Laboratory Fermenter Research Articles

Measuring process control automation benefits through a human‐machine function allocation analysis

AbstractOften technology is introduced into manufacturing and process control environments to partially automate tasks too complex to fully automate. However, it is not always clear how to measure the benefits of such projects. More often than not, automation eliminates but also shifts human work, and broad productivity measures can fail to capture such changes. To this end, this effort describes a manufacturing case study that looks at human‐machine allocation metrics involving a laboratory fermentation unit upgrade. Using a workflow monitoring and function allocation analytic approach, it was determined that upgrading an older microbial culture bioreactor (fermentor) to a new design with intelligent monitoring capabilities resulted in an approximate 17% reduction in dedicated human supervision. This workload reduction allowed scientists to spend less time on repetitive tasks and more time concentrating on other, more open‐ended problems that require more expertize. However, the new technology increased human efforts across other functions, suggesting potential mitigation paths for future technology development. This effort illustrates that the impact of new technology on human‐machine tasking can be quantified through a function allocation analysis and also provide diagnostic information, both of which are critical in understanding any overall added benefit of intelligent systems.

Microbiologic assessment of accelerated solid-state fermentation of agricultural organic wastes

Livestock and poultry wastes, when effectively managed, become feedstock for organic fertiliser production. Researchers from the All-Russian Research Institute of Reclaimed Lands, the branch of Federal Research Center “V.V. Dokuchaev Soil Science Institute”, proposed an accelerated regimen of cattle manure solid-phase fermentation with peat: 48 h at 37 °C, then 48 h at 60 °C and 24 h at 37 °C, terminating with nat-ural cooling of the fermented mass. A distinctive feature of the proposed accelerated fermentation is maintenance of set-point temperatures. The aim of the work is to perform a microbiological evaluation of the process of accelerated solid-phase fermentation. An experiment was carried out in a 1.75 dm3 laboratory fermenter. During the fermentation, we studied the number of microorganisms, which use organic and mineral nitrogen forms, using the limiting dilution method, as well as the species membership by mass spectrometry. The experimental findings showed that the temperature regime of the main fermentation steps yielded the maximum number of mesophilic and thermophilic nitrogen-transforming microorganisms. Their active growth caused the intensive transformation of the fermented mixture, as evidenced by mesophilic and thermophilic mineralisation coefficients. At the end of the process, the linear mineralisation coefficients were used to assess the completion of the fermentation product transformation and stabilisation. The fermentation product comprised a high number of nitrogen-transforming microorganisms (on average, 3.5±0.3•108 COE/g on a dry weight basis). The determination of the microbiota species membership in the fermented mass and the final product confirmed that the process temperature regime ensured the elimination of the sanitary-indicatory microorganisms present in the original mixture (E. coli, Citrobacter, Proteus). In addition, during pasteurisation, this regime led to the active development of non-pathogenic Bacillus bacteria (B. megaterium, B. subtilis, B. licheniformic, B. pumilus and B. altitudinis). The fermentation product is recommended for use as an environmentally safe organic fertiliser based on the microbiological evaluation.

Streptomyces griseus KJ623766: A Natural Producer of Two Anthracycline Cytotoxic Metabolites β- and γ-Rhodomycinone.

Background: This study aimed to produce, purify, structurally elucidate, and explore the biological activities of metabolites produced by Streptomyces (S.) griseus isolate KJ623766, a recovered soil bacterium previously screened in our lab that showed promising cytotoxic activities against various cancer cell lines. Methods: Production of cytotoxic metabolites from S. griseus isolate KJ623766 was carried out in a 14L laboratory fermenter under specified optimum conditions. Using a 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium-bromide assay, the cytotoxic activity of the ethyl acetate extract against Caco2 and Hela cancer cell lines was determined. Bioassay-guided fractionation of the ethyl acetate extract using different chromatographic techniques was used for cytotoxic metabolite purification. Chemical structures of the purified metabolites were identified using mass, 1D, and 2D NMR spectroscopic analysis. Results: Bioassay-guided fractionation of the ethyl acetate extract led to the purification of two cytotoxic metabolites, R1 and R2, of reproducible amounts of 5 and 1.5 mg/L, respectively. The structures of R1 and R2 metabolites were identified as β- and γ-rhodomycinone with CD50 of 6.3, 9.45, 64.8 and 9.11, 9.35, 67.3 µg/mL against Caco2, Hela and Vero cell lines, respectively. Values were comparable to those of the positive control doxorubicin. Conclusions: This is the first report about the production of β- and γ-rhodomycinone, two important scaffolds for synthesis of anticancer drugs, from S. griseus.

