Use of pervaporation process for the recovery of aroma compounds produced by P. fermentans in sugarcane molasses.

Natural fruity aroma was produced during submerged fermentation by Pichia fermentans using sugarcane molasses as a cultivation broth. The aroma compounds were recovered from the fermentation by a pervaporation process using a polydimethylsiloxane membrane (Pervap 4060-Sulzer). Isoamyl acetate, a characteristic compound associated with fruity aromas, was the major compound produced. The pervaporation module was fed at three different temperatures to test the best conditions to recover the natural fruity aroma. The total flux (J T), partial fluxes of each component (J i), and enrichment factors (ß) were determined within the tested ranges. The process was performed at 45 °C, a feed flow of 1.5 mL/min and 0.1 kPa, for a duration of 13 h to concentrate the natural flavor. The pervaporation process can concentrate the isoamyl acetate from fermented broth from 9 to 61.8 mg/L in the first hour of pervaporation. As a single step of downstream operation, pervaporation was efficient for recovering and concentrating the natural fruity aroma. The obtained product was colorless and had a characteristic banana flavor.

Hydrogen production by dark fermentation using a new low-cost culture medium composed of corn steep liquor and cassava processing water: Process optimization and scale-up.

The use of effluents for hydrogen production through dark fermentation is promising because it results in the generation of value-added products and reduction of the effluent's organic load. A low-cost medium using agroindustrial effluents, corn steep liquor (CSL) and cassava processing wastewater (CPW) was evaluated for hydrogen production with microbial consortia (Vir and Gal). Four variables were evaluated for their impact on biohydrogen production through a Plackett Burman design. Subsequently, the significant variables were optimized using a central composite design, resulting in two mathematical models with regression coefficients R2 > 0.92. The maximum yields were validated and resulted in 107 and 83.1 mL of biohydrogen/g COD removed for Vir and Gal, respectively. The lower medium cost for biohydrogen production was 81.5 USD/m3, approximately 80% more economical than some supplemented media. Finally, the scale-up of the biohydrogen production by consortia to 5L resulted in an increase of more than 40%.

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.

Biohydrogen production in cassava processing wastewater using microbial consortia: Process optimization and kinetic analysis of the microbial community.

Biohydrogen production was evaluated using cassava processing wastewater (CPW) and two microbial consortia (Vir and Gal) from different Brazilian environments. The biohydrogen production was optimized using a Box-Behnken design (T, pH, C/N, and % v/v inoculum). Maximum yields were obtained with hydrolyzed substrate: 4.12 and 3.80 mol H2 / for Vir and Gal, respectively. Similarly, the kinetic parameters µ, k, and q were higher with hydrolyzed CPW in both consortia. The molecular analysis of the consortia through Illumina high-throughput sequencing showed the presence of bacteria from the families Porphyromonadaceae, Clostridiaceae, Ruminococcaceae, and Enterococcaceae. The relative abundance of microbial families varies as fermentation progresses. In both consortia, Clostridiaceae reached the maximum relative abundance in the media between 16 and 24 h, interval in which approximately 90% of the biohydrogen is generated.

Biological hydrogen production from palm oil mill effluent (POME) by anaerobic consortia and Clostridium beijerinckii.

Palm oil mill effluent (POME) was tested as a substrate to produce hydrogen by dark fermentation. Two microbial consortia and a pure culture of Clostridium beijerinckii (ATCC 8260) were cultured anaerobically in raw, diluted and hydrolyzed POME to compare biohydrogen production yields in all three media. Experiments were done in 15 mL Hungate tubes containing 5 mL of medium and 1 mL of inoculum. When Clostridium beijerinckii was cultivated at 30 °C in the hydrolyzed POME (P003), containing 7.5 g/L of sucrose, during 8 days of fermentation and 20 % of the inoculum, the maximum biohydrogen production yield was 4.62 LH2/Lmed. Consortium C3 also showed the best production in hydrolyzed POME while consortium C6 achieved its maximum production in raw POME. This effluent is a potential substrate for biohydrogen production.

Microalgal biomass pretreatment for integrated processing into biofuels, food, and feed.

