Laboratory-scale Bioreactor Research Articles

Medium Formulation and Fed-Batch Cultivation of <i>Methylosinus trichosporium</i>

Methanotrophs display the ability to consume methane as a carbon source and produce a wide-range of high-value products, e.g. ectoine/hydroxyectoine, poly-b-hydroxybutyrate (PHB), single cell protein, extracellular polysaccharides and lipids. Usually methanotrophs show low specific substrate consumption rates, which restricts their application at pilot and industrial scale. Thus, in order to reduce the time and costs of the cultivation process, it is vital to accelerate the growth of applied organisms. Usually, methanotrophic bacteria cultivations are carried out using fully synthetic mineral mediums (nitrate mineral salts medium (NMS)) without the addition of any growth factors. Potentially, higher biomass growth and substrate uptake rates can be achieved by supplementing the growth medium with vitamins, amino acids etc. or by using more bioavailable substrates.The aim of our research was to study the influence of growth factors such as vitamins, and different nitrogen sources (yeast extract, yeast nitrogen base with/without amino acids and tryptone) on the growth of Methylomonas methanica, Methylomicrobium alcaliphilum and Methylosinus trishosporium.Experiments for studying the influence of growth factors were carried out in shake flasks by varying the medium compositions and analyzing the effects of said variations on the kinetics of the cultivation, e.g. specific biomass growth rate and biomass yield from substrate.Subsequent tests of the developed nutrient medium, which promotes higher biomass growth rates, were carried out in laboratory 5 L bioreactor Methylosinus trishosporium cultivations to study the main process parameters.From the statistical analysis of experimental data it was observed, that supplementation of the growth medium with yeast extract or tryptone, seems to promote the growth rate of methanotrophs, when methanol is used as the main substrate. Furthermore, specific growth rates observed during cultivations in mediums containing vitamins (including cobolamin) also seem to positively affect the biomass growth rate. Based on the results of lab-scale bioreactor cultivations, using the identified medium composition it was possible to achieve a maximal biomass specific growth rate of 0.15 L⸱h‑1 and productivity of 0.16 g⸱L-1⸱h-1.

Efficient bioremediation of laboratory wastewater co-contaminated with PAHs and dimethylformamide by a methylotrophic enrichment culture

A methylotrophic enrichment culture, MM34X, has been assessed for its exceptional ability in biodegradation of dimethylformamide (DMF) and bioremediation of laboratory wastewater (LWW) co-contaminated with polycyclic aromatic hydrocarbons (PAHs). The culture MM34X tolerated high concentrations of DMF and efficiently degraded 98% of 20,000 mg L−1 DMF within 120 h. LWW bioremediation was performed in stirred bottle laboratory-scale bioreactor. After 35 days of incubation, 2760.8 ± 21.1 mg L−1 DMF, 131.8 ± 9.7 mg L−1 phenanthrene, 177.3 ± 7.5 mg L−1 pyrene and 39.5 ± 2.7 mg L−1 BaP in LWW were removed. Analysis of post-bioremediation residues indicated the absence of any known toxic intermediates. The efficacy of bioremediation was further evaluated through cyto-genotoxicity assays using Allium cepa. The roots of A. cepa exposed to bioremediated LWW showed improved mitotic index, whereas original LWW completely arrested cell growth. Similarly, the alkaline comet assay indicated alleviation of genotoxicity in bioremediated LWW, as evidenced by significantly lower DNA damage in terms of tail DNA and Olive tail moment. In addition, oxidative stress assays, performed using fluorescent probes 2′,7′-dichlorodihydrofluorescein diacetate, C11-BODIPY and dihydrorhodamine 123, revealed significant mitigation of oxidative stress potential in bioremediated LWW. Our findings suggest that the enrichment MM34X may prime the development of inexpensive and efficient large-scale bioremediation of LWW co-contaminated with PAHs and DMF.

