Modeling the influence of propionic acid concentration and pH on the kinetics of Salmonella Typhimurium.

Salmonella Typhimurium is a foodborne pathogen often found in the poultry production chain. Antibiotics have been used to reduce S. Typhimurium contamination in poultry aviaries and improve chicken growth. However, antibiotics were banned in several countries. Alternatively, organic acids, such as propionic acid (PA), can control pathogens. This study determined the PA minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and mathematically modeled S. Typhimurium growth/inactivation kinetics under the influence of PA at different pH values (4.5, 5.5, and 6.5) which are within the pH range of the chicken gastrointestinal tract. The PA MIC against S. Typhimurium was pH-dependent, resulting in 5.0, 3.5 and 9.0 mM undissociated PA at pH 4.5, 5.5, and 6.5, respectively. The Baranyi and Roberts and the Weibull model fit growth and inactivation data well, respectively. Secondary models were proposed. The validated model predicted 3-log reduction of S. Typhimurium in 3 h at 68.2 mM of undissociated PA and pH 4.5. The models presented a good capacity to describe the kinetics of S. Typhimurium subjected to PA, representing a useful tool to predict PA antibacterial action depending on the pH.

First- and second-generation integrated process for bioethanol production: Fermentation of molasses diluted with hemicellulose hydrolysate by recombinant Saccharomyces cerevisiae.

The integration of first- (1G) and second-generation (2G) ethanol production by adding sugarcane juice or molasses to lignocellulosic hydrolysates offers the possibility to overcome the problem of inhibitors (acetic acid, furfural, hydroxymethylfurfural and phenolic compounds), and add nutrients (such as salts, sugars and nitrogen sources) to the fermentation medium, allowing the production of higher ethanol titers. In this work, an 1G2G production process was developed with hemicellulosic hydrolysate (HH) from a diluted sulfuric acid pretreatment of sugarcane bagasse and sugarcane molasses. The industrial Saccharomyces cerevisiae CAT-1 was genetically modified for xylose consumption and used for co-fermentation of sucrose, fructose, glucose, and xylose. The fed-batch fermentation with high cell density that mimics an industrial fermentation was performed at bench scale fermenter, achieved high volumetric ethanol productivity of 1.59 g L-1 h-1, 0.39 g g-1 of ethanol yield, and 44.5 g L-1 ethanol titer, and shown that the yeast was able to consume all the sugars present in must simultaneously. With the results, it was possible to establish a mass balance for the global process: from pretreatment to the co-fermentation of molasses and HH, and it was possible to establish an effective integrated process (1G2G) with sugarcane molasses and HH co-fermentation employing a recombinant yeast.

Potential of Mortierellaceae for polyunsaturated fatty acids production: mini review.

The health benefits of polyunsaturated fatty acids (PUFAs) have encouraged the search for rich sources of these compounds. However, the supply chain of PUFAs from animals and plants presents environmental concerns, such as water pollution, deforestation, animal exploitation and interference in the trophic chain. In this way, a viable alternative has been found in microbial sources, mainly in single cell oil (SCO) production by yeast and filamentous fungi. Mortierellaceae is a filamentous fungal family world-renowned for PUFA-producing strains. For example, Mortierella alpina can be highlighted due to be industrially applied to produce arachidonic acid (20:4 n6), an important component of infant supplement formulas. Thus, the state of the art of strategies to increase PUFAs production by Mortierellaceae strains is presented in this review. Firstly, we have discussed main phylogenetic and biochemical characteristics of these strains for lipid production. Next, strategies based on physiological manipulation, using different carbon and nitrogen sources, temperature, pH and cultivation methods, which can increase PUFA production by optimizing process parameters are presented. Furthermore, it is possible to use metabolic engineering tools, controlling the supply of NADPH and co-factors, and directing the activity of desaturases and elongase to the target PUFA. Thus, this review aims to discuss the functionality and applicability of each of these strategies, in order to support future research for PUFA production by Mortierellaceae species.

