s with 0.7 g of glass beads. For Lipomyces k. the optimum extraction conditions make use of the Folch method applying seven cycles of 42 s with 0.54 g of glass beads. These results reinforce the idea that, for each yeast, different extraction processes may be needed to correctly determine the lipid yield. The extraction procedure was further evaluated with less harmful solvents. Toluene was tested as a possible substitute of chloroform, and ethanol as a possible substitute of methanol. With the optimized extraction process, better results for Lipomyces k. were obtained using toluene and ethanol, while for Rhodotorula g. toluene proved to be a valid substitute of chloroform but ethanol is far less effective than methanol.

Hydrodynamic cavitation as a strategy to enhance the efficiency of lignocellulosic biomass pretreatment.

Hydrodynamic cavitation (HC) is a process technology with potential for application in different areas including environmental, food processing, and biofuels production. Although HC is an undesirable phenomenon for hydraulic equipment, the net energy released during this process is enough to accelerate certain chemical reactions. The application of cavitation energy to enhance the efficiency of lignocellulosic biomass pretreatment is an interesting strategy proposed for integration in biorefineries for the production of bio-based products. Moreover, the use of an HC-assisted process was demonstrated as an attractive alternative when compared to other conventional pretreatment technologies. This is not only due to high pretreatment efficiency resulting in high enzymatic digestibility of carbohydrate fraction, but also, by its high energy efficiency, simple configuration, and construction of systems, besides the possibility of using on the large scale. This paper gives an overview regarding HC technology and its potential for application on the pretreatment of lignocellulosic biomass. The parameters affecting this process and the perspectives for future developments in this area are also presented and discussed.

Sugarcane bagasse hydrolysate as a potential feedstock for red pigment production by Monascus ruber.

Sugarcane bagasse (SCB) hydrolysate could be an interesting source for red pigment production by Monascus ruber Tieghem IOC 2225. The influence of different wavelength of light-emitting diode (LED) at 250 µmol.m-2.s-1 of photon flux density on red pigment production by M. ruber in glucose-based medium was evaluated. Then, SCB hydrolysate was used as carbon source under the previously selected light incidence conditions. In glucose-based medium, the highest pigment production was achieved in fermentation assisted with orange LED light (8.28 UA490nm), white light (8.26 UA490nm) and under dark condition (7.45 UA490nm). By using SCB hydrolysate-based medium, the highest red pigment production (18.71 AU490nm) was achieved under dark condition and the glucose and cellobiose present in the hydrolysate were metabolized. SCB enzymatic hydrolysate was demonstrated to be a promising carbon source for high thermal stability red pigment production (activation energy of 10.5 kcal.mol-1), turning an interesting alternative for implementation in biorefineries.

Relationship between starch and lipid accumulation induced by nutrient depletion and replenishment in the microalga Parachlorella kessleri.

Photosynthetic carbon partitioning into starch and neutral lipids, as well as the influence of nutrient depletion and replenishment on growth, pigments and storage compounds, were studied in the microalga, Parachlorella kessleri. Starch was utilized as a primary carbon and energy storage compound, but nutrient depletion drove the microalgae to channel fixed carbon into lipids as secondary storage compounds. Nutrient depletion inhibited both cellular division and growth and caused degradation of chlorophyll. Starch content decreased from an initial value of 25, to around 10% of dry weight (DW), while storage lipids increased from almost 0 to about 29% of DW. After transfer of cells into replenished mineral medium, growth, reproductive processes and chlorophyll content recovered within 2 days, while the content of both starch and lipids decreased markedly to 3 or less % of DW; this suggested that they were being used as a source of energy and carbon.

Optimization of CO2 bio-mitigation by Chlorella vulgaris.

Biofixation of CO2 by microalgae has been recognized as an attractive approach to CO2 mitigation. The main objective of this work was to maximize the rate of CO2 fixation ( [Formula: see text] ) by the green microalga Chlorella vulgaris P12 cultivated photoautotrophically in bubble column photobioreactors under different CO2 concentrations (ranging from 2% to 10%) and aeration rates (ranging from 0.1 to 0.7 vvm). Results showed that the maximum [Formula: see text] (2.22 gL(-1)d(-1)) was obtained by using 6.5% CO2 and 0.5 vvm after 7 days of cultivation at 30°C. Although final biomass concentration and maximum biomass productivity of microalgae were affected by the different cultivation conditions, no significant differences were obtained in the biochemical composition of microalgal cells for the evaluated levels of aeration and CO2. The present study demonstrated that optimization of microalgal cultivation conditions can be considered a useful strategy for maximizing CO2 bio-mitigation by C. vulgaris.

