Bacillus genus industrial applications and innovation: First steps towards a circular bioeconomy.

In recent decades, environmental concerns have directed several policies, investments, and production processes. The search for sustainable and eco-friendly strategies is constantly increasing to reduce petrochemical product utilization, fossil fuel pollution, waste generation, and other major ecological impacts. The concepts of circular economy, bioeconomy, and biorefinery are increasingly being applied to solve or reduce those problems, directing us towards a greener future. Within the biotechnology field, the Bacillus genus of bacteria presents extremely versatile microorganisms capable of producing a great variety of products with little to no dependency on petrochemicals. They are able to grow in different agro-industrial wastes and extreme conditions, resulting in healthy and environmentally friendly products, such as foods, feeds, probiotics, plant growth promoters, biocides, enzymes, and bioactive compounds. The objective of this review was to compile the variety of products that can be produced with Bacillus cells, using the concepts of biorefinery and circular economy as the scope to search for greener alternatives to each production method and providing market and bioeconomy ideas of global production. Although the genus is extensively used in industry, little information is available on its large-scale production, and there is little current data regarding bioeconomy and circular economy parameters for the bacteria. Therefore, as this work gathers several products' economic, production, and environmentally friendly use information, it can be addressed as one of the first steps towards those sustainable strategies. Additionally, an extensive patent search was conducted, focusing on products that contain or are produced by the Bacillus genus, providing an indication of global technology development and direction of the bacteria products. The Bacillus global market represented at least $18 billion in 2020, taking into account only the products addressed in this article, and at least 650 patent documents submitted per year since 2017, indicating this market's extreme importance. The data we provide in this article can be used as a base for further studies in bioeconomy and circular economy and show the genus is a promising candidate for a greener and more sustainable future.

Pentose-rich hydrolysate from oil palm empty fruit bunches for ß-glucan production using Pichia jadinii and Cyberlindnera jadinii.

Pentose-rich hydrolysate obtained from dilute acid pretreatment of oil palm empty fruit bunches was successfully consumed by pentose-consuming yeasts: Cyberlindnera jadinii (Cj) and Pichia jadinii (Pj). Nitrogen supplementation and no additional detoxification step were required. Pj produced 5.87 g/L of biomass using a C/N ratio of 14 after 120 h of fermentation, with xylose consumption of 71%. Cj produced 10.50 g/L of biomass after 96 h of fermentation with C/N ratio of 11.5, with maximum xylose consumption of 85%. ß-glucans, high value-added macromolecules, were further extracted from the yeast biomass, achieving yields of 3.1 and 3.0% from Pj and Cj, respectively. The isolated polysaccharides showed a chemical structure of ß-(1,3)-glucan with residues of other molecules. Additionally, ß-(1,6) branches seems to have been broken during isolation process. Further studies assessing ß-glucans production at industrial scale should be carried out looking for nitrogen sources and optimizing the ß-glucan isolation method.

Current developments and challenges of green technologies for the valorization of liquid, solid, and gaseous wastes from sugarcane ethanol production.

The sugarcane industry is one of the largest in the world and processes huge volumes of biomass, especially for ethanol and sugar production. These processes also generate several environmentally harmful solid, liquid, and gaseous wastes. Part of these wastes is reused, but with low-added value technologies, while a large unused fraction continues to impact the environment. In this review, the classic waste reuse routes are outlined, and promising green and circular technologies that can positively impact this sector are discussed. To remain competitive and reduce its environmental impact, the sugarcane industry must embrace technologies for bagasse fractionation and pyrolysis, microalgae cultivation for both CO2 recovery and vinasse treatment, CO2 chemical fixation, energy generation through the anaerobic digestion of vinasse, and genetically improved fermentation yeast strains. Considering the technological maturity, the anaerobic digestion of vinasse emerges as an important solution in the short term. However, the greatest environmental opportunity is to use the pure CO2 from fermentation. The other opportunities still require continued research to reach technological maturity. Intensifying the processes, the exploration of driving-change technologies, and the integration of wastes through biorefinery processes can lead to a more sustainable sugarcane processing industry.

Lignocellulosic biomass: Acid and alkaline pretreatments and their effects on biomass recalcitrance - Conventional processing and recent advances.