Development of a strategy induction AOX1 promoter methylotrophic yeast Komagataella phaffii

Currently, there is a significant increase in interest in the industrial production of enzyme preparations (and other recombinant proteins) using various microorganisms, including methylotrophic yeasts such as Komagataella phaffii. At the same time, the most significant productivity of the target proteins is achieved by methanol induction of heterologous genes cloned under the control of the AOX1 promoter. Thus, the efficiency of biosynthesis is largely determined by the metabolism of methanol. In this connection, the aim of the work is to develop an optimal strategy for methanol induction of the AOX1 promoter of Komagataella phaffii. The object of the study is the culture of the recombinant phospholipase A2 producing strain Komagataella phaffii. The studies were carried out in a laboratory fermenter Infors Minifors (Switzerland) on a liquid nutrient medium BSM (Basal Salt Medium) We used the generally accepted methods of studying the characteristics of metabolic activity, including the calculation of specific characteristics and productivity of the strain. The result of the study is the determination of the specific rate of consumption of methanol used as a carbon source, which was 19.2±1.8 mg/g*h. Also, the specific growth rate of Komagataella phaffii was determined and amounted to 0.24 h-1.Based on the data obtained during the research, a strategy for the induction of the AOX1 promoter in the cultivation of the methylotrophic yeast Komagataella phaffii was developed by maintaining the methanol concentration in the range of 0.6 to 2% based on the concentration of dissolved oxygen in the medium. The developed strategy of induction of the AOX1 promoter made it possible to obtain at least 1.87 g / l of recombinant protein (phospholipase A2) during cultivation of Komagataella phaffii for 96 h, which is 3.7 times higher than the known results.

Understanding the differences in 2G ethanol fermentative scales through omics data integration.

In this work, we evaluated the fermentative performance and metabolism modifications of a second generation (2G) industrial yeast by comparing an industrial condition during laboratory and industrial scale fermentations. Fermentations were done using industrial lignocellulosic hydrolysate and a synthetic medium containing inhibitors and analyses were carried out through transcriptomics and proteomics of these experimental conditions. We found that fermentation profiles were very similar, but there was an increase in xylose consumption rate during fermentations using synthetic medium when compared to lignocellulosic hydrolysate, likely due to the presence of unknown growth inhibitors contained in the hydrolysate. We also evaluated the bacterial community composition of the industrial fermentation setting and found that the presence of homofermentative and heterofermentative bacteria did not significantly change the performance of yeast fermentation. In parallel, temporal differentially expressed genes (tDEG) showed differences in gene expression profiles between compared conditions, including heat shocks and the presence of up-regulated genes from the TCA cycle during anaerobic xylose fermentation. Thus, we indicate HMF as a possible electron acceptor in this rapid respiratory process performed by yeast, in addition to demonstrating the importance of culture medium for the performance of yeast within industrial fermentation processes, highlighting the uniquenesses according to scales.

Saccharomyces cerevisiae Gene Expression during Fermentation of Pinot Noir Wines at an Industrially Relevant Scale.