Hydrothermal pretreatment for the production of prebiotic oligosaccharides from tobacco stem.

Tobacco stem is an abundant and inexpensive renewable source to produce prebiotics by circular economy. In this study, hydrothermal pretreatments were evaluated on the release of xylooligosaccharides (XOS) and cello-oligosaccharides (COS) from the tobacco stem by a central composite rotational design associated with response surface methodology to evaluate the effects of temperature (161.72 to 218.3 °C) and solid load (SL) (2.93 to 17.07%). XOS were the main compounds released to the liquor. Desirability function was performed to maximize the production of XOS and minimize the effects of release of monosaccharides and degradation compounds. The result indicated yield of 96% w[XOS]/w[xylan] for 190 °C-2.93% SL. The highest value for COS and total oligomers content (COS + XOS) was 6.42 g/L and 17.7 g/L, respectively, for 190 °C-17.07% SL. The mass balance for the best yield XOS condition predicted 132 kg of XOS (X2-X6) from 1000 kg of tobacco stem.

An overview on fermentation strategies to overcome lignocellulosic inhibitors in second-generation ethanol production using cell immobilization.

The development of technologies to ferment carbohydrates (mainly glucose and xylose) obtained from the hydrolysis of lignocellulosic biomass for the production of second-generation ethanol (2G ethanol) has many economic and environmental advantages. The pretreatment step of this biomass is industrially performed mainly by steam explosion with diluted sulfuric acid and generates hydrolysates that contain inhibitory compounds for the metabolism of microorganisms, harming the next step of ethanol production. The main inhibitors are: organic acids, furan, and phenolics. Several strategies can be applied to decrease the action of these compounds in microorganisms, such as cell immobilization. Based on data published in the literature, this overview will address the relevant aspects of cell immobilization for the production of 2G ethanol, aiming to evaluate this method as a strategy for protecting microorganisms against inhibitors in different modes of operation for fermentation. This is the first overview to date that shows the relation between inhibitors, cells immobilization, and fermentation operation modes for 2G ethanol. In this sense, the state of the art regarding the main inhibitors in 2G ethanol and the most applied techniques for cell immobilization, besides batch, repeated batch and continuous fermentation using immobilized cells, in addition to co-culture immobilization and co-immobilization of enzymes, are presented in this work.

Fermentation strategies to improve propionic acid production with propionibacterium ssp.: a review.

Propionic acid (PA) is a carboxylic acid applied in a variety of processes, such as food and feed preservative, and as a chemical intermediate in the production of polymers, pesticides and drugs. PA production is predominantly performed by petrochemical routes, but environmental issues are making it necessary to use sustainable processes based on renewable materials. PA production by fermentation with the Propionibacterium genus is a promising option in this scenario, due to the ability of this genus to consume a variety of renewable carbon sources with higher productivity than other native microorganisms. However, Propionibacterium fermentation processes present important challenges that must be faced to make this route competitive, such as: a high fermentation time, product inhibition and low PA final titer, which increase the cost of product recovery. This article summarizes the state of the art regarding strategies to improve PA production by fermentation with the Propionibacterium genus. Firstly, strategies associated with environmental fermentation conditions and nutrition requirements are discussed. Subsequently, advantages and disadvantages of various strategies proposed to improve process performance (high cell concentration by immobilization or recycle, co-culture fermentation, genome shuffling, evolutive and metabolic engineering, and in situ recovery) are evaluated.

Comparison of Spathaspora passalidarum and recombinant Saccharomyces cerevisiae for integration of first- and second-generation ethanol production.

