Effect of ultrasonication on the metabolome and transcriptome profile changes in the fermentation of Ganoderma lucidum.

Development of an efficient liquid fermentation method is helpful for food and pharmaceutical applications. This study investigated the effect of ultrasonication on the liquid fermentation of Ganoderma lucidum, a popular edible and medical fungi. Significant changes at both metabolic and transcriptional levels in mycelia were induced by ultrasound treatment. Compared with the control, 857 differential metabolites were identified (578 up- and 279 down-regulated metabolites), with more metabolites biosynthesis after sonication; 569 differentially expressed genes (DEGs) (267 up- and 302 down-) and 932 DEGs (378 up- and 554 down-) were identified in ultrasound-treated samples with recovery time of 0.5 and 3 h, respectively. Furthermore, 334 DEGs were continuously induced within the recovery time of 3 h, indicating the lasting influence of sonication on mycelia. The DEGs and differential metabolites were mainly involved in pathways of carbohydrate, energy metabolism, amino acids, terpenoids biosynthesis and metabolism and membrane transport, suggesting that ultrasound induced multifaceted effects on primary and secondary metabolism. Ultrasonication enhanced the triterpenoids production of G. lucidum (34.96 %) by up-regulating the expression of terpenoids synthase genes. This study shows that the application of ultrasound in liquid fermentation of G. lucidum is an efficient approach to produce more metabolites.

Physico-chemical aspects of Thai fermented fish viscera, Tai-Pla, curry powder processed by hot air drying and hybrid microwave-infrared drying.

The objective of this research was to comparatively investigate the effect of hot air drying (HA) and hybrid microwave-infrared drying (MI) on physico-chemical characteristics of Thai fermented fish viscera, Tai-Pla, curry powder (TCP). HA was carried out at 60°C, 70°C, and 80°C and MI was carried out at a microwave power of 740, 780, and 810 W with a constant infrared heating power (500 W) for different drying times to obtain the final moisture content ≤ 12.0% and the water activity (aw) ≤ 0.6. The quality characteristics of TCP were governed by HA temperature and MI output power. TCP dried using HA and MI at all conditions had similar contents of protein, lipid, ash, fiber, and carbohydrate (p>0.05). The fastest drying rate was detected when MI at 810 W for 40 min was applied (p<0.05). In this condition, TCP had the lowest browning index (A294 and A420) and the highest lightness (L* value) (p<0.05). TCP dried with MI at all powers had higher phenolic content and lower TBARS compared to HA (p<0.05). However, no significant differences in DPPH• scavenging activity were observed among TPC made by HA and MI (p>0.05). Similar Fourier transform infrared (FTIR) spectra with different peak intensities were observed in all samples, indicating the same functional groups with different contents were found. The bulk density of all TCP ranged from 0.51 g/mL to 0.61 g/mL and the wettability ranged from 24.02% to 26.70%. MI at 810 W for 40 min effectively reduced the drying time (5-fold faster) and lowered the specific energy consumption (18-fold lower) compared to the HA at 60°C for 210 min. Therefore, MI is a promising drying technique to reduce the drying time and improve the overall quality of TCP.

Optogenetic Amplification Circuits for Light-Induced Metabolic Control.

Dynamic control of microbial metabolism is an effective strategy to improve chemical production in fermentations. While dynamic control is most often implemented using chemical inducers, optogenetics offers an attractive alternative due to the high tunability and reversibility afforded by light. However, a major concern of applying optogenetics in metabolic engineering is the risk of insufficient light penetration at high cell densities, especially in large bioreactors. Here, we present a new series of optogenetic circuits we call OptoAMP, which amplify the transcriptional response to blue light by as much as 23-fold compared to the basal circuit (OptoEXP). These circuits show as much as a 41-fold induction between dark and light conditions, efficient activation at light duty cycles as low as ∼1%, and strong homogeneous light-induction in bioreactors of at least 5 L, with limited illumination at cell densities above 40 OD600. We demonstrate the ability of OptoAMP circuits to control engineered metabolic pathways in novel three-phase fermentations using different light schedules to control enzyme expression and improve production of lactic acid, isobutanol, and naringenin. These circuits expand the applicability of optogenetics to metabolic engineering.