Saccharomyces cerevisiae metabolism produces ethanol and other compounds during the fermentation of grape must into wine. Thousands of genes change expression over the course of a wine fermentation, allowing S. cerevisiae to adapt to and dominate the fermentation environment. Investigations into these gene expression patterns previously revealed genes that underlie cellular adaptation to the grape must and wine environments, involving metabolic specialization and ethanol tolerance. However, the majority of studies detailing gene expression patterns have occurred in controlled environments that may not recapitulate the biological and chemical complexity of fermentations performed at production scale. Here, an analysis of the S. cerevisiae RC212 gene expression program is presented, drawing from 40 pilot-scale fermentations (150 liters) using Pinot noir grapes from 10 California vineyards across two vintages. A core gene expression program was observed across all fermentations irrespective of vintage, similar to that of laboratory fermentations, in addition to novel gene expression patterns likely related to the presence of non-Saccharomyces microorganisms and oxygen availability during fermentation. These gene expression patterns, both common and diverse, provide insight into Saccharomyces cerevisiae biology critical to fermentation outcomes under industry-relevant conditions.IMPORTANCE This study characterized Saccharomyces cerevisiae RC212 gene expression during Pinot noir fermentation at pilot scale (150 liters) using industry-relevant conditions. The reported gene expression patterns of RC212 are generally similar to those observed under laboratory fermentation conditions but also contain gene expression signatures related to yeast-environment interactions found in a production setting (e.g., the presence of non-Saccharomyces microorganisms). Key genes and pathways highlighted by this work remain undercharacterized, indicating the need for further research to understand the roles of these genes and their impact on industrial wine fermentation outcomes.

Dynamics of growth of microbial cell titer of Bradyrhizobium japonicum and Sinorhizobium fredii strains during submerged cultivation in a laboratory fermenter

Dynamics of growth of microbial cell titer of Bradyrhizobium japonicum and Sinorhizobium fredii strains during submerged cultivation in a laboratory fermenter

Вплив подрібнення соломи на інтенсивність виходу біогазу

Straw has a high energy capacity, but it is not ideal for the operation of biogas plants, since it contains a large amount of nitrogen-free substances, and only 4-6% nitrogen and mineral salts, but if it is properly pretreated, it is possible to reduce the fermentation time and increase the biogas yield. The positive results of pretreatment of biological raw materials are a high degree of grinding and homogenization of raw materials; reduction of the biomass fermentation period; the release of natural enzymes that are biological catalysts for the fermentation of biomass; stabilization of biological processes, which leads to the absence of foam and floating crust in the upper part of the bioreactor; the percentage of methane in biogas increases to 70-75 %. The aim of the study is to determine the intensity of methane fermentation of crushed buckwheat, rapeseed, soybean straw and corn stalks, depending on the size of crushed particles and the method of grinding. The article discusses the issue of determining the dynamics of biogas output depending on the method and geometric dimensions of grinding buckwheat straw, soybeans, rapeseed and corn chaff in a mesophilic mode of fermentation. The following devices were used to prepare the straw: shears, hammer mill, feed chopper and extruder. Additionally, soaking was carried out in water at a certain temperature. The prepared biomass together with the seed in a ratio of 1: 4 was put into laboratory fermenter bags and a thermostat. The biogas yield was measured for 42 days using special methods, which are patented in Ukraine. Based on the measurement results, graphs were built that characterize the dynamics of biogas output.

Cloning and expression of gellan gum biosynthetic genes gelQ, gelB, gelL and gel K of Sphingomonas paucimobilis, production and characterization of the recombinant gellan gum

Abstract Gellan gum, a linear exopolysaccharide produced by Sphingomonas paucimobilis, is one of the most multifunctional polymers used in food, cosmetics and pharmaceutical industries. In the present study, for the first time, the gellan gum biosynthetic genes gel Q, gel B, gel L and gel K of Sphingomonas paucimobilis were cloned and successfully expressed in the same bacterium using the vector, pBBR122. The recombinant proteins were purified using Ni-NTA column chromatography and the molecular weight was predicted using SDS-PAGE. The biomass and gellan gum production of the strains harboring the expressed genes were analyzed in a laboratory fermentor and the production was higher with recombinant gel Q gene (Biomass: 7.03 ± 0.1 g/l; Gellan gum: 11.71 ± 0.2 g/l) compared to recombinant strains expressed with gel B, gel K and gel L genes. The optimum gellan gum production was obtained at 30 °C, pH 6.5, 400 rpm agitation speed, 90% Dissolved oxygen level and carbon-nitrogen ratio 3. The viscosity of polymer decreased with an increased shear rate which substantiates the use of this exopolysaccharide as a better gelling and thickening agent. The glucose: glucuronic acid: rhamnose ratio of the recombinant polymer was around 2:1:1 which is similar to the structural property of its native gellan. Different shapes like disc, membrane, fibres, particles and beads of gellan were made which may have wide applications in tissue engineering and drug delivery. The water holding capacity of the gellan gum gel was maximum in 100 mM calcium and minimum in gel with 5 mM Ca2+at 60 min. Compression test results illustrated that there was no significant change in the textural property of the recombinant gellan for different storage intervals compared to native gellan.