First-generation ethanol (E1G) is based on the fermentation of sugars released from saccharine or starch sources, while second-generation ethanol (E2G) is focused on the fermentation of sugars released from lignocellulosic feedstocks. During the fractionation process to release sugars from hemicelluloses (mainly xylose), some inhibitor compounds are released hindering fermentation. Thus, the biggest challenge of using hemicellulosic hydrolysate is selecting strains and processes able to efficiently ferment xylose and tolerate inhibitors. With the aim of diluting inhibitors, sugarcane molasses (80% of sucrose content) can be mixed to hemicellulosic hydrolysate in an integrated E1G-E2G process. Cofermentations of xylose and sucrose were evaluated for the native xylose consumer Spathaspora passalidarum and a recombinant Saccharomyces cerevisiae strain. The industrial S. cerevisiae strain CAT-1 was modified to overexpress the XYL1, XYL2 and XKS1 genes and a mutant ([4-59Δ]HXT1) version of the low-affinity HXT1 permease, generating strain MP-C5H1. Although S. passalidarum showed better results for xylose fermentation, this yeast showed intracellular sucrose hydrolysis and low sucrose consumption in microaerobic conditions. Recombinant S. cerevisiae showed the best performance for cofermentation, and a batch strategy at high cell density in bioreactor achieved unprecedented results of ethanol yield, titer and volumetric productivity in E1G-E2G production process.

Secretome analysis as a tool to elucidate bacterial contamination influence during second-generation ethanol production in a Melle-Boinot process.

Melle-boinot fermentation process can be used to increase the ethanol productivity in second-generation ethanol process (2G). However, bacterial contamination can result in decreased ethanol production and sugars consumption. The available literature on microbial contamination in the 2G at the secretome level, microbial interactions and their impacts on ethanol production are scarce. In this context, the cultivation of Spathaspora passalidarum was studied in pure and co-culture with Lactobacillus fermentum under conditions that mimic the Melle-boinot process. Glucose consumption and ethanol production by S. passalidarum were not affected by bacterial contamination. Xylose consumption was higher in pure culture (11.54 ± 2.62, 16.23 ± 1.76 and 6.50 ± 1.68 g) than in co-culture fermentation (11.89 ± 0.38, 7.29 ± 0.49 and 5.54 ± 2.63 g) in cycle 2. The protein profile of the fermented broth was similar in pure and co-culture fermentation. The low effect of L. fermentum on fermentation and protein profile may be associated with the inhibition of the bacteria by the low nutrient fermentation broth, with centrifugation and/or with sulfuric acid washing. Thereby, considering that research on microbial contamination in the 2G fermentation process is very limited, particularly at the omics level, these findings may contribute to the lignocellulosic biomass fermentation industry.

Redox potential as a key parameter for monitoring and optimization of xylose fermentation with yeast Spathaspora passalidarum under limited-oxygen conditions.

The determination of optimum values of volumetric oxygen transfer coefficient (kLa) for Spathaspora passalidarum is an important aspect for the optimization of ethanol production from pentoses since oxygen plays a key role on yeast metabolism. By studying the fermentation of a xylose and glucose mixture, the highest ethanol volumetric productivity was achieved at a kLa of 45 h-1 (1.12 gethanol L-1 h-1), reducing the fermentation time to half when compared to other oxygen-limiting conditions that were considered optimum for other native strains, besides increasing xylose consumption rates. The high cell density fermentation showed to be a good strategy to be applied in industrial processes with S. passalidarum, enabling the complete exhaustion of a high initial substrate concentration (90 g L-1) in less than 24 h, with a final ethanol titer of 28.61 (± 0.42) g L-1. By performing a detailed investigation on oxidation-reduction potential (ORP), it was possible to conclude that the highest ethanol formation rates were registered at oxireduction potential values around - 100 mV, becoming an important parameter to be controlled when oxygen-limiting conditions are applied in industrial fermentations. The oxygen availability also affected the activity of enzyme XR and its preference for NADH or NADPH, directly affecting the activity of enzyme XDH and the redox imbalance on the xylose pathway. In addition, respirometric parameters were determined for the yeast S. passalidarum under an aerobic growth condition.