Dynamical Modeling of Optogenetic Circuits in Yeast for Metabolic Engineering Applications.

Dynamic control of engineered microbes using light via optogenetics has been demonstrated as an effective strategy for improving the yield of biofuels, chemicals, and other products. An advantage of using light to manipulate microbial metabolism is the relative simplicity of interfacing biological and computer systems, thereby enabling in silico control of the microbe. Using this strategy for control and optimization of product yield requires an understanding of how the microbe responds in real-time to the light inputs. Toward this end, we present mechanistic models of a set of yeast optogenetic circuits. We show how these models can predict short- and long-time response to varying light inputs and how they are amenable to use with model predictive control (the industry standard among advanced control algorithms). These models reveal dynamics characterized by time-scale separation of different circuit components that affect the steady and transient levels of the protein under control of the circuit. Ultimately, this work will help enable real-time control and optimization tools for improving yield and consistency in the production of biofuels and chemicals using microbial fermentations.

Effects of GSM 1800 band radiation on composition, structure and bioactivity of exopolysaccharides produced by yoghurt starter cultures.

In this study, the effects of GSM 1800 band radiation on composition, structure and bioactivity of exopolysaccharides (EPSs) produced by Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus were determined. For this, GSM 1800 band radiation was applied to both cultures and characteristics of EPSs extracted from the control groups (K) and the radiation stressed groups (R) were determined. An alteration in the chemical composition of the EPSs was observed and EPS production levels and molecular weights of the EPSs increased following the GSM 1800 band radiation application. Alterations in the functional groups, thermal and morphological characteristics of EPSs following the GSM 1800 band radiation application were confirmed by FTIR, TGA and SEM analysis, respectively. Importantly no alterations in the antioxidant and antibacterial activity of the EPSs were observed following the radiation application. These results suggested the effects of the GSM radiation on final characteristics of EPSs from yogurt starter cultures.

Role of ultrasound in assisted fermentation technologies for process enhancements.

Biological molecules are widely produced by fermentation technology using bacteria, fungi or yeast. Fermentation is a biochemical process wherein the rate of bioconversion is governed by the organisms involved. The growth of the organism is mainly limited by mass transfer rates of nutrients and gases that directly affect the product formation in fermentation. Attempts have been made to enhance the growth rate and yield using mutational, recombinant strain development approach at microbial level as well as fed batch and continuous processing approach at bioprocess level in the past. The growth rate of microbes can be accelerated by increased mass transfer rates and cell wall permeability with the use of controlled low frequency ultrasound irradiation. The present review provides insights into the application of acoustic cavitation in process intensification of fermentation approaches and the role of various factors involved are highlighted with typical examples.

Metabolomics approach used for understanding temperature-related pectinase activity in Bacillus licheniformis DY2.

Pectinases are enzymes that catalyze pectin degradation. There is a global demand for pectinases because of their wide utility and catalytic efficiency. Optimization of the fermentation process to increase the pectolytic enzyme activity is generally practiced to lower process costs, but whether temperature influences the metabolome, enhancing pectinase activity, is not known. Here, we developed a metabolomics approach to explore it. The activity of P-DY2 pectinase produced by Bacillus licheniformis DY2 was higher in cells grown at 30°C than those grown at 37°C. Differential metabolome analysis revealed fluctuating tricarboxylic acid (TCA) cycle at 30°C. Consistently, the transcripts of TCA cycle genes and activities of pyruvate dehydrogenase and α-Ketoglutaric dehydrogenase were lower at 30°C than 37°C. Furthermore, inhibition of pyruvate dehydrogenase and succinate dehydrogenase enhanced the activity of P-DY2, supporting the conclusion that the inactivated pyruvate metabolism and TCA cycle were required for pectinase activity, and that P-DY2 was TCA cycle-independent. Collectively, these findings indicated that fermentation temperature affected P-DY2 activity by metabolic modulation, with an inactivated TCA cycle as a characteristic feature of high P-DY2 activity. More importantly, the present study highlights an approach of promoting pectinase activity through metabolic modulation by using metabolic pathway inhibitors.