Microalgae are sources of nutritional products and biofuels. However, their economical processing is challenging, because of (i) the inherently low concentration of biomass in algal cultures, below 0.5%, (ii) the high-water content in the harvested biomass, above 70%; and (iii) the variable intracellular content and composition. Cell wall structure and strength vary enormously among microalgae, from naked Dunaliella cells to robust Haematococcus cysts. High-value products justify using fast and energy-intensive processes, ranging from 0.23 kWh/kg dry biomass in high-pressure homogenization, to 6 kWh/kg dry biomass in sonication. However, in biofuels production, the energy input must be minimized, requiring slower, thermal or chemical pretreatments. Whichever the primary fraction of interest, the spent biomass can be processed into valuable by-products. This review discusses microalgal cell structure and composition, how it affects pretreatment, focusing on technologies tested for large scale or promising for industrial processes, and how these can be integrated into algal biorefineries.

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.

Biotransformation of limonene by an endophytic fungus using synthetic and orange residue-based media.

Aroma and fragrances have high commercial value for use in food, cosmetics and perfumes. The biotransformation of terpenes by microorganisms represents an attractive alternative method for production of flavourings. Endophytic fungi offer a great potential for the production of several groups of compounds; however, few studies have evaluated the biotransformation of limonene. Following preliminary studies on the biotransformation of limonene, submerged fermentation was carried out using an endophytic fungus isolated from Pinus taeda and identified as Phomopsis sp. The presence of several biotransformation products was detected and identified by mass spectrometry (GC-MS). The studied strain showed a divergent metabolic behaviour, as compounds of interest such as α-terpineol, carvone, and limoneno-1,2-diol were produced under different conditions. In addition to the minor metabolites terpinen-4-ol, menthol and carveol, this strain also produced major metabolites, including 0.536 g L-1 carvone and 2.08 g L-1 limonene-1,2-diol in synthetic medium and 2.10 g L-1 limonene-1,2-diol in a natural orange extract medium with single fed-batch, while the cyclic fed-batch resulted in concentrations less than 1 g L-1. Therefore, our study produced a wide variety of limonene derivatives at a high concentration using a natural medium and a newly isolated endophytic fungal strain.

Pilot scale biodiesel production from microbial oil of Rhodosporidium toruloides DEBB 5533 using sugarcane juice: Performance in diesel engine and preliminary economic study.

A successful pilot-scale process for biodiesel production from microbial oil (Biooil) produced by Rhodosporidium toruloides DEBB 5533 is presented. Using fed-batch strategy (1000L working volume), a lipid productivity of 0.44g/L.h was obtained using a low-cost medium composed by sugarcane juice and urea. The microbial oil was used for biodiesel production and its performance was evaluated in diesel engine tests, showing very good performance, especially for the blend B20 SCO, when operating at 2500rpm with lower pollutant emissions (CO2 - 220% less; CO - 7-fold less; NOX 50% less and no detectable HC emissions (<0.11ppm)) when compared with the blends of standard biofuel from soybean oil. A preliminary analysis showed that microbial biodiesel is economically competitive (US$ 0.76/L) when compared to the vegetable biodiesel (US$ 0.81/L). Besides, the yield of biodiesel from microbial oil is higher (4172L/ha of cultivated sugarcane) that represents 6.3-fold the yield of standard biodiesel (661L/ha of cultivated soybean).

Conducting starter culture-controlled fermentations of coffee beans during on-farm wet processing: Growth, metabolic analyses and sensorial effects.

In this study, the potential use of Pichia fermentans YC5.2 as a starter culture to conduct controlled coffee bean fermentations during on-farm wet processing was investigated. Inoculated fermentations were conducted with or without the addition of 2% (w/v) sucrose, and the resultant microbial growth and metabolism, bean chemistry and beverage quality were compared with spontaneous (control) fermentation. In both inoculated treatments, P. fermentans prevailed over indigenous microbiota and a restricted microbial composition was observed at the end of fermentation process. The inoculation also increased the production of specific volatile aroma compounds (e.g., ethanol, acetaldehyde, ethyl acetate and isoamyl acetate) and decreased the production of lactic acid during the fermentation process. Sucrose supplementation did not significantly interfere with the growth and frequency of P. fermentans YC5.2 inoculum but maintained high levels of wild bacteria population and lactic acid production similar to the spontaneous process. In roasted beans, the content of sugars and organic acids were statistically (p<0.05) similar for all the treatments. However, the inoculated fermentations were shown to influence the volatile fraction of roasted coffee beans by increasing the concentration of yeast-derived metabolites compared to control. Sensory analysis of coffee beverages demonstrated that the use of the YC5.2 strain was favorable for the production of high-quality coffees with distinctive characteristics, e.g., intense perception of 'vanilla' taste and 'floral' aromas. In conclusion, the use of P. fermentans YC5.2 in coffee processing was shown to be a viable alternative to control the fermentation step and to ensure consistent quality of finished products.