Online monitoring of the redox potential in microaerobic and anaerobic Scheffersomyces stipitis fermentations.

OBJECTIVE: A correlation among different volumetric oxygen transfer coefficients (kLa) and the oxireduction potential (ORP) in batch fermentations using Scheffersomyces stipitis was evaluated. Experiments were performed using a mixture of xylose and glucose as the substrates. RESULTS: Microaerophilic condition (kLa = 4.9 h-1) have shown to be suitable when compared to complete anaerobiosis (kLa = 0), providing an ethanol yield and a productivity after 48 h of 64.3% and 0.45 g ethanol L-1 h-1, respectively; the maximum ethanol titer obtained was 21.50 g ethanol L-1. Values of ORP varying from - 270 to - 330 mV resulted in high ethanol production from xylose by S. stipitis. CONCLUSIONS: Different ORP values were found in anaerobiosis and kLa 4.9 h-1, suggesting that for ethanol production by S. stipitis, values from - 270 to - 330 mV are favorable under the studied circumstances. In this ORP range, the greatest rates of xylose consumption and ethanol production were registered. ORP monitoring was demonstrated to be a suitable option for online control throughout the fermentation processes, which may provide a more efficient bioprocess operation with a very low O2 concentration.

Effect of contamination with Lactobacillus fermentum I2 on ethanol production by Spathaspora passalidarum.

Second-generation bioethanol is a promising source of renewable energy. In Brazilian mills, the production of ethanol from sugarcane (first generation, 1G) is a consolidated process performed by Saccharomyces cerevisiae and characterized by high substrate concentrations, high cell density, and cell recycle. The main bacterial contaminants in 1G fermentation tanks are lactic acid bacteria, especially bacteria from the Lactobacillus genus, which is associated with a decrease in ethanol yield and yeast cell viability, among other negative effects. Second-generation (2G) bioethanol production is characterized by the conversion of glucose and xylose into ethanol by genetically modified or non-Saccharomyces yeasts. Spathaspora passalidarum is a promising non-Saccharomyces yeast for 2G ethanol production due to its ability to effectively convert xylose into ethanol. The effect of bacterial contamination on the fermentation of this yeast is unknown; therefore, L. fermentum, a common bacterium found in Brazilian 1G processes, was studied in coculture with S. passalidarum in a fed-batch fermentation process similar to that used in 1G mills. Individually, L. fermentum I2 was able to simultaneously consume glucose and xylose in nutrient-rich broth (Man, Rogosa, and Sharpe (MRS + xylose) but failed to grow in a glucose- and xylose-based synthetic broth. In coculture with S. passalidarum, the bacteria remained at a concentration of 108 UFC/mL throughout cell recycling, but no flocculation was observed, and it did not affect the fermentative parameters or the cellular viability of the yeast. Under both conditions, the maximum ethanol production was 21 g L-1 with volumetric productivity ranging from 0.65 to 0.70 g L-1 h-1. S. passalidarum was thus shown to be resistant to L. fermentum I2 under the conditions studied.

Sugarcane must fed-batch fermentation by Saccharomyces cerevisiae: impact of sterilized and non-sterilized sugarcane must.