Lignocellulosic biomass is one of the most abundant organic resources worldwide and is a promising source of renewable energy and bioproducts. It basically consists of three fractions, cellulose, hemicelluloses and lignin, which confer a recalcitrant structure. As such, pretreatment steps are required to make each fraction available for further use, with acidic, alkaline and combined acidic-alkaline treatments being the most common techniques. This review focuses on recent strategies for lignocellulosic biomass pretreatment, with a critical discussion and comparison of their efficiency based on the composition of the materials. Mild pretreatments usually allow the recovery of the three biomass fractions for further transformation and valorisation. An insight is provided of newly developed technologies from recently filed patents on lignocellulosic biomass pretreatment and the transformation of agro-industrial residues into high value-added products, such as biofuels and organic acids.

Sequential chemical and enzymatic pretreatment of palm empty fruit bunches for Candida pelliculosa bioethanol production.

Second-generation bioethanol production process was developed using pretreated empty fruit bunches (EFB). Consecutive acid/alkali EFB pretreatment was performed, first with HCl and then with NaOH with final washing steps for phenolic compounds elimination. Scanning electron microscopy images showed that EFB chemical treatments indeed attacked the cellulose fibers and removed the silica from surface pores. The optimization of enzymatic hydrolysis of EFB's cellulosic fraction was performed with 0.5%-4% v/v of Cellic® CTec2/Novozymes, different EFB concentrations (5%-15%, w/v), and hydrolysis time (6-72 H). Optimization essays were carried out in Erlenmeyer flasks and also in a 1 L stirred tank reactor. After enzymatic hydrolysis, a hydrolysate with 66 g/L of glucose was achieved with 2.2% (v/v) Cellic® CTec2, 15% (m/v) acid/alkaline pretreated EFB after 39 H of hydrolysis. A gain of 11.2% was then obtained in the 1 L stirred tank promoted by the agitation (72.2 g/L glucose). The hydrolysate was employed in bioethanol production by a new isolate Candida pelliculosa CCT 7734 in a separate hydrolysis and fermentation process reaching 16.6 and 23.0 g/L of bioethanol through batch and fed-batch operation, respectively. An integrated biorefinery process was developed for EFB processing chain.

L-lysine production improvement: a review of the state of the art and patent landscape focusing on strain development and fermentation technologies.

L-lysine is an essential amino acid used in various industrial sectors but mainly in food and animal feed. Intense research has been directed toward increasing its productivity. This literature review presents the state of the art and patent landscape of the industrial production of L-lysine, with a focus on the strain development and fermentation technologies, through geographic, social, and chronological analysis, using the text mining technique. The geographic analysis showed a greater tendency for countries with industrial plants with large production capacity to submit patents or publish articles, while the social analysis reflected the close relationship between educational units and companies. The technologies of each document were divided into optimization of fermentation parameters, conventional mutation, and genetic engineering. Corynebacterium glutamicum and Escherichia coli present the most attractive industrial phenotypes, and their cultivation occurs mainly in fed-batch processes with control parameters carefully selected to enhance metabolism. These strains are generally modified by conventional approaches (e.g., mutagenesis and selection of auxotrophic and/or regulatory mutants) or by genetic engineering technologies. The combination of both these approaches enables genomic breeding and the construction of strains with industrial potential, capable of accumulating more than 120 g/L of L-lysine. From the analysis of these approaches, we developed a descriptive flow of substrate uptake, amino acid metabolism, and mechanisms of excretion of a lysine-producing model cell. It is expected that the various mechanisms of L-lysine production, here shown and described, will become a guide that aids in increasing amino acid productivity without interfering with the strain stability.

Microalgal biorefineries: Integrated use of liquid and gaseous effluents from bioethanol industry for efficient biomass production.

A new method for CO2 recovery was proposed for cultivation of different microalgae. First, a chemical fixation, where CO2 was injected in alkalinized vinasse to form (bi)carbonate salts, was performed. In addition, biological fixation with CO2-enriched air injection was also accomplished for evaluation of the most promising results. Two bioreactor systems, a stirred-tank reactor and a bubble column reactor, were employed. A higher carbon transfer rate (43.35 g.L-1.h-1) was achieved in the bubble column reactor using NaOH-alkalinized vinasse, along with reductions of the chemical oxygen demand (COD), biological oxygen demand (BOD) and turbidity (TD). This allowed the cultivation of microalgae and cyanobacteria at vinasse concentrations between 70 and 100%, reaching a biomass production of 2.25 g.L-1 in 15 days of culture. The viability of chemical CO2 fixation together with the use of 100% treated vinasse from a bioethanol production unit for microalgae cultivation has been demonstrated in a successfully integrated biorefinery approach.

Statistical Optimization of Laccase Production and Delignification of Sugarcane Bagasse by Pleurotus ostreatus in Solid-State Fermentation.