Isolation, selection and evaluation of yeasts for use in fermentation of coffee beans by the wet process.

During wet processing of coffee, the ripe cherries are pulped, then fermented and dried. This study reports an experimental approach for target identification and selection of indigenous coffee yeasts and their potential use as starter cultures during the fermentation step of wet processing. A total of 144 yeast isolates originating from spontaneously fermenting coffee beans were identified by molecular approaches and screened for their capacity to grow under coffee-associated stress conditions. According to ITS-rRNA gene sequencing, Pichia fermentans and Pichia kluyveri were the most frequent isolates, followed by Candida Candida glabrata, quercitrusa, Saccharomyces sp., Pichia guilliermondii, Pichia caribbica and Hanseniaspora opuntiae. Nine stress-tolerant yeast strains were evaluated for their ability to produce aromatic compounds in a coffee pulp simulation medium and for their pectinolytic activity. P. fermentans YC5.2 produced the highest concentrations of flavor-active ester compounds (viz., ethyl acetate and isoamyl acetate), while Saccharomyces sp. YC9.15 was the best pectinase-producing strain. The potential impact of these selected yeast strains to promote flavor development in coffee beverages was investigated for inoculating coffee beans during wet fermentation trials at laboratory scale. Inoculation of a single culture of P. fermentans YC5.2 and co-culture of P. fermentans YC5.2 and Saccharomyces sp. YC9.15 enhanced significantly the formation of volatile aroma compounds during the fermentation process compared to un-inoculated control. The sensory analysis indicated that the flavor of coffee beverages was influenced by the starter cultures, being rated as having the higher sensory scores for fruity, buttery and fermented aroma. This demonstrates a complementary role of yeasts associated with coffee quality through the synthesis of yeast-specific volatile constituents. The yeast strains P. fermentans YC5.2 and Saccharomyces sp. YC9.15 have a great potential for use as starter cultures in wet processing of coffee and may possibly help to control and standardize the fermentation process and produce coffee beverages with novel and desirable flavor profiles.

Microscale direct transesterification of microbial biomass with ethanol for screening of microorganisms by its fatty acid content

Abstract We present an improved method of direct transesterification suitable for the quantitative analysis of multiple dry samples for its fatty acid content, using a minimal amount of biomass and reactants. The method features an acid-catalyzed direct alcoholysis of microgram samples of dry biomass; the rationale behind the solvent and reagent proportions chosen is discussed. The method was validated using seven microbial strains with diverse lipid content (Saccharomyces cerevisiae, Saccharomyces boulardii, Candida tropicalis, Haematococcus pluvialis, Chlorella vulgaris, Spirulina platensis and Schizochytrium limacinum), and compared with a macroscale direct transesterification method, and with gravimetric analysis of lipids extracted with solvents. The microscale method showed a conversion of 98.06 ± 0.87% of the lipids, using approximately 3 mg of dry biomass, 1mL of 0.2M H2SO4 dissolved in anhydrous ethanol (the acid is the catalyzer and ethanol the reactant)). The mixture was maintained at 70 °C for 20 h with periodic mixing, and then extracted with 2mL n-heptane and analyzed by GC-FID. The lipid content was then calculated considering dilution and sample mass. This method is effective, reliable, and technically attractive for analytical and comparative purposes.

Bioethanol from lignocelluloses: Status and perspectives in Brazil.

The National Alcohol Program--PróAlcool, created by the government of Brazil in 1975 resulted less dependency on fossil fuels. The addition of 25% ethanol to gasoline reduced the import of 550 million barrels oil and also reduced the emission CO(2) by 110 million tons. Today, 44% of the Brazilian energy matrix is renewable and 13.5% is derived from sugarcane. Brazil has a land area of 851 million hectares, of which 54% are preserved, including the Amazon forest (350 million hectares). From the land available for agriculture (340 million hectares), only 0.9% is occupied by sugarcane as energy crop, showing a great expansion potential. Studies have shown that in the coming years, ethanol yield per hectare of sugarcane, which presently is 6000 L/ha, could reach 10,000 L/ha, if 50% of the produced bagasse would be converted to ethanol. This article describes the efforts of different Brazilian institutions and research groups on second generation bioethanol production, especially from sugarcane bagasse.