The presence of microbial contaminants is common in the sugarcane ethanol industry and can decrease process yield, reduce yeast cell viability and induce yeast cell flocculation. To evaluate the effect of microbial contamination on the fermentation process, we compared the use of sterilized and non-sterilized sugarcane must in the performance of Saccharomyces cerevisiae with similar fermentation conditions to those used in Brazilian mills. Non-sterilized sugarcane must had values of 103 and 108 CFU mL-1 of wild yeast and bacterial contamination, respectively; decreased total reducing sugar (TRS); and increased lactic and acetic acids, glycerol and ethanol concentrations during storage. During fermentation cycles with sterilized and non-sterilized sugarcane must, S. cerevisiae viability did not change, whereas ethanol yield varied from 74.1 to 80.2%, but it did not seem to be related to must microbial contamination. Ethanol productivity decreased throughout the fermentation cycles and was more pronounced in the last two fermentation cycles with non-sterilized must, but that may be related to the decrease in must TRS. High values of the ratio of total acid production per ethanol were reported at the end of the last two fermentation cycles conducted with non-sterilized must. Additionally, the values of wild yeast contamination increased from 102 to 103 CFU mL-1 and bacterial contamination increased from 104 to 106 CFU mL-1 when comparing the first and last fermentation cycles with non-sterilized must. In addition to the increase in microbial contamination and acid concentration, ethanol yield and yeast viability rates were not directly affected by the microbial contamination present in the non-sterilized sugarcane must.

Fermentation strategy for second generation ethanol production from sugarcane bagasse hydrolyzate by Spathaspora passalidarum and Scheffersomyces stipitis.

Alcoholic fermentation of released sugars in pretreatment and enzymatic hydrolysis of biomass is a central feature for second generation ethanol (E2G) production. Saccharomyces cerevisiae used industrially in the production of first generation ethanol (E1G) convert sucrose, fructose, and glucose into ethanol. However, these yeasts have no ability to ferment pentose (xylose). Therefore, the present work has focused on E2G production by Scheffersomyces stipitis and Spathaspora passalidarum. The fermentation strategy with high pitch, cell recycle, fed-batch mode, and temperature decrease for each batch were performed in a hydrolyzate obtained from a pretreatment at 130°C with NaOH solution (1.5% w/v) added with 0.15% (w/w) of anthraquinone (AQ) and followed by enzymatic hydrolysis. The process strategy has increased volumetric productivity from 0.35 to 0.38 g · L-1 · h-1 (first to third batch) for S. stipitis and from 0.38 to 0.81 g · L-1 · h-1 for S. passalidarum (first to fourth batch). Mass balance for the process proposed in this work showed the production of 177.33 kg ethanol/ton of sugar cane bagasse for S. passalidarum compared to 124.13 kg ethanol/ton of sugar cane bagasse for S. stipitis fermentation. The strategy proposed in this work can be considered as a promising strategy in the production of second generation ethanol. Biotechnol. Bioeng. 2017;114: 2211-2221. © 2017 Wiley Periodicals, Inc.

The Coptotermes gestroi aldo-keto reductase: a multipurpose enzyme for biorefinery applications.

BACKGROUND: In nature, termites can be considered as a model biological system for biofuel research based on their remarkable efficiency for lignocellulosic biomass conversion. Redox enzymes are of interest in second-generation ethanol production because they promote synergic enzymatic activity with classical hydrolases for lignocellulose saccharification and inactivate fermentation inhibitory compounds produced after lignocellulose pretreatment steps. RESULTS: In the present study, the biochemical and structural characteristics of the Coptotermes gestroi aldo-keto reductase (CgAKR-1) were comprehensively investigated. CgAKR-1 displayed major structural differences compared with others AKRs, including the differences in the amino acid composition of the substrate-binding site, providing basis for classification as a founding member of a new AKR subfamily (family AKR1 I). Immunolocalization assays with anti-CgAKR-1 antibodies resulted in strong fluorescence in the salivary gland, proventriculus, and foregut. CgAKR-1 supplementation caused a 32% reduction in phenolic aldehydes, such as furfural, which act as fermentation inhibitors of hemicellulosic hydrolysates, and improved ethanol fermentation by the xylose-fermenting yeast Scheffersomyces stipitis by 45%. We observed synergistic enzymatic interactions between CgAKR-1 and commercial cellulosic cocktail for sugarcane bagasse saccharification, with a maximum synergism degree of 2.17 for sugar release. Our data indicated that additive enzymatic activity could be mediated by reactive oxygen species because CgAKR-1 could produce hydrogen peroxide. CONCLUSION: In summary, we identified the founding member of an AKRI subfamily with a potential role in the termite digestome. CgAKR-1 was found to be a multipurpose enzyme with potential biotechnological applications. The present work provided a basis for the development and application of integrative and multipurpose enzymes in the bioethanol production chain.

