Sequential Fermentation Process Research Articles

Recombinant Production of Pseudomonas aeruginosa Rhamnolipids in P. putida KT2440 on Acetobacterium woodii Cultures Grown Chemo-Autotrophically with Carbon Dioxide and Hydrogen.

The establishment of sustainable processes for the production of commodity chemicals is one of today's central challenges for biotechnological industries. The chemo-autotrophic fixation of CO2 and the subsequent production of acetate by acetogenic bacteria via anaerobic gas fermentation represents a promising platform for the ecologically sustainable production of high-value biocommodities via sequential fermentation processes. In this study, the applicability of acetate-containing cell-free spent medium of the gas-fermenting acetogenic bacterium A. woodii WP1 as the feeder strain for growth and the recombinant production of P. aeruginosa PAO1 mono-rhamnolipids in the well-established nonpathogenic producer strain P. putida KT2440 were investigated. Additionally, the potential possibility of a simplified production process without the necessary separation of feeder strain cells was elucidated via the cultivation of P. putida in cell-containing A. woodii culture broth. For these cultures, the content of both strains was investigated by examining the relative quantification of strain-exclusive genes via qPCR. The recombinant production of mono-rhamnolipids was successfully achieved with maximum titers of approximately 360-400 mg/L for both cell-free and cell-containing A. woodii spent medium. The reported processes therefore represent a successful proof of principle for gas fermentation-derived acetate as a potential sustainable carbon source for future recombinant rhamnolipid production processes by P. putida KT2440.

Sequential Bioprocess with Gluconobacter oxydans and Candida tropicalis for Gluconic Acid and Single-Cell Protein Production from Enzymatic Hydrolysate

To meet the growing global demand for gluconic acid as a cement and concrete retarder, inexpensive and abundant lignocellulosic materials are regarded as the most suitable alternatives to starchy materials. However, their enzymatic hydrolysate contains not only glucose but also xylose, which negatively affects the performance of gluconic acid as a retarder. Notably, glucose is preferentially bio-oxidized into gluconic acid by Gluconobacter oxydans, but gluconic acid cannot be metabolized by Candida tropicalis. Given this, an artificially designed biological cascade process, respectively employing Gluconobacter oxydans and Candida tropicalis, was established to successfully carry out glucose conversion into gluconic acid, and xylose into a single-cell protein, using the enzymatic hydrolysate of corncobs as a feedstock. This sequential fermentation process produced 95.8 g/L gluconic acid and 9.0 g/L single-cell protein from one liter of the enzymatic hydrolysate that initially contained 98.1 g/L of glucose and 25.4 g/L of xylose. The mass-balance calculation showed that approximately 280 grams of gluconic acid and 27 grams of the single-cell protein could be harvested from 1000 grams of the corncob feedstock. The results suggest that the above-mentioned two-step bioconversion method is efficient in utilizing glucose and xylose from lignocellulosic hydrolysates.

Recycled Lactobacillus pentosus biomass can regenerate biosurfactants after various fermentative and extractive cycles

In this work cells of Lactobacillus pentosus, growing on 20g/L of glucose for 15h at 31°C, were subjected to sequential fermentation and extraction processes with phosphate buffered saline (PBS) in order to study the capacity of L. pentosus to regenerate biosurfactants after various fermentative and extractive cycles. After a first extraction cycle, it was observed that, in absence of nutrients, L. pentosus, dissolved in PBS, does not have the capacity to induce the formation of new biosurfactants, although the extraction process almost did not affect the cell viability. However, when this biomass was recycled and subjected to three sequential fermentative processes, in presence of fresh fermentative medium, with their respective extraction procedures, L. pentosus cells recovered its capacity to produce more biosurfactants, observing an increase in the biosurfactant/biomass ratio, achieving a total biosurfactant concentration of 2.7g/L, being the highest reported at the moment in the literature for L. pentosus growing on glucose.