Modulation of the bacterial population in commercial cucumber fermentations by brining salt type.

Differences in the bacterial population of cucumber fermentations brined with no salt, 100mmoll-1 (1·1%) calcium chloride (CaCl2 ) or 1·03moll-1 (6%) sodium chloride (NaCl) were studied. Changes in the microbiology and chemistry of commercial and laboratory scale cucumber fermentations occurring as a function of time were monitored using colony counts and metagenetic analysis, and a pH probe and high-performance liquid chromatography analysis respectively. Dissolved oxygen and carbon dioxide content were monitored in commercial fermentations. Fermentations brined with calcium chloride (CaCl2 ) or no salt sustained faster microbial growth and reduction in pH than those brined with 1·03moll-1 NaCl. Leuconostoc, Lactococcus and Weissella dominated in fermentations brined with no salt or 100mmoll-1 CaCl2 on day 1 as compared to Weissella and enterobacteria in fermentations containing 1·03moll-1 NaCl. Lactobacilli dominated all fermentations by the third day, regardless of salt type, and was followed, in relative abundance by Pediococcus, Leuconostoc, Lactococcus and Weissella. From 84 to 96% of the population was composed of Lactobacillus by day 7 of the fermentations, except in the no salt fermentations in which a mixed population of LAB remained. The population of LAB found in commercial cucumber fermentations brined with 100mmoll-1 CaCl2 (n=18) or 1·03moll-1 NaCl (n=9) mimicked that of laboratory fermentations. A declining population of aerobes was detected in commercial fermentations brined with CaCl2 on day 1. A reduced NaCl content in cucumber fermentation enhances microbial diversity. This study fills a knowledge gap and aids in the design of improved reduced NaCl cucumber fermentations.

Modern Biogas Generation Method Based on Ultrasonic and Anaerobic Fermentation of Municipal Wastewater Biological Sludge

Classical biogas generation during the municipal wastewater treatment flow is based on anaerobically fermentation of biological sludge.The efficiency of organic matter conversion into higher biogas volume depends on organic load (COD/BOD) of active biological sludge, especially in soluble form. Soluble COD amount can be rise by non-conventional pretreatment methods as ultrasonic application. Ultrasonic pretreatment of biological sludge increases the amount of biogas from anaerobic digestion phase comparing with traditional process. This paper shows the influence of ultrasonic field on biological sludge fermentation. Ultrasonic treatment step was performed in a field, in a medium size wastewater treatment plant, at industrial pilot scale level using biological sludge, before anaerobic fermentation phase. Laboratory fermentation tests have proved that ultrasonic pretreatment lead to biogas higher production with 84%. Keywords: ultrasonic, wastewater treatment plant, biological sludge, anaerobic, biogas

A Co-Culturing Approach Enables Discovery and Biosynthesis of a Bioactive Indole Alkaloid Metabolite.

Whole-genome sequence data of the genus Streptomyces have shown a far greater chemical diversity of metabolites than what have been discovered under typical laboratory fermentation conditions. In our previous natural product discovery efforts on Streptomyces sp. MA37, a bacterium isolated from the rhizosphere soil sample in Legon, Ghana, we discovered a handful of specialised metabolites from this talented strain. However, analysis of the draft genome of MA37 suggested that most of the encoded biosynthetic gene clusters (BGCs) remained cryptic or silent, and only a small fraction of BGCs for the production of specialised metabolites were expressed when cultured in our laboratory conditions. In order to induce the expression of the seemingly silent BGCs, we have carried out a co-culture experiment by growing the MA37 strain with the Gram-negative bacterium Pseudomonas sp. in a co-culture chamber that allows co-fermentation of two microorganisms with no direct contact but allows exchange of nutrients, metabolites, and other chemical cues. This co-culture approach led to the upregulation of several metabolites that were not previously observed in the monocultures of each strain. Moreover, the co-culture induced the expression of the cryptic indole alkaloid BGC in MA37 and led to the characterization of the known indolocarbazole alkaloid, BE-13793C 1. Neither bacterium produced compound 1 when cultured alone. The structure of 1 was elucidated by Nuclear Magnetic Resonance (NMR), mass spectrometry analyses and comparison of experimental with literature data. A putative biosynthetic pathway of 1 was proposed. Furthermore, BE-13793C 1 showed strong anti-proliferative activity against HT-29 (ATCC HTB-38) cells but no toxic effect to normal lung (ATCC CCL-171) cells. To the best of our knowledge, this is the first report for the activity of 1 against HT-29. No significant antimicrobial and anti-trypanosomal activities for 1 were observed. This research provides a solid foundation for the fact that a co-culture approach paves the way for increasing the chemical diversity of strain MA37. Further characterization of other upregulated metabolites in this strain is currently ongoing in our laboratory.