XANTHAN PRODUCTION ON CRUDE GLYCEROL BY LAB-SCALE BIOREACTOR CULTIVATION OF LOCAL Xanthomonas ISOLATE

Intensive development of the global biodiesel industry has led to the generation of a large excess of crude glycerol, which is impure, and its disposal into the environment is unacceptable without previous purification. Since purification costs are high, the application of crude glycerol in biotechnological production of valueadded products represents a promising solution for a sustainable utilization of this effluent. The aim of this study was to examine xanthan biosynthesis by the Xanthomonas PL 3 strain on a medium containing crude glycerol from biodiesel production in a laboratory-scale bioreactor in order to further scale up this bioprocess. Xanthan was produced by submerged cultivation in a crude glycerol-based medium (glycerol content of 20 g/L) in a 3 L lab-scale bioreactor (working volume of 2 L), under aerobic conditions for 168 h (0-48 h: 25°C, 1 vvm and 200 rpm; 48-168 h: 30 °C, 2 vvm, and agitation rate adjusted according to the dissolved oxygen concentration which was maintained at values higher than 30%). The bioprocess was monitored by the analysis of cultivation medium samples in predetermined time intervals, and its success was estimated based on the xanthan concentration in the medium, separated biopolymer average molecular weight and degree of nutrient conversion. In the applied experimental conditions, 11.10 g/L of xanthan with the average molecular weight of 2.85·105 g/moL was biosynthesized. At the end of this bioprocess, the degree of total glycerol, nitrogen and phosphorous conversion was 62.82%, 41.54% and 24.80%, respectively. Results obtained in this study suggest that the Xanthomonas PL 3 strain has the ability to produce xanthan of good quality on cultivation media containing crude glycerol from the biodiesel industry.

Distinct mechanisms underlying assembly processes and interactions of microbial communities in two single-stage bioreactors coupling anammox with denitrification

Coupling anammox with denitrification processes are promising technologies for total nitrogen removal from wastewater. However, the assembly processes and interactions of functional bacteria in the coupling systems are still poorly understood. Herein, two lab-scale up-flow bioreactors were separately operated to achieve the simultaneous anammox and denitrification (SAD) and the partial denitrification and anammox (PDA) systems under different substrate loading conditions. Although the two bioreactors seemed to have similar composition of functional bacteria, denitrifying bacteria Denitratisoma and Thauera exhibited higher abundances in the PDA bioreactor. In contrast, the genus Candidatus Jettenia was more abundant in the SAD bioreactor. The null model showed that stochastic processes dominantly governed the community assembly in both bioreactors across different substrate loadings. However, homogeneous selection appeared to be more important for shaping communities in the SAD system under high and moderate substrate-loading conditions. Particularly, drift was the most important process in driving the assembly of dominant denitrifying bacteria (e.g. genera Denitratisoma and Thauera), whereas anammox bacteria were primarily governed by the homogenous selection under high substrate-loading conditions. Moreover, anammox and denitrifying bacteria in the PDA bioreactor established simple and efficient networks, probably exerting impacts on the assembly processes of their co-occurring species. By contrast, the networks in the SAD bioreactor showed low modularity and interconnectivity. This study highlighted the specificity of community assembly processes and interactions in SAD and PDA systems, which would advance our understanding of the fundamental ecological laws underpinning the collaboration of anammox and denitrifying bacteria.

Experimental and model-based characterisation of Bacillus spizizenii growth under different temperature, pH and salinity conditions prior to aquacultural wastewater treatment application

Aquacultural production generates large amounts of wastewater with all its negative effects on life and the environment, particularly caused by elevated amounts of salt and organic matter. Within the natural microflora of such wastewaters, Bacillus spp. are largely present and therefore also actively used within biological treatment. The study conducted here followed work done in Ho Chi Minh City, Vietnam, that examined local aquacultural wastewater samples and identified different strains including species of Bacillus. More specifically in this study, the recently reclassified species Bacillus spizizenii was investigated in closer detail to quantitatively characterise its growth under different temperature, pH and salinity conditions. The strain was grown in flask and lab-scale batch bioreactor cultures. The experimental data obtained was subjected to mathematical modelling to estimate specific growth parameters. The modelling covers, in a newly combined form, logistic and delayed growth as well as growth dependency on temperature, pH and salinity. Major results obtained quantify the best growth conditions at temperature 38.7∘C, pH 7.5 and salinity 6.6 g l−1, although the organism was experimentally shown here to grow delayed at salinities of 54.7 g l−1, with a likely growth at even higher salinities. The results obtained can be used to grow Bacillus spizizenii under best conditions in bioreactors to high biomass to finally apply it to aquacultural wastewater sites.