Laccases are oxidative enzymes related to the degradation of phenolic compounds, including lignin units, with concomitant reduction of oxygen to water. Delignification is a necessary pretreatment step in the process of converting plant biomass into fermentable sugars. The objective of this work was to optimize the production of laccases and to evaluate the delignification of sugarcane bagasse by Pleurotus ostreatus in solid-state fermentation. Among eight variables (pH, water activity, temperature, and concentrations of CuSO4, (NH4)2SO4, KH2PO4, asparagine, and yeast extract), copper sulfate and ammonium sulfate concentrations were demonstrated to significantly influence laccase production. The replacement of ammonium sulfate by yeast extract and the addition of ferulic acid as inducer provided increases of 5.7- and 2.0-fold, respectively, in laccase activity. Optimization of laccase production as a function of yeast extract, copper sulfate, and ferulic acid concentrations was performed by response surface methodology and optimal concentrations were 6.4 g/L, 172.6 µM, and 1.86 mM, respectively. Experimentally, the maximum laccase activity of 151.6 U/g was produced at the 5th day of solid-state fermentation. Lignin content in sugarcane bagasse was reduced from 31.89% to 26.36% after 5 days and to 20.79% after 15 days by the biological treatment of solid-state fermentation.

Callus Growth Kinetics of Physic Nut (Jatropha curcas L.) and Content of Fatty Acids from Crude Oil Obtained In Vitro.

The callus growth kinetics allows identifying the appropriate moment for callus pealing and monitoring the accumulation of primary and secondary metabolites. The physic nut (Jatropha curcas L.) is a plant species used for biofuel production due to its high oil content; however, this plant presents a great amount of bioactive compounds which can be useful for industry. The aim of this research was to establish a calli growth curve and to evaluate the fatty acid profile of crude oil extracted from callus. The callus growth kinetics presented a sigmoid standard curve with six distinct phases: lag, exponential, linear, deceleration, stationary, and decline. Total soluble sugars were higher at the inoculation day. Reducing sugars were higher at the inoculation day and at the 80th day. The highest percentage of ethereal extract (oil content) was obtained at the 120th day of culture, reaching 18 % of crude oil from the callus. The calli produced medium-chain and long-chain fatty acids (from 10 to 18 carbon atoms). The palmitic acid was the fatty acid with the highest proportion in oil (55.4 %). The lipid profile obtained in callus oil was different from the seed oil profile.

Monitoring fermentation parameters during phytase production in column-type bioreactor using a new data acquisition system.

Fermentation parameters for phytase production in column-type bioreactor were monitored using a new data acquisition system. There are a number of studies reporting phytase production in flasks, but a lack of data about microorganism respiration behaviour during phytase production using column bioreactor. The objectives of this work were the monitoration of fermentation parameters during phytase production and its relation with fungal growth and forced air. Phytase production by A. niger FS3 increased with forced air. The O(2) consumption and CO(2) production during solid-state fermentation were monitored by sensors (in the bottom and top of the columns) linked to controllers, recorded by acquisition software and processed by Fersol2(®) software tool. Phytase synthesis was associated with fungal growth. Therefore, phytase could be used to estimate FS3 biomass formed in citric pulp degradation.

A simplified model for A. Niger FS3 growth during phytase formation in solid State fermentation

A simplified model to describe fungal growth during citric pulp fermentation for phytase production was described for the first time. Experimental data for biomass growth were adjusted to classical mathematical growth models (Monod and Logistic). The Monod model predictions showed good agreement with the experimental results for biomass concentration during 96 hours of fermentation. Parameters such as yield of biomass from oxygen (Y X/O), maintenance coefficient (m) and specific growth rate (µ) were compared showing a good correlation between the data and the model. An alternative method for biomass determination in this process was developed since a great correlation was found between biomass growth and enzyme formation.

Um modelo simplificado para descrever o crescimento fúngico durante a fermentação em polpa citric para a produção da fitase foi descrita pela primeira vez. Dados experimentais para a formação de biomassa foram ajustados a modelos clássicos de crescimento microbiano (Monod e Logístico). O modelo Monod previsto mostrou boa correlação aos resultados experimentais para a concentração de biomassa até 96 horas de fermentação. Parâmetros como rendimento de biomassa a partir de oxigênio (Y X/O), coeficiente de manutenção (m) e taxa específica de crescimento (µ) foram comparados mostrando uma boa correlação entre os dados e o modelo. Um método alternativo para a determinação de biomassa neste processo foi desenvolvido a partir de uma excelente correlação encontrado entre o crescimento microbiano e a formação da enzima.