Effect of a novel method for in-house cellulase production on 2G ethanol yields

Abstract Significant enzyme loadings are needed in the production of second-generation (2G) ethanol from biomass, and the cost of these enzymes remains a critical challenge for implementation of industrial plants at commercial scale. Aiming to contribute to 2G ethanol viability, its production yield was investigated in terms of the fermentability of sugarcane bagasse hydrolysates obtained using enzymatic cocktails produced in-house. For that, two different cultivation methods (sequential solid-state and submerged fermentation and conventional submerged fermentation) were compared. Sequential and submerged fermentation methods resulted in enzymatic cocktails with different compositions in terms of endoglucanase, xylanase, and β-glucosidase activities. At the same protein loading, use of the sequential fermentation cocktail resulted in 11% hydrolysis increase, relative to that achieved with the submerged fermentation cocktail. Sugarcane bagasse hydrolysis was increased by 8–19% with the addition of low loadings of in-house produced cocktails to a commercial enzyme. Furthermore, the yield of 2G ethanol production (78%) from the hydrolysate obtained using the sequential fermentation cocktail was higher than the yields achieved using the conventional submerged fermentation cocktail hydrolysate (71%) and the commercial enzyme hydrolysate (74%). The sequential fermentation method was superior to conventional submerged fermentation, resulting in a cocktail with higher enzyme activities and better performance in sugarcane bagasse hydrolysis. Moreover, this work shows the improvement on 2G ethanol production by the sequential fermentation process developed for in-house enzyme production. These findings contribute to existing knowledge of 2G ethanol production by demonstrating the effect of the enzymatic cocktails composition on the fermentability of sugarcane bagasse hydrolysates.

Secretome analysis of Trichoderma reesei and Aspergillus niger cultivated by submerged and sequential fermentation processes: Enzyme production for sugarcane bagasse hydrolysis

Cellulases and hemicellulases from Trichoderma reesei and Aspergillus niger have been shown to be powerful enzymes for biomass conversion to sugars, but the production costs are still relatively high for commercial application. The choice of an effective microbial cultivation process employed for enzyme production is important, since it may affect titers and the profile of protein secretion. We used proteomic analysis to characterize the secretome of T. reesei and A. niger cultivated in submerged and sequential fermentation processes. The information gained was key to understand differences in hydrolysis of steam exploded sugarcane bagasse for enzyme cocktails obtained from two different cultivation processes. The sequential process for cultivating A. niger gave xylanase and β-glucosidase activities 3- and 8-fold higher, respectively, than corresponding activities from the submerged process. A greater protein diversity of critical cellulolytic and hemicellulolytic enzymes were also observed through secretome analyses. These results helped to explain the 3-fold higher yield for hydrolysis of non-washed pretreated bagasse when combined T. reesei and A. niger enzyme extracts from sequential fermentation were used in place of enzymes obtained from submerged fermentation. An enzyme loading of 0.7 FPU cellulase activity/g glucan was surprisingly effective when compared to the 5-15 times more enzyme loadings commonly reported for other cellulose hydrolysis studies. Analyses showed that more than 80% consisted of proteins other than cellulases whose role is important to the hydrolysis of a lignocellulose substrate. Our work combined proteomic analyses and enzymology studies to show that sequential and submerged cultivation methods differently influence both titers and secretion profile of key enzymes required for the hydrolysis of sugarcane bagasse. The higher diversity of feruloyl esterases, xylanases and other auxiliary hemicellulolytic enzymes observed in the enzyme mixtures from the sequential fermentation could be one major reason for the more efficient enzyme hydrolysis that results when using the combined secretomes from A. niger and T. reesei.

Sequential solid-state and submerged cultivation of Aspergillus niger on sugarcane bagasse for the production of cellulase

Sequential solid-state and submerged cultivation with sugarcane bagasse as substrate for cellulase production by Aspergillus niger A12 was assessed by measuring endoglucanase activity. An unconventional pre-culture with an initial fungal growth phase under solid-state cultivation was followed by a transition to submerged fermentation by adding the liquid culture medium to the mycelium grown on solid substrate. For comparison, control experiments were conducted using conventional submerged cultivation. The cultures were carried out in shake flasks and in a 5-L bubble column bioreactor. An endoglucanase productivity of 57±13IU/L/h was achieved in bubble column cultivations prepared using the new method, representing an approximately 3-fold improvement compared to conventional submerged fermentation. Therefore, the methodology proposed here of a sequential fermentation process offers a promising alternative for cellulase production.