Ultrasound assisted process intensification of uricase and alkaline protease enzyme co-production in Bacillus licheniformis.

Low energy ultrasound irradiation was used to enhance co-production of enzymes uricase and alkaline protease using Bacillus licheniformis NRRL 14209. Production of uricase and alkaline protease was evaluated for different ultrasound parameters such as ultrasound power, time of irradiation, duty cycle and growth stage of organisms at which irradiation is carried out. Maximum uricase production of 0.825 U/mL and alkaline protease of 0.646 U/mL have been obtained when fermentation broth was irradiated at 6 h of growth stage with 60 W power for 15 min of duration having 40% of duty cycle. The enzyme yield was found to be enhanced by a factor of 1.9-3.8 and 1.2-2.2 for uricase and alkaline protease respectively. Nevertheless, intracellular uricase was also observed in a fermentation broth after ultrasonic process intensification. The results indicate the effectiveness of low frequency ultrasound in improving enzyme yields with a vision of commercial applicability of the process.

Optogenetic regulation of engineered cellular metabolism for microbial chemical production.

The optimization of engineered metabolic pathways requires careful control over the levels and timing of metabolic enzyme expression. Optogenetic tools are ideal for achieving such precise control, as light can be applied and removed instantly without complex media changes. Here we show that light-controlled transcription can be used to enhance the biosynthesis of valuable products in engineered Saccharomyces cerevisiae. We introduce new optogenetic circuits to shift cells from a light-induced growth phase to a darkness-induced production phase, which allows us to control fermentation with only light. Furthermore, optogenetic control of engineered pathways enables a new mode of bioreactor operation using periodic light pulses to tune enzyme expression during the production phase of fermentation to increase yields. Using these advances, we control the mitochondrial isobutanol pathway to produce up to 8.49 ± 0.31 g l-1 of isobutanol and 2.38 ± 0.06 g l-1 of 2-methyl-1-butanol micro-aerobically from glucose. These results make a compelling case for the application of optogenetics to metabolic engineering for the production of valuable products.

Low-melanin containing pullulan production from sugarcane bagasse hydrolysate by Aureobasidium pullulans in fermentations assisted by light-emitting diode.

Pullulan is a polymer produced by Aureobasidium pullulans and the main bottleneck for its industrial production is the presence of melanin pigment. In this study, light-emitting diodes (LEDs) of different wavelengths were used to assist the fermentation process aiming to produce low-melanin containing pullulan by wild strain of A. pullulans LB83 with different carbon sources. Under white light using glucose-based medium, 11.75g.L-1 of pullulan with high melanin content (45.70UA540nm.g-1) was obtained, this production improved in process assisted by blue LED light, that resulted in 15.77g.L-1 of pullulan with reduced content of melanin (4.46UA540nm.g-1). By using sugarcane bagasse (SCB) hydrolysate as carbon source, similar concentration of pullulan (about 20g.L-1) was achieved using white and blue LED lights, with lower melanin contents in last. Use of LED light was found as a promising approach to assist biotechnological process for low-melanin containing pullulan production.

Use of ultrasounds in the food industry-Methods and effects on quality, safety, and organoleptic characteristics of foods: A review.