Insights into the microbial response mechanisms to ciprofloxacin during sulfur-mediated biological wastewater treatment using a metagenomics approach

The fate and removal of ciprofloxacin, a class of fluoroquinolone antibiotic, during sulfur-mediated biological wastewater treatment has been recently well documented. However, little is known regarding the genetic response of microorganisms to ciprofloxacin. Here, a lab-scale anaerobic sulfate-reducing bioreactor was continuously operated over a long term for ciprofloxacin-contaminated wastewater treatment to investigate the response of the microorganisms to ciprofloxacin by adopting a metagenomics approach. It was found that total organic carbon (TOC) removal and sulfate reduction were promoted by approximately 10% under ciprofloxacin stress, along with the enrichment of functional genera (e.g., Desulfobacter, Geobacter) involved in carbon and sulfur metabolism. The metagenomic analytical results demonstrated that ciprofloxacin triggered the microbial SOS response, as demonstrated by the up-regulation of the multidrug efflux pump genes (8–125-fold higher than that of the control) and ciprofloxacin-degrading genes (4–33-fold higher than that of the control). Moreover, the contents of ATP, NADH, and cytochrome C, as well as related functional genes (including genes involved in energy generation, electron transport, carbon metabolism, and sulfur metabolism) were markedly increased under ciprofloxacin stress. This demonstrated that the carbon and sulfur metabolisms were enhanced for energy (ATP) generation and electron transport in response to ciprofloxacin-induced stress. Interestingly, the microbes tended to cooperate while being subjected exposure to exogenous ciprofloxacin according to the reconstructed metabolic network using the NetSeed model. Particularly, the species with higher complementarity indices played more pivotal roles in strengthening microbial metabolism and the SOS response under long-term ciprofloxacin stress. This study characterized the response mechanisms of microorganisms to ciprofloxacin at the genetic level in sulfur-mediated biological wastewater treatment. These new understandings will contribute the scientific basis for improving and optimizing the sulfur-mediated bioprocess for antibiotics-laden wastewater treatment.

Differential colonization and functioning of microbial community in response to phosphate levels

Microbes play a major role in phosphate cycling and regulate its availability in various environments. The metagenomic study highlights the microbial community divergence and interplay of phosphate metabolism functional genes in response to phosphate rich (100 mgL−1), limiting (25 mgL−1), and stressed (5 mgL−1) conditions at lab-scale bioreactor. Total five core phyla were found responsive toward different phosphate (Pi) levels. However, major variations were observed in Proteobacteria and Actinobacteria with 33–81% and 5–56% relative abundance, respectively. Canonical correspondence analysis reflects the colonization of Sinorhizobium (0.8–4%), Mesorhizobium (1–4%), Rhizobium (0.5–3%) in rich condition whereas, Pseudomonas (1–2%), Rhodococcus (0.2–2%), Flavobacterium (0.2–1%) and Streptomyces (0.3–4%) colonized in limiting and stress condition. The functional profiling demonstrates that Pi limiting and stress condition subjected biomass were characterized by abundant PQQ-Glucose dehydrogenase, alkaline phosphatase, 5′-nucleotidase, and phospholipases C genes. The finding implies that the major abundant genera belonging to phosphate solubilization enriched in limiting/stressed conditions decide the functional turnover by modulating the metabolic flexibility for Pi cycling. The study gives a better insight into intrinsic ecological responsiveness mediated by microbial communities in different Pi conditions that would help to design the microbiome according to the soil phosphate condition. Furthermore, this information assists in sustainably maintaining the ecological balance by omitting excessive chemical fertilizers and eutrophication.

