Manuka Honey with Varying Levels of Active Manuka Factor (AMF) Ratings as an Anaerobic Fermentation Substrate for Limosilactobacillus reuteri DPC16

Manuka honey is known for its strong antibacterial effect against pathogens but can promote probiotic growth in certain conditions. In a two-factor ANOVA study, AMFTM Manuka honey (Active Manuka Factor: 05+, 10+, 15+ and 20+) was utilised as a substrate for probiotic Limosilactobacillus reuteri DPC16 in an anaerobic batch fermenter for 36 h. The biomass growth in MRS broth was noticeably higher with AMF Manuka honey than invert syrup and control samples without any additional sweetener source. The pH value was significantly lowered below 4.0 only in the AMF samples with the formation of lactic acid as the major metabolite. Other beneficial short-chain fatty acids (SCFA), such as acetic, succinic, and propionic acids, produced during the fermentation, along with the honey saccharides, were quantified by two-dimensional (2-D) nuclear magnetic resonance (NMR) spectroscopy. A significantly (p < 0.05) high biomass in AMF 20+ sample after 36 h, can partly be attributed to the high total sugar and oligosaccharide content in the honey. Importantly, however, no statistically significant difference was observed in the recorded major fermentation outcomes for the different AMF levels. The results, nevertheless, indicate the potential prebiotic efficacy of Manuka honey as a fermentation substrate for the lactobacilli probiotic strain.

Screening for optimal protease producing Bacillus licheniformis strains with polymer-based controlled-release fed-batch microtiter plates

Background Substrate-limited fed-batch conditions have the favorable effect of preventing overflow metabolism, catabolite repression, oxygen limitation or inhibition caused by elevated substrate or osmotic concentrations. Due to these favorable effects, fed-batch mode is predominantly used in industrial production processes. In contrast, screening processes are usually performed in microtiter plates operated in batch mode. This leads to a different physiological state of the production organism in early screening and can misguide the selection of potential production strains. To close the gap between screening and production conditions, new techniques to enable fed-batch mode in microtiter plates have been described. One of these systems is the ready-to-use and disposable polymer-based controlled-release fed-batch microtiter plate (fed-batch MTP). In this work, the fed-batch MTP was applied to establish a glucose-limited fed-batch screening procedure for industrially relevant protease producing Bacillus licheniformis strains. Results To achieve equal initial growth conditions for different clones with the fed-batch MTP, a two-step batch preculture procedure was developed. Based on this preculture procedure, the standard deviation of the protease activity of glucose-limited fed-batch main culture cultivations in the fed-batch MTP was ± 10%. The determination of the number of replicates revealed that a minimum of 6 parallel cultivations were necessary to identify clones with a statistically significant increased or decreased protease activity. The developed glucose-limited fed-batch screening procedure was applied to 13 industrially-relevant clones from two B. licheniformis strain lineages. It was found that 12 out of 13 clones (92%) were classified similarly as in a lab-scale fed-batch fermenter process operated under glucose-limited conditions. When the microtiter plate screening process was performed in batch mode, only 5 out of 13 clones (38%) were classified similarly as in the lab-scale fed-batch fermenter process. Conclusion The glucose-limited fed-batch screening process outperformed the usual batch screening process in terms of the predictability of the clone performance under glucose-limited fed-batch fermenter conditions. These results highlight that the implementation of glucose-limited fed-batch conditions already in microtiter plate scale is crucial to increase the precision of identifying improved protease producing B. licheniformis strains. Hence, the fed-batch MTP represents an efficient high-throughput screening tool that aims at closing the gap between screening and production conditions.

Fermentation as a Strategy for Bio-Transforming Waste into Resources: Lactic Acid Production from Agri-Food Residues

Lactic acid (LA) obtained by fermentation of carbohydrates is well-known and widely used in the food sector. This process is as an alternative to the chemical synthesis and ensures several advantages especially in terms of environmental sustainability. In particularly, the opportunity to use agro-food residues as fermentable raw materials could improve the overall process sustainability, without considering the indisputable advantages in terms of waste reduction and residual biomass valorization, in a bio- and circular economy perspective. This research deals with the study and development of the fermentation processes of various waste biomasses from the agro-food industries, including milk whey (MW), ricotta cheese whey (RCW), pear processing residues (PPR), potato pomace (PP), tomato pomace (PT), in order to obtain an experimental protocol applicable to the production of LA. Lactobacillus casei DSM 20011 (ATCC 393), a homofermentative L(+)-LA producing bacterium has been used, starting from small-scale tests to verify of the microorganism to grow in complex medium with different carbon sources and the possible presence of potentially toxic substances for microbial growth. Yields from 27.0 ± 0.3% to 46.0 ± 0.7% have been obtained. Then, a scaling-up was performed in a 1 L batch fermenter, using a mixed medium of RCW and PPR in different ratio. The best LA yield was 78.3% with a volumetric productivity of 1.12 g/L·h in less than 60 h.