Bioethanol production by recycled Scheffersomyces stipitis in sequential batch fermentations with high cell density using xylose and glucose mixture.

Here, it is shown three-step investigative procedures aiming to improve pentose-rich fermentations performance, involving a simple system for elevated mass production by Scheffersomyces stipitis (I), cellular recycle batch fermentations (CRBFs) at high cell density using two temperature strategies (fixed at 30°C; decreasing from 30 to 26°C) (II), and a short-term adaptation action seeking to acclimatize the microorganism in xylose rich-media (III). Cellular propagation provided 0.52gdrycellweightgRS(-1), resulting in an expressive value of 45.9gdrycellweightL(-1). The yeast robustness in CRBF was proven by effective ethanol production, reaching high xylose consumption (81%) and EtOH productivity (1.53gL(-1)h(-1)). Regarding the short-term adaptation, S. stipitis strengthened its robustness, as shown by a 6-fold increase in xylose reductase (XR) activity. The short fermentation time (20h for each batch) and the fermentation kinetics for ethanol production from xylose are quite promising.

Poly(3-Hydroxybutyrate) Production in Repeated fed-Batch with Cell Recycle Using a Medium with low Carbon Source Concentration.

Among approaches applied to obtain high productivity and low production costs in bioprocesses are high cell density and the use of low cost substrates. Usually low cost substrates, as waste/agroindustrial residues, have low carbon concentration, which leads to a difficulty in operating bioprocesses. Real time control of process for intracellular products is also difficult. The present study proposes a strategy of repeated fed-batch with cell recycle to attain high cell density of Cupriavidus necator and high poly(3-hydroxybutyrate) (P(3HB)) productivity, using a substrate with low carbon source concentration (90 g l(-1)). Also, the use of the oxygen uptake rate data was pointed out as an on line solution for process control, once P(3HB) is an intracellular product. The results showed that total biomass (X), residual biomass (Xr) and P(3HB) values at the end of the culture were 61.6 g l(-1), 19.3 g l(-1) and 42.4 g l(-1) respectively, equivalent to 68.8 % of P(3HB) in the cells, and P(3HB) productivity of 1.0 g l(-1) h(-1). Therefore, the strategy proposed was efficient to achieve high productivity and high polymer content from a medium with low carbon source concentration.

High-cell-density culture strategies for polyhydroxyalkanoate production: a review.

This article gives an overview of high-cell-density cultures for polyhydroxyalkanoate (PHA) production and their modes of operation for increasing productivity. High cell densities are very important in PHA production mainly because this polymer is an intracellular product accumulated in various microorganisms, so a high cellular content is needed for the polymer production. This review describes relevant results from fed-batch, repeated batch, and continuous modes of operation without and with cell recycle for the production of these polymers by microorganisms. Finally, recombinant microorganisms for PHA production, as well future directions for PHA production, are discussed.

Carbon source pulsed feeding to attain high yield and high productivity in poly(3-hydroxybutyrate) (PHB) production from soybean oil using Cupriavidus necator.

Poly(3-hydroxybutyrate) (PHB) biosynthesis from soybean oil by Cupriavidus necator was studied using a bench scale bioreactor. The highest cell concentration (83 g l(-1)) was achieved using soybean oil at 40 g l(-1) and a pulse of the same concentration. The PHB content was 81% (w/w), PHB productivity was 2.5 g l(-1) h(-1), and the calculated Y(p/s) value was 0.85 g g(-1). Growth limitation and the onset of PHB biosynthesis took place due to exhaustion of P, and probably also Cu, Ca, and Fe.