Evaluation of degradation potential of Free cell and Immobilized cell using Shake flask technique and lab scale Bioreactor technique for remediation of Nitroaromatics in aqueous phase

In this study, Free cell and Immobilized cell using Shake flask technique and lab scale Bioreactor techniquewas evaluated for the remediation of TNT in aqueous phase. In shake flask study, all samples with Freeand Immobilized cell culture were kept in a shaker for 24 hours at 120 rpm at room temperature whereasthe immobilized cell bioreactor was set at airflow 1 ml/l, agitation 50-60 rpm for 8 hours at 25 °C. Thequantitative analysis by HPLC in shake flask study revealed 82.37% and 98.12% of TNT degradation byFree cell culture and immobilized cell culture respectively after 24 hours and 81.19% of TNT degradationwas found in Bioreactor. The results depicted that Immobilized cell culture had better performance thanfree cell culture and immobilized cell bioreactor proved more effective than shake flask culture technique.

Designing an Efficient Consortium for Improved Crop Productivity using Phosphate Stress Adapted Bacteria with Multiple Growth-Promoting Attributes

An efficient consortium NEER-PHOS was designed using high phosphate solubilizing microbes isolated from lab-scale bioreactor operated under phosphate (Pi) rich, limiting, and stress conditions to meet the current agronomy demands in an eco-friendly manner. Individual microbes were screened for higher phosphate solubilizing potential with additional growth-promoting abilities like nitrogen fixation, potassium solubilization, auxin, and HCN production. The best-selected multipotent microbes were used to design the consortium. The selected microbes T166, X225, X303, X308, X298, and PVN-1 were identified as Proteus mirabilis, Pseudomonas sp., Pseudomonas aeruginosa, Chryseobacterium sp., and Klebsiella pneumoniae, respectively and results into more than 250 mg/L released PO4 3 in liquid medium. A total of 63 consortium combinations were assayed, and the best solubilizing combination, number 58 (557.8 mg/L-PO4 3−), was selected for the pot experiment. The performance of the designed consortium was studied on Vigna radiata, Cicer arietinum, and Triticum aestivum. The overall analyzed growth parameters suggest that the designed consortium enhances growth significantly (p < 0.05) and reduces fertilizers by 50–100% in V. radiata and C. arietinum. Hence, the designed consortium NEER-PHOS can reduce the chemical fertilizer application and simultaneously improve plant productivity.

Fusion Tag Design Influences Soluble Recombinant Protein Production in Escherichia coli.

Fusion protein technologies to facilitate soluble expression, detection, or subsequent affinity purification in Escherichia coli are widely used but may also be associated with negative consequences. Although commonly employed solubility tags have a positive influence on titers, their large molecular mass inherently results in stochiometric losses of product yield. Furthermore, the introduction of affinity tags, especially the polyhistidine tag, has been associated with undesirable changes in expression levels. Fusion tags are also known to influence the functionality of the protein of interest due to conformational changes. Therefore, particularly for biopharmaceutical applications, the removal of the fusion tag is a requirement to ensure the safety and efficacy of the therapeutic protein. The design of suitable fusion tags enabling the efficient manufacturing of the recombinant protein remains a challenge. Here, we evaluated several N-terminal fusion tag combinations and their influence on product titer and cell growth to find an ideal design for a generic fusion tag. For enhancing soluble expression, a negatively charged peptide tag derived from the T7 bacteriophage was combined with affinity tags and a caspase-2 cleavage site applicable for CASPase-based fusiON (CASPON) platform technology. The effects of each combinatorial tag element were investigated in an integrated manner using human fibroblast growth factor 2 as a model protein in fed-batch lab-scale bioreactor cultivations. To confirm the generic applicability for manufacturing, seven additional pharmaceutically relevant proteins were produced using the best performing tag of this study, named CASPON-tag, and tag removal was demonstrated.

Microsensor in Microbioreactors: Full Bioprocess Characterization in a Novel Capillary-Wave Microbioreactor.

Microbioreactors (MBRs) with a volume below 1 mL are promising alternatives to established cultivation platforms such as shake flasks, lab-scale bioreactors and microtiter plates. Their main advantages are simple automatization and parallelization and the saving of expensive media components and test substances. These advantages are particularly pronounced in small-scale MBRs with a volume below 10 µL. However, most described small-scale MBRs are lacking in process information from integrated sensors due to limited space and sensor technology. Therefore, a novel capillary-wave microbioreactor (cwMBR) with a volume of only 7 µL has the potential to close this gap, as it combines a small volume with integrated sensors for biomass, pH, dissolved oxygen (DO) and glucose concentration. In the cwMBR, pH and DO are measured by established luminescent optical sensors on the bottom of the cwMBR. The novel glucose sensor is based on a modified oxygen sensor, which measures the oxygen uptake of glucose oxidase (GOx) in the presence of glucose up to a concentration of 15 mM. Furthermore, absorbance measurement allows biomass determination. The optical sensors enabled the characterization of an Escherichia coli batch cultivation over 8 h in the cwMBR as proof of concept for further bioprocesses. Hence, the cwMBR with integrated optical sensors has the potential for a wide range of microscale bioprocesses, including cell-based assays, screening applications and process development.