Isolation and screening of microorganisms with potential for biotransformation of terpenic substrates

The objective of the present work was to isolate and select strains with potential to perform the biotransformation of terpenic substrates. Microorganisms obtained from a collection culture and also isolated from a natural source of terpene substrate were tested. Seventeen strains were selected by their resistance to terpenes in potato dextrose agar containing up to 1 percent of limonene or α-pinene and β-pinene (1:1). Subsequently, 10 strains were selected by their capacity of using these terpenes as sole carbon source in a mineral medium. The biotransformation capacity of these strains was tested and the products obtained were identified by GC-MS.

Lab-Scale production of Bacillus atrophaeus' spores by solid state fermentation in fifferent types of bioreactors

Studies were conducted to evaluate Bacillus atrophaeus spores' production by solid-state fermentation (SSF) using sugarcane bagasse as support and soybean molasses as substrate at lab-scale in column bioreactors (forced aeration), plastic bags and Erlenmeyer flasks (aeration by diffusion). Different moisture contents (84 percent, 86 percent and 88 percent; 89 percent, 91 percent and 93 percent) and aeration rates (30mL/min, 45mL/min, 60mL/min and 90mL/min) were studied. The best condition for spore production (3.3x10(10) CFU.g-¹dry matter) in column bioreactor was 80 percent of initial humidity and no aeration. In Erlenmeyer flasks and plastic bags the best sporulation production reached 1.7 up to 4.7x10(10) CFU.g-1dry matter with 88-93 percent of initial moisture. The aeration rate had no significant effect on the spore yield. The initial moisture had a significant effect depending on the bioreactor type. Sporulation kinetic's assay was carried out and it showed the possibility to reduce the time of spore formation in two days.

Estudos foram conduzidos para avaliar a produção de esporos de Bacillus atrophaeus, em escala laboratorial, por fermentação em estado sólido (FES) em biorreatores de coluna (aeração forçada), sacos plásticos e frascos tipo Erlenmeyer (aeração por difusão), usando bagaço de cana como suporte e melaço de soja como substrato. Diferentes teores de umidade (84 por cento, 86 por cento e 88 por cento, 89 por cento, 91 por cento e 93 por cento) e taxas de aeração (30mL/min, 45mL/min, 60mL/min e 90mL/min) foram estudados. A melhor condição para a produção de esporos no biorreator de coluna (3.3 x 10(10) CFU.g-1 matéria seca) foi 80 por cento de umidade inicial, sem aeração. Em frascos Erlenmeyer e sacos de plástico a melhor esporulação foi na faixa de 1.7 a 4.7 x 10(10) CFU.g-1 matéria seca, com 88-93 por cento de umidade inicial. A taxa de aeração não teve efeito significativo sobre o rendimento da esporulação. A umidade inicial apresentou efeito significativo relacionado ao tipo do biorreator. O estudo da cinética da esporulação demonstrou a possibilidade de reduzir o tempo de incubação para esporulação em dois dias.

Coffee residues as substrates for aroma production by Ceratocystis fimbriata in solid state fermentation

Neste trabalho duas diferentes cepas de Ceratocystis fimbriata foram testadas para a producão de aromas frutais em fermentacão no estado sólido (FES) utilizando como substratos casca e polpa de café, suplementados com glicose. Os experimentos foram realizados em frascos Erlenmeyer de 250 mL. As condicões experimentais foram: umidade inicial de 70%, adicão de 20% de glicose e pH 6,0. Os frascos foram cobertos com gaze e a aeracão ocorreu por difusão passiva. A análise do headspace da cultura foi feita por cromatografia gasosa e 12 compostos foram detectados utilizando a casca de café. A análise respirométrica foi realizada para o acompanhamento do crescimento do microrganismo pela determinacão do dióxido de carbono produzido. A producão de ésteres caracterizou o aroma frutal da cultura. A concentracão máxima de voláteis totais foi alcancada após 72 h de cultivo em casca de café (28 µmol.L-1.g-1). Os principais compostos produzidos foram acetato de etila, etanol e acetaldeído, representando 84,7%, 7,6% and 2,0% dos voláteis totais, respectivamente.