Mixotrophic cultivation of Chlorella vulgaris using industrial dairy waste as organic carbon source.

Growth parameters and biochemical composition of the green microalga Chlorella vulgaris cultivated under different mixotrophic conditions were determined and compared to those obtained from a photoautotrophic control culture. Mixotrophic microalgae showed higher specific growth rate, final biomass concentration and productivities of lipids, starch and proteins than microalgae cultivated under photoautotrophic conditions. Moreover, supplementation of the inorganic culture medium with hydrolyzed cheese whey powder solution led to a significant improvement in microalgal biomass production and carbohydrate utilization when compared with the culture enriched with a mixture of pure glucose and galactose, due to the presence of growth promoting nutrients in cheese whey. Mixotrophic cultivation of C. vulgaris using the main dairy industry by-product could be considered a feasible alternative to reduce the costs of microalgal biomass production, since it does not require the addition of expensive carbohydrates to the culture medium.

Brazilian kefir: structure, microbial communities and chemical composition

Microbial ecology and chemical composition of Brazilian kefir beverage was performed. The microorganisms associated with Brazilian kefir were investigated using a combination of phenotypic and genotypic methods. A total of 359 microbial isolates were identified. Lactic acid bacteria (60.5 percent) were the major isolated group identified, followed by yeasts (30.6 percent) and acetic acid bacteria (8.9 percent). Lactobacillus paracasei (89 isolates), Lactobacillus parabuchneri (41 isolates), Lactobacillus casei (32 isolates), Lactobacillus kefiri (31 isolates), Lactococcus lactis (24 isolates), Acetobacter lovaniensis (32 isolates), Kluyveromyces lactis (31 isolates), Kazachstania aerobia (23 isolates), Saccharomyces cerevisiae (41 isolates) and Lachancea meyersii (15 isolates) were the microbial species isolated. Scanning electron microscopy showed that the microbiota was dominated by bacilli (short and curved long) cells growing in close association with lemon-shaped yeasts cells. During the 24 h of fermentation, the protein content increased, while lactose and fat content decreased. The concentration of lactic acid ranged from 1.4 to 17.4 mg/ml, and that of acetic acid increased from 2.1 to 2.73 mg/ml. The production of ethanol was limited, reaching a final mean value of 0.5 mg/ml.

Brazilian kefir: structure, microbial communities and chemical composition.

Microbial ecology and chemical composition of Brazilian kefir beverage was performed. The microorganisms associated with Brazilian kefir were investigated using a combination of phenotypic and genotypic methods. A total of 359 microbial isolates were identified. Lactic acid bacteria (60.5%) were the major isolated group identified, followed by yeasts (30.6%) and acetic acid bacteria (8.9%). Lactobacillus paracasei (89 isolates), Lactobacillus parabuchneri (41 isolates), Lactobacillus casei (32 isolates), Lactobacillus kefiri (31 isolates), Lactococcus lactis (24 isolates), Acetobacter lovaniensis (32 isolates), Kluyveromyces lactis (31 isolates), Kazachstania aerobia (23 isolates), Saccharomyces cerevisiae (41 isolates) and Lachancea meyersii (15 isolates) were the microbial species isolated. Scanning electron microscopy showed that the microbiota was dominated by bacilli (short and curved long) cells growing in close association with lemon-shaped yeasts cells. During the 24 h of fermentation, the protein content increased, while lactose and fat content decreased. The concentration of lactic acid ranged from 1.4 to 17.4 mg/ml, and that of acetic acid increased from 2.1 to 2.73 mg/ml. The production of ethanol was limited, reaching a final mean value of 0.5 mg/ml.

Fermentative behavior of Saccharomyces strains during microvinification of raspberry juice (Rubus idaeus L.).