Phosphate-inducible poly-hydroxy butyrate production dynamics in CO2 supplemented upscaled cultivation of engineered Phaeodactylum tricornutum

Diatoms such as Phaeodactylum tricornutum are emerging as sustainable alternatives to traditional eukaryotic microbial cell factories. In order to facilitate a viable process for production of heterologous metabolites, a rational genetic design specifically tailored to metabolic requirements as well as optimised culture conditions are required. In this study we investigated the effect of constitutive and inducible expression of the heterologous poly-3-hydroxybutyrate (PHB) pathway in P. tricornutum using non-integrative episomes in 3 different configurations. Constitutive expression led to downregulation of at least one individual gene out of three (phaA, phaB and phaC) and was outperformed by inducible expression. To further asses and optimise the dynamics of PHB accumulation driven by the inducible alkaline phosphatase 1 promoter, we upscaled the production to lab-scale bioreactors and tested the effect of supplemented CO2 on biomass and PHB accumulation. While ambient CO2 cultivation resulted in a maximum PHB yield of 2.3% cell dry weight (CDW) on day 11, under elevated CO2 concentrations PHB yield peaked at 1.7% CDW on day 8, coincident with PHB titres at 27.9 mg L−1 that were approximately threefold higher than ambient CO2. With other more valuable bio-products in mind, these results highlight the importance of the genetic design as well as substrate availability to supply additional reduction equivalents to boost biomass accumulation and relieve potential enzymatic bottlenecks for improved product accumulation.

Denitrification Performance in Packed-Bed Reactors Using Novel Carbon-Sulfur-Based Composite Filters for Treatment of Synthetic Wastewater and Anaerobic Ammonia Oxidation Effluent.

To avoid nitrate pollution in water bodies, two low-cost and abundant natural organic carbon sources were added to make up the solid-phase denitrification filters. This study compared four novel solid-phase carbon-sulfur-based composite filters, and their denitrification abilities were investigated in laboratory-scale bioreactors. The filter F4 (mixture of elemental sulfur powder, shell powder, and peanut hull powder with a mass ratio of 6:2.5:1.5) achieved the highest denitrification ability, with an optimal nitrate removal rate (NRR) of 723 ± 14.2 mg NO3–-N⋅L–1⋅d–1 when the hydraulic retention time (HRT) was 1 h. The HRT considerably impacted effluent quality after coupling of anaerobic ammonium oxidation (ANAMMOX) and solid-phase-based mixotrophic denitrification process (SMDP). The concentration of suspended solids (SS) of the ANAMMOX effluent may affect the performance of the coupled system. Autotrophs and heterotrophs were abundant and co-existed in all reactors; over time, the abundance of heterotrophs decreased while that of autotrophs increased. Overall, the SMDP process showed good denitrification performance and reduced the sulfate productivity in effluent compared to the sulfur-based autotrophic denitrification (SAD) process.

Optimized expression of large fragment DNA polymerase I from Geobacillus stearothermophilus in Escherichia coli expression system

Bst DNA polymerase is a DNA polymerase derived from Geobacillus stearothermophilus, has a strand-displacement activity, and is used in loop-mediated isothermal amplification (LAMP) for rapid detection of COVID-19. Despite its potential to be employed in the detection of COVID-19, using commercially available enzymes is not economically feasible. The use of noncommercial enzyme for routine use is desirable. However, research on Bst DNA polymerase is still limited in Indonesia. For those reasons, a preliminary study of scale-up production of recombinant Bst polymerase was conducted. Therefore, the optimization of expression conditions was performed. The optimum conditions for Bst polymerase expression were as follows: 1 mM of IPTG, post-induction incubation time of 6 h, and induction at OD600 1.1. Employing optimum conditions could result in 2.8 times increase in protein yield compared to the initial conditions. Subsequently, an operation in 1 L working volume by a lab-scale bioreactor had been performed, followed by purification and dialysis. The optimum result for a 1 L lab-scale bioreactor was achieved by applying 100 rpm and 3 vvm, giving 11.7 mg/L of protein yield. Bst polymerase was successfully purified showing 813.56 U/mg of polymerase activity.