Peningkatan Populasi, Pertumbuhan dan Serapan Nitrogen Tanaman Kedelai dengan Pemberian Azotobacter Penghasil Eksopolisakarida

Nitrogen-fixing Azotobacter is widely used as biofertilizer in sustainable agriculture. The bacteria produce exopolysaccharide which might have a significant role in enhancing soybean nitrogen uptake and growth. The objective of this research was to obtain growth media of Exopolysaccharide–producing Azotobacter; and increase shoot and root growth as well as nitrogen uptake of soybean var. Anjasmoro at early vegetative phase following inoculation of Azotobacter chroococcum liquid. Research consist of two phase, 1) determination of organic-based media for A. chroococcum liquid inoculant production, and 2) pot experiment for application of liquid inoculant on soybean. The first experiment was performed in a series of batch fermenter consisted of several organic media for 72 hours. The second experiment was set in completely randomized design consisted of three density of liquid inoculant. The results verified that the best media which induced exopolysachharide production of A. chroococcum was 1% molase enriched with 0.1% NH4Cl. Liquid inoculant clearly enhanced population of Azotobacter in soybean rhizosphere, plant height, roots dry weight and N uptake of 21 day old soybean. This research implied that A. chroococcum might be used as biofertilizer at early growth of soybean. Keywords: Azotobacter chroococcum, biofertilizer, liquid inoculat

Response Surface Optimization of Bioethanol Production from Sugarcane Molasses by Pichia veronae Strain HSC-22

Pichia veronae strain HSC-22 (accession number KP012558) showed a good tolerance to relatively high temperature, ethanol and sugar concentrations. Response surface optimization based on central composite design of experiments predicted the optimal values of the influencing parameters that affect the production of bioethanol from sugarcane molasses to be as follows: initial pH 5, 25% (w : v) initial molasses concentration, 35°C, 116 rpm, and 60 h. Under these optimum operating conditions the maximum bioethanol production on a batch fermenter scale was recorded as 32.32 g/L with 44% bioethanol yield.

Fermentative Production of Thymidine by a Metabolically Engineered Escherichia coli Strain

ABSTRACT Thymidine is an important precursor in the production of various antiviral drugs, including azidothymidine for the treatment of AIDS. Since thymidine-containing nucleotides are synthesized only by the de novo pathway during DNA synthesis, it is not easy to produce a large amount of thymidine biologically. In order to develop a host strain to produce thymidine, thymidine phosphorylase, thymidine kinase, and uridine phosphorylase genes were deleted from an Escherichia coli BL21 strain to develop BLdtu. Since the genes coding for the enzymes related to the nucleotide salvage pathway were disrupted, BLdtu was unable to utilize thymidine or thymine, and thymidine degradation activity was completely abrogated. We additionally expressed T4 thymidylate synthase, T4 nucleotide diphosphate reductase, bacteriophage PBS2 TMP phosphohydrolase, E. coli dCTP deaminase, and E. coli uridine kinase in the BLdtu strain to develop a thymidine-producing strain (BLdtu24). BLdtu24 produced 649.3 mg liter−1 of thymidine in a 7-liter batch fermenter for 24 h, and neither thymine nor uridine was detected. However, the dUTP/dTTP ratio was increased in BLdtu24, which could lead to increased double-strand breakages and eventually to cell deaths during fermentation. To enhance thymidine production and to prevent cell deaths during fermentation, we disrupted a gene (encoding uracil-DNA N-glycosylase) involved in DNA excision repair to suppress the consumption of dTTP and developed BLdtug24. Compared with the thymidine production in BLdtu24, the thymidine production in BLdtug24 was increased by ∼1.2-fold (740.3 mg liter−1). Here, we show that a thymidine-producing strain with a relatively high yield can be developed using a metabolic engineering approach.

Hydrogen concentrations in methane-forming cells probed by the ratios of reduced and oxidized coenzyme F420

Coenzyme F420 is the central low-redox-potential electron carrier in methanogenic metabolism. The coenzyme is reduced under hydrogen by the action of F420-dependent hydrogenase. The standard free-energy change at pH 7 of F420 reduction was determined to be −15 kJ mol−1, irrespective of the temperature (25–65 °C). Experiments performed with methane-forming cell suspensions of Methanothermobacter thermautotrophicus incubated under various conditions demonstrated that the ratios of reduced and oxidized F420 were in thermodynamic equilibrium with the gas-phase hydrogen partial pressures. During growth in a fed-batch fermenter, ratios changed in connection with the decrease in dissolved hydrogen. For most of the time, the changes were as expected for thermodynamic equilibrium between the oxidation state of F420 inside the cells and extracellular hydrogen. Also, methanol-metabolizing, but not acetate-converting, cells of Methanosarcina barkeri maintained the ratios of reduced and oxidized coenzyme F420 in thermodynamic equilibrium with external hydrogen. The results of the study demonstrate that F420 is a useful probe to assess in situ hydrogen concentrations in H2-metabolizing methanogens.