BIOETHANOL AND XYLITOL PRODUCTION FROM DIFFERENT LIGNOCELLULOSIC HYDROLYSATES BY SEQUENTIAL FERMENTATION

ABSTRACTSugarcane bagasse, Agave tequilana bagasse and coffee husks were hydrolyzed by steam explosion followed by enzymatic treatment without detoxifying process prior to fermentation. The hydrolysates underwent sequential fermentations using three different microorganisms to produce bioethanol and xylitol. Sugarcane bagasse hydrolysate was found to be the best media for bioethanol (0.44 g/g yield, 85.5% efficiency) and xylitol (0.29 g/g yield) production and S. cerevisiae ITV01 performed efficient alcoholic fermentation in the presence of a high acetic acid concentration (5.0 g/L, initial hydrolysate concentration). For all hydrolysates, Candida tropicalis grew less; however, it produced more xylitol than Candida magnolia (0.29 and 0.22 g/g, respectively). The effect of oxygen on xylitol production in Candida tropicalis was also investigated, xylitol production improving as oxygen concentration level increased (flask volume/medium volume ratios of 1.25, 3.3 and 6.6 yielded 0.143, 0.320 and 0.406 g/g, respectively). The maximum calculated efficiency for the conversion of consumed xylose to xylitol was 44.6%, with a maximum xylitol production of 12.18 g/L.PRACTICAL APPLICATIONSBioethanol and xylitol production by sequential fermentation process using different lignocellulosic materials like sugarcane bagasse, Agave tequilana bagasse and coffee husks, is a new simple and interesting process because it takes into account the total utilization of the principal component, the lignocellulosic material (glucose and xylose) as well as the use of waste material. One fermentation step produces two important products, bioethanol and xylitol, which have various applications in different kinds of industry.

Microbial production of D-mannitol and D-fructose from glycerol.

AbstractIn view of the recent development that some petrochemical products are efficiently available as substrates for the fermentation industry, glycerol manufactured from propylene by chemical synthesis would also be hoped for the purpose. This paper describes some of the factors influencing mannitol production from glycerol by Torulopsis yeasts and a microbial conversion of glycerol to D‐fructose via mannitol, in which two sequential steps of yeast and Acetobacter fermentation are involved. Torulopsis mannitofaciens CBS 5981 and Torulopsis vcrsatilis CBS 1752, exceptionally good mannitol producers, were selected for the study. High concentrations of nitrogen sources and KH2PO4 in the medium markedly decreased mannitol yield in spite of good utilization of the substrate. T. mannitofaciens produced mannitol in yield of 31% of the glycerol consumed at optimal condition. The fermentation by washed yeast cells gave much higher mannitol yield of more than 50%. A sequential fermentation process was carried out without isolation and purification of the intermediate and yielded.51.7%. D‐fructose from the glycerol.

Microbial production of xylitol from glucose.

A microbiological method is described for the production of xylitol, which is used as a sugar substitute for diabetics. A sequential fermentation process yielded 9.0 g of xylitol from 77.5 g of glucose via D-arabitol and D-xylulose. Candida guilliermondii var. soya (ATCC 20216) consumed 5.1 g of D-xylulose and produced 2.8 g of xylitol per 100 ml. Pentitol production from D-xylulose by yeasts was divided into three types: I, yeast-produced xylitol; II, yeast-produced D-arabitol; and III, yeast-produced xylitol and D-arabitol. D-Xylulose, but not glucose, was dissimilated to xylitol by yeasts under aerobic conditions.

Microbial Production of Xylitol from Glucose1

A microbiological method is described for the production of xylitol, which is used as a sugar substitute for diabetics. A sequential fermentation process yielded 9.0 g of xylitol from 77.5 g of glucose via D-arabitol and D-xylulose. Candida guilliermondii var. soya (ATCC 20216) consumed 5.1 g of D-xylulose and produced 2.8 g of xylitol per 100 ml. Pentitol production from D-xylulose by yeasts was divided into three types: I, yeast-produced xylitol; II, yeast-produced D-arabitol; and III, yeast-produced xylitol and D-arabitol. D-Xylulose, but not glucose, was dissimilated to xylitol by yeasts under aerobic conditions.