Residual brewing yeasts as a source of beta-glucans

Residual brewing yeast is one of the main solid wastes in brewing. Using residual brewing biomass as a source of biologically active substances is an important way of recycling these brewing by-products. According to the literature S. cerevisiae is considered as the promising source of polysaccharides, particularly beta-glucans. Beta-glucans are structural polysaccharides of the yeast cell and perform immune stimulating properties. At the same time, there is too little information about the content of these polysaccharides in brewing yeast of the genus Brettanomyces. The objects of this study were yeast cultures of Saccharomyces cerevisiae and Brettanomyces bruxellensis. In this work, the cultivations of the yeasts were carried out to compare them as possible sources of beta-glucans. The yeasts were cultivated in a simple periodic culture using a laboratory fermenter (Biostat A, Sartorius). As a result, the content of beta-glucans in the yeasts S. cerevisiae and B. bruxellensis biomass was measured by enzymatic method (Megazyme, Ireland). According to the obtained data, the yeast B. bruxellensis contains a higher amount of beta-glucans than the yeast S. cerevisiae.

Optimisation of yeast vitality measurement to better predict fermentation performance

Brewers are increasingly aware of the importance of yeast health and need appropriate methods to measure its vitality. Many of the current methods are time consuming, expensive or impractical at an industrial scale. Here, the measurement of CO2 production using the ANKOM RF Gas Production Measurement System was optimised. Using response surface methodology, the optimal temperature, yeast biomass and media composition were established. The performance of the method was assessed by measuring the vitality of yeast from laboratory (20 L) and brewery (400-600 hL) fermentations. The drop in pH in early fermentation, diacetyl reduction and fermentation rate were correlated to measurement of yeast vitality. It was shown that the method can be used to assess yeast health and to predict its performance in brewery fermentation. The measurement time was 20 minutes and a minimum vitality of approximately 200 mbar was found to be sufficient for brewery fermentations. The method can be used to optimise yeast propagation processes or to determine the optimal harvesting/cropping time to improve yeast quality, fermentation consistency and beer quality together with reducing cycle time and time in fermenter. © 2020 The Institute of Brewing & Distilling

Modeling Yeast in Suspension during Laboratory and Commercial Fermentations to Detect Aberrant Fermentation Processes

Understanding yeast dynamics during fermentation is important for quality control, whether monitoring fermentation consistency or identifying aberrant events, such as premature yeast flocculation (PYF). Previous models of fermentation dynamics tend to be parameter rich and require large time series, which are rare in industry. This research investigates five simpler models to 1) describe fermentation dynamics, 2) refine quality control sampling regimes to improve model fit, and 3) identify PYF fermentations. The ability of these models to describe yeast dynamics was evaluated using model fitting with time series data and Akaike Information Criterion (AIC) model selection. Data simulated from large time series was used with this model fitting approach to improve sampling schedules without increasing sampling effort. Lastly, PYF was identified in fermentations of fungal-contaminated malt using linear discriminant analysis (LDA). For large data sets, a four-parameter extension of the normal curve performed best while smaller data sets were better described by the 2-parameter gamma model. Moving sampling effort nearer the population peak improved model fits. Lastly, all models detected PYF, however the two-parameter gamma model provided a simple metric for distinguishing PYF. This research provides guidelines on appropriate model use, improving sampling regimes, and identifying PYF.