Sixteen different strains of Saccharomyces cerevisiae and Saccharomyces bayanus were evaluated in the production of raspberry fruit wine. Raspberry juice sugar concentrations were adjusted to 16° Brix with a sucrose solution, and batch fermentations were performed at 22 °C. Various kinetic parameters, such as the conversion factors of the substrates into ethanol (Y(p/s)), biomass (Y(x/s)), glycerol (Y(g/s)) and acetic acid (Y(ac/s)), the volumetric productivity of ethanol (Q(p)), the biomass productivity (P(x)), and the fermentation efficiency (E(f)) were calculated. Volatile compounds (alcohols, ethyl esters, acetates of higher alcohols and volatile fatty acids) were determined by gas chromatography (GC-FID). The highest values for the E(f), Y(p/s), Y(g/s), and Y(x/s) parameters were obtained when strains commonly used in the fuel ethanol industry (S. cerevisiae PE-2, BG, SA, CAT-1, and VR-1) were used to ferment raspberry juice. S. cerevisiae strain UFLA FW 15, isolated from fruit, displayed similar results. Twenty-one volatile compounds were identified in raspberry wines. The highest concentrations of total volatile compounds were found in wines produced with S. cerevisiae strains UFLA FW 15 (87,435 µg/L), CAT-1 (80,317.01 µg/L), VR-1 (67,573.99 µg/L) and S. bayanus CBS 1505 (71,660.32 µg/L). The highest concentrations of ethyl esters were 454.33 µg/L, 440.33 µg/L and 438 µg/L for S. cerevisiae strains UFLA FW 15, VR-1 and BG, respectively. Similar to concentrations of ethyl esters, the highest concentrations of acetates (1927.67 µg/L) and higher alcohols (83,996.33 µg/L) were produced in raspberry wine from S. cerevisiae UFLA FW 15. The maximum concentration of volatile fatty acids was found in raspberry wine produced by S. cerevisiae strain VR-1. We conclude that S. cerevisiae strain UFLA FW 15 fermented raspberry juice and produced a fruit wine with low concentrations of acids and high concentrations of acetates, higher alcohols and ethyl esters.

Production of fermented cheese whey-based beverage using kefir grains as starter culture: evaluation of morphological and microbial variations.

Whey valorization concerns have led to recent interest on the production of whey beverage simulating kefir. In this study, the structure and microbiota of Brazilian kefir grains and beverages obtained from milk and whole/deproteinised whey was characterized using microscopy and molecular techniques. The aim was to evaluate its stability and possible shift of probiotic bacteria to the beverages. Fluorescence staining in combination with Confocal Laser Scanning Microscopy showed distribution of yeasts in macro-clusters among the grain's matrix essentially composed of polysaccharides (kefiran) and bacteria. Denaturing gradient gel electrophoresis displayed communities included yeast affiliated to Kluyveromyces marxianus, Saccharomyces cerevisiae, Kazachatania unispora, bacteria affiliated to Lactobacillus kefiranofaciens subsp. Kefirgranum, Lactobacillus kefiranofaciens subsp. Kefiranofaciens and an uncultured bacterium also related to the genus Lactobacillus. A steady structure and dominant microbiota, including probiotic bacteria, was detected in the analyzed kefir beverages and grains. This robustness is determinant for future implementation of whey-based kefir beverages.

WITHDRAWN: Technological trends, global market, and challenges of bio-ethanol production.

The Publisher regrets that this article is an accidental duplication of an article that has already been published, doi:10.1016/j.biotechadv.2010.07.001. The duplicate article has therefore been withdrawn.

Technological trends, global market, and challenges of bio-ethanol production.

Ethanol use as a fuel additive or directly as a fuel source has grown in popularity due to governmental regulations and in some cases economic incentives based on environmental concerns as well as a desire to reduce oil dependency. As a consequence, several countries are interested in developing their internal market for use of this biofuel. Currently, almost all bio-ethanol is produced from grain or sugarcane. However, as this kind of feedstock is essentially food, other efficient and economically viable technologies for ethanol production have been evaluated. This article reviews some current and promising technologies for ethanol production considering aspects related to the raw materials, processes, and engineered strains development. The main producer and consumer nations and future perspectives for the ethanol market are also presented. Finally, technological trends to expand this market are discussed focusing on promising strategies like the use of microalgae and continuous systems with immobilized cells.

Light regime characterization in an airlift photobioreactor for production of microalgae with high starch content.

The slow development of microalgal biotechnology is due to the failure in the design of large-scale photobioreactors (PBRs) where light energy is efficiently utilized. In this work, both the quality and the amount of light reaching a given point of the PBR were determined and correlated with cell density, light path length, and PBR geometry. This was made for two different geometries of the downcomer of an airlift PBR using optical fiber technology that allows to obtain information about quantitative and qualitative aspects of light patterns. This is important since the ability of microalgae to use the energy of photons is different, depending on the wavelength of the radiation. The results show that the circular geometry allows a more efficient light penetration, especially in the locations with a higher radial coordinate (r) when compared to the plane geometry; these observations were confirmed by the occurrence of a higher fraction of illuminated volume of the PBR for this geometry. An equation is proposed to correlate the relative light intensity with the penetration distance for both geometries and different microalgae cell concentrations. It was shown that the attenuation of light intensity is dependent on its wavelength, cell concentration, geometry of PBR, and the penetration distance of light.