System performance and functional analysis for the methanogenic bioreactor of a two-phase anaerobic digestion system: The effect of influent sulfate

In this study, a lab-scale bioreactor was built to investigate the influence of sulfate on the overall performance, microbial community, and metabolic pathways in the methanogenic bioreactor of a two-phase anaerobic digestion system. The results demonstrated that by gradually cultivating the sludge of the methanogenic bioreactor, a chemical oxygen demand (COD) degradation efficiency of over 65% was achieved for sulfate contents from 10,000 mg/L to 30,000 mg/L. The sulfate reduction performance could only be maintained at a high level for influent COD/sulfate ratios no less than 1.88. In addition, the methane production rate (MPR) decreased from 0.92 L/L/d to 0.28 L/L/d, and the proportion of methane in biogas dropped from 51% to 33% when the influent sulfate exceeded 30,000 mg/L. High-throughput sequencing coupled with PICRUSt2 revealed that the dissimilatory sulfate process could be enhanced by sulfate within 30,000 mg/L. While an amount of greater than 5000 mg/L sulfate remarkably inhibited the assimilatory sulfate reduction process, and this inhibition was induced by the down-regulation of the cysNC, cysC, cysI and cysJ genes. For the methanogenic process, sulfate contents within 30,000 mg/L had no obvious impact on all of the acetoclastic, hydrogenotrophic, and methylotrophic methanogenesis processes, but serious suppression was observed for sulfate amounts greater than 30,000 mg/L. This inhibition primarily originated from the decrease in mcrA, mcrB, and mcrG genes that regulate the conversion of methylcoenzyme M to methane, the last step of all three methanogenesis pathways.

Microbial Mediation of Carbon, Nitrogen, and Sulfur Cycles During Solid Waste Decomposition.

Landfills are a unique "terrestrial ecosystem" and serve as a significant carbon sink. Microorganisms convert biodegradable substances in municipal solid waste (MSW) to CH4, CO2, and microbial biomass, consisting of the carbon cycling in landfills. Microbial-mediated N and S cycles are also the important biogeochemical process during MSW decomposition, resulting in N2O and H2S emission, respectively. Meanwhile, microbial-mediated N and S cycles affect carbon cycling. How microbial community structure and function respond to C, N, and S cycling during solid waste decomposition, however, are not well-characterized. Here, we show the response of bacterial and archaeal community structure and functions to C, N, and S cycling during solid waste decomposition in a long-term (265days) operation laboratory-scale bioreactor through 16S rRNA-based pyrosequencing and metagenomics analysis. Bacterial and archaeal community composition varied during solid waste decomposition. Aerobic respiration was the main pathway for CO2 emission, while anaerobic C fixation was the main pathway in carbon fixation. Methanogenesis and denitrification increased during solid waste decomposition, suggesting increasing CH4 and N2O emission. In contract, fermentation decreased along solid waste decomposition. Interestingly, Clostridiales were abundant and showed potential for several pathways in C, N, and S cycling. Archaea were involved in many pathways of C and N cycles. There is a shift between bacteria and archaea involvement in N2 fixation along solid waste decomposition that bacteria Clostridiales and Bacteroidales were initially dominant and then Methanosarcinales increased and became dominant in methanogenic phase. These results provide extensive microbial mediation of C, N, and S cycling profiles during solid waste decomposition.

Modeling free surface gas transfer in agitated lab-scale bioreactors

A physics-based approach for modeling convective gas-liquid mass transfer across free surfaces in lab-scale agitated vessels is presented. This approach, which combines numerical modeling with semi-empirical turbulence theory, is validated across multiple operating scales and conditions. The findings show that, regardless of the mechanical action driving motion, free surface mass transfer rate in lab-scale systems is consistent with empirical relationships. The role and importance of free surface convective mass transfer in lab scale devices is also discussed. The computational approach is shown to be practical within the context of industrial analysis and design timescales.