PSXII-41 Evaluation of grain sorghum hybrids reveals potential for improving ruminal fermentation

Abstract In vitro incubations were used to compare fermentation characteristics of corn (uncharacterized hybrid) to 25 sorghum parental lines and hybrids (Clemson University; Richardson Seed Inc., Lubbock, TX; Scott Seed Company, Hereford, TX). Two experiments were conducted as randomized complete block designs using ruminal contents from two ruminally-fistulated steers (blocks). Grains were ground by a 1-mm screen and used as substrates (2 g, dry matter basis) in laboratory fermenters containing strained ruminal contents and bicarbonate buffer. Fermenters were equipped with pressure monitoring devices (ANKOM Technology, Macedon, NY) to quantify gas production as an indicator of microbial digestion. Cultures were incubated for 24 (experiment 1) to 30 hours (experiment 2), and gas production was recorded at 15-minute intervals. Dry matter disappearance was determined, and organic acid concentrations in the spent culture media were analyzed by gas chromatography. Experiment 1 compared corn to 24 sorghum cultivars, and experiment 2 compared corn to six sorghum cultivars, five of which were represented in experiment 1, plus one waxy hybrid. Data were analyzed using mixed models with cultivar as a fixed effect and block as a random effect. For gas production data, time and the interaction between time and cultivar also were used as fixed effects. For both experiments, there was an interaction between cultivar and time (P < 0.0001) for gas production, revealing large differences among cultivars with respect to their relative susceptibility to microbial fermentation. Similarly, dry matter disappearance; production of acetate, propionate, and butyrate; and acetate:propionate varied substantially among cultivars (P < 0.01), and in many cases exceeding measurements obtained with control corn. Sorghum cultivars used in these experiments varied widely in their susceptibility to digestion by ruminal microorganisms, revealing potential for development of hybrids that can compete with corn as energy sources while contributing to improved sustainability of feedlot production.

Определение параметров глубинного культивирования микромицета Trichoderma asperellum ВКПМ F-1323 для получения споровых форм культуры

The basic physical parameters of the cultivation of micromycete Trichoderma asperellum VKPM F-1323 are determined on complex nutrient media. The choice of the object of study was due to its antagonistic activity with attitude to many phytopathogenic fungi such as Ascochyta pisi, Cercospora beticola, Claviceps purpurea, Sclerotinia sclerotiorum, Alternaria alternata, Botrytis cinerea, Fusarium graminearum, Passalora fulva, Verticillum dahliae, as well as growth-stimulating activity with attitude to plants, suggesting a motivation for the use of Trichoderma asperellum VKPM F-1323 as the basis for biological products used in agriculture. During the experiment, the nutrient medium composition (g/l) was used: molasses – 20, yeast extract – 7, NaNO3 – 2, K2HPO4 – 1, KCl – 0.5, MgSO4 – 0.5, FeSO4 – 0.01. Before sterilization, the pH was adjusted to 7.5. Cultivation was carried out at a temperature of 27 °C with constant stirring and aeration in autoclaved laboratory fermenters with a working volume of 2 l. The cultivation of micromycete in a liquid nutrient medium was chosen in connection with the rapid progress of the process. In addition, unlike solid-phase cultivation, the deep one allows accumulating in the finished product form numerous secondary metabolites with antagonistic activity against phytopathogens, as well as growth-stimulating activity with attitude to plants. In the work, the values of mixing speed, aeration intensity, and initial pH of the medium are determined, which determine the maximum productivity of the process according to the conidia concentration (titer) of the studied culture. During the cultivation process, the pH level was monitored without maintaining it during the process, since it is known that a change in pH values characterizes the course of the cultivation process with the formation of spore forms, and the initial pH value is an important factor in the conidia formation of Trichoderma fungi. It is shown that during cultivation for 72 hours the maximum concentration of conidia is achieved for a stirring speed of 700 rpm, without reflective baffles (chippers) in the design of the fermenter, aeration intensity of 0.25 l/l of medium×min and the initial pH of the nutrient medium 7.0. Subject to the above conditions, the concentration of conidia is 1.35 ± 0.09 conidia /ml.

Utilization of a Model Cocoa Fermentation System and Roasting to Assess Differences Among Bioactive Compounds and Bioactivity of Cocoa Through Variable Processing Conditions (FS07-04-19)

ObjectivesCocoa beans (Theobroma cacao) are a highly concentrated source of dietary flavanols that undergo processing steps, to enhance sensory characteristics. Although it is widely accepted that preservation of native cocoa flavanols is critical for retaining bioactivity, it is possible that reactions occurring throughout cocoa processing may generate compounds with novel activities, potentially preserving or enhancing bioactivity. Our objective was to evaluate the impact of fermentation and roasting on levels of flavanols and bioactivity of cocoa. MethodsCombinations of fermentation and roasting parameters were employed via a controlled laboratory fermentation model system and roasting operation. Cocoa powders were produced from unfermented cocoa beans using different treatments representing extremes of fermentation and roasting. Cocoa beans were fermented in a simulated pulp media for 168 h. Fermentation and roasting temperatures varied depending on treatment group. Further processing (winnowing, grinding, pressing) produced cocoa powders for each treatment. Extracts were made from the final beans and powders. Total polyphenols were quantified by the Folin-Ciocalteu assay. HILIC UPLC-MS/MS was employed to analyze flavanols based on their degree of polymerization (DP). ResultsFermentation and roasting contributed to losses of native flavanols. Total polyphenol levels and individual flavanols (DP 1–10) were approximately 27–90% lower in hot fermented/hot roasted cocoa powder and approximately 11–54% lower in cool fermented/cool roasted cocoa powder when compared to the unfermented/unroasted (control) treatment. Hot roasting had the most significant impact on flavanol degradation. There was also a direct relationship between increasing flavanol concentration in cocoa powder and DP. ConclusionsCompared to fermentation, roasting demonstrated a more significant impact on flavanol degradation and composition among cocoa bean treatments. Further research in vitro and in vivo is being performed currently to determine whether these process-induced flavanol losses correlate with reduced anti-obesity and anti-inflammatory activities. Funding SourcesThis work was supported by the Agriculture and Food Research Initiative (AFRI) of the US Department of Agriculture.

Scale Up Studies for Polyhydroxyalkanoate Production by a Bacillus flexus Strain with Industrial Potential.

Polyhydroxyalkanoates (PHA) are synthesized by bacteria under unfavourable growth conditions like excess of carbon over nitrogen, coupled with oxygen limitation. The PHA polymers of microbial origin are diverse in chemical composition and material properties. A bioprocess for PHA production by indigenously isolated Bacillus flexus MTCC 12841 was devised and optimized at a laboratory fermentor scale. Fermentation strategies that involved modifications in some parameters like aeration, agitation, temperature, nutrient feeding or changes in C:N ratio led to substantial improvement of 59% in PHA production reaching highest concentration of 9.73g/L. Biomass too was enhanced to 15.70g/L equivalent to 126% increase over the optimized shake flask runs. PHA (Yp/s) and biomass (Yx/s) yields were found to be 0.32 and 0.51g/g respectively, indicating good carbon utilization efficiency. The characterization of polymer by GC-MS revealed that the culture produced poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) as a co-polymer. The novelty of the research findings lies in the demonstration of increased production of PHA at lab fermentor level coupled with the identification of the natural ability of the strain to also produce PHBV without any need for exogenous addition of precursors. The fermentation process as well as the strain may be subjected to further optimization to increase the PHA production as well as to increase the % of HVcontent in the co-polymer.

Factorial Design Based Bench-scale Production of Collagenase by Pseudomonas sp. Found in Protein Waste of Himalayan Region

The current study was focused on factorial design based bench-scale production of collagenase by Pseudomonas sp. Chemical and fermentation conditions including medium components (carbon, nitrogen, and growth supplements) were optimized. The medium containing sucrose, tryptone and gelatin substrate was found to enhance the production of collagenase. The physical parameters (agitation speed and aeration rate) were also optimized. Moreover, the interactive effect of optimized physicochemical parameters using two levels of six factors (26) of factorial design was studied for the maximum collagenase production. Among 64 combinations, the 57th combination was shown maximum 1.43 U/mL collagenase activity. The bench-scale production of collagenase was achieved in a 6 L working volume laboratory fermenter. The bench-scale fermenter produced 2.3-fold enhanced collagenase activity at reduced cultivation time (14th h) in comparison to the shake flask (24th h). The lead combination can be used for the large scale collagenase production in industrial fermenters.