Integrated Whole-Cell Biocatalysis for Trehalose Production from Maltose Using Permeabilized Pseudomonas monteilii Cells and Bioremoval of Byproduct.

Trehalose is a non-conventional sugar with potent applications in the food, healthcare and biopharma industries. In this study, trehalose was synthesized from maltose using whole-cell Pseudomonas monteilii TBRC 1196 producing trehalose synthase (TreS) as the biocatalyst. The reaction condition was optimized using 1% Triton X-100 permeabilized cells. According to our central composite design (CCD) experiment, the optimal process was achieved at 35°C and pH 8.0 for 24 h, resulting in the maximum trehalose yield of 51.60 g/g after 12 h using an initial cell loading of 94 g/l. Scale-up production in a lab-scale bioreactor led to the final trehalose concentration of 51.91 g/l with a yield of 51.60 g/g and productivity of 4.37 g/l/h together with 8.24 g/l glucose as a byproduct. A one-pot process integrating trehalose production and byproduct bioremoval showed 53.35% trehalose yield from 107.4 g/l after 15 h by permeabilized P. moteilii cells. The residual maltose and glucose were subsequently removed by Saccharomyces cerevisiae TBRC 12153, resulting in trehalose recovery of 99.23% with 24.85 g/l ethanol obtained as a co-product. The present work provides an integrated alternative process for trehalose production from maltose syrup in bio-industry.

A Robust Fermentation Process for Natural Chocolate-like Flavor Production with Mycetinis scorodonius.

Submerged fermentation of green tea with the basidiomycete Mycetinis scorodonius resulted in a pleasant chocolate-like and malty aroma, which could be a promising chocolate flavor alternative to current synthetic aroma mixtures in demand of consumer preferences towards healthy natural and ‘clean label’ ingredients. To understand the sensorial molecular base on the chocolate-like aroma formation, key aroma compounds of the fermented green tea were elucidated using a direct immersion stir bar sorptive extraction combined with gas chromatography–mass spectrometry–olfactometry (DI-SBSE-GC-MS-O) followed by semi-quantification with internal standard. Fifteen key aroma compounds were determined, the most important of which were dihydroactinidiolide (odor activity value OAV 345), isovaleraldehyde (OAV 79), and coumarin (OAV 24), which were also confirmed by a recombination study. Furthermore, effects of the fermentation parameters (medium volume, light protection, agitation rate, pH, temperature, and aeration) on the aroma profile were investigated in a lab-scale bioreactor at batch fermentation. Variation of the fermentation parameters resulted in similar sensory perception of the broth, where up-scaling in volume evoked longer growth cycles and aeration significantly boosted the concentrations yet added a green note to the overall flavor impression. All findings prove the robustness of the established fermentation process with M. scorodonius for natural chocolate-like flavor production.

Isolation of Glucoamylase from Aspergillus Niger to Produce Liquid Sugar from Tapioca Flour

This study aims to isolate the glucoamylase enzyme from Aspergillus niger to produce liquid sugar from tapioca flour. Enzymes are produced in a batch system laboratory-scale bioreactor. The fermentation substrate in an agar medium modified with the addition of starch. Fermentation was carried out for 168 hours at room temperature with an agitation speed of 120 rpm. The manufacture of liquid sugar is done by adding an enzyme as much as 0.3; 0.4; and 0.5 ml while the substrate concentration was varied by 20%; 30%; and 40%. The results showed that A. niger produce extracellular enzymes such as amylase and glucoamylase, particulary at exponential phase and the initial stationary phase. Enzymatic hydrolysis of starch consists of two stages, namely liquification and saccharification. In the liquification process, the enzyme α-amylase is used to break down starch containing amylose and amylopectin into dextrins. Next in the second stage is the saccharification process, where dextrin is hydrolyzed into glucose with the help of the glucoamylase enzyme. Optimal liquid sugar production can be done by adding 0.5 mL of crude enzyme extract at 30% (w/v) tapioca concentration. Further research is recommended to carry out enzyme purification to produce liquid sugar products with higher sugar purity.