The use of ultrasounds has recently gained significant interest in the food industry mainly due to the new trends of consumers toward functional foods. Offering several advantages, this form of energy can be applied for the improvement of qualitative characteristics of high-quality foods as well as for assuring safety of a vast variety of foodstuffs, and at the same time minimizing any negative effects of the sensory characteristics of foods. Furthermore, the non-destructive nature of this technology offers several opportunities for the compositional analysis of foods. However, further research is required for the improvement of related techniques and the reduction of application costs in order to render this technology efficient for industrial use. This review paper covers the main applications of ultrasounds as well as several advantages of the use of the technology in combination with conventional techniques. The effects of ultrasounds on the characteristics, microbial safety, and quality of several foods are also detailed.

Enhancement of pectinase production by ultraviolet irradiation and diethyl sulfate mutagenesis of a Fusarium oxysporum isolate.

Fusarium oxysporum strain BM-201 was treated with ultraviolet (UV) radiation to obtain a high pectinase-producing strain. Mutant UV-10-41 was obtained and then treated by diethyl sulfate. Next, the mutant UV-diethyl sulfate-43 derived from UV-10-41 was selected as high pectinase-producing strain. Mutant UV-diethyl sulfate-43 was incubated on slant for 10 generations, demonstrating that the pectinase-producing genes were stable. Pectinase activity reached 391.2 U/mL, which is 73.6% higher than that of the original strain.

Study of a High-Yield Cellulase System Created by Heavy-Ion Irradiation-Induced Mutagenesis of Aspergillus niger and Mixed Fermentation with Trichoderma reesei.

The aim of this study was to evaluate and validate the efficiency of 12C6+ irradiation of Aspergillus niger (A. niger) or mutagenesis via mixed Trichoderma viride (T. viride) culturing as well as a liquid cultivation method for cellulase production via mixed Trichoderma reesei (T. reesei) and A. niger culture fermentation. The first mutagenesis approach was employed to optimize yield from a cellulase-producing strain via heavy-ion mutagenesis and high-throughput screening, and the second was to effectively achieve enzymatic hydrolysis of cellulase from a mixed culture of mutant T. viride and A. niger. We found that 12C6+-ion irradiation induced changes in cellulase biosynthesis in A. niger but had no effect on the time course of the synthesis. It is notable that the exoglucanases (CBH) activities of A. niger strains H11-1 and H differed (6.71 U/mL vs. 6.01 U/mL) and were significantly higher than that of A. niger mutant H3-1. Compared with strain H, the filter paper assay (FPA), endoglucanase (EG) and ß-glucosidase (BGL) activities of mutant strain H11-1 were increased by 250.26%, 30.26% and 34.91%, respectively. A mixed culture system was successfully optimized, and the best ratio of T. reesei to A. niger was 5:1 for 96 h with simultaneous inoculation. The BGL activity of the mixed culture increased after 72 h. At 96 h, the FPA and BGL activities of the mixed culture were 689.00 and 797.15 U/mL, respectively, significantly higher than those of monocultures, which were 408.70 and 646.98 U/mL for T. reesei and 447.29 and 658.89 U/mL for A. niger, respectively. The EG activity of the mixed culture was 2342.81 U/mL, a value that was significantly higher than that of monocultures at 2206.57 U/mL for T. reesei and 1727.62 U/mL for A. niger. In summary, cellulose production and hydrolysis yields were significantly enhanced by the proposed combination scheme.

A New Hydrogen-Producing Strain and Its Characterization of Hydrogen Production.

A newly isolated photo non-sulfur (PNS) bacterium was identified as Rhodopseudomonas palustris PB-Z by sequencing of 16S ribosomal DNA (rDNA) genes and phylogenetic analysis. Under vigorous stirring (240 rpm), the hydrogen production performances were greatly improved: The maximum hydrogen production rate and cumulative hydrogen production increased by 188.9 ± 0.07 % and 83.0 ± 0.06 %, respectively, due to the hydrogen bubbles were immediately removed from the culture medium. The effects of different wavelength of light on hydrogen production with stirring were much different from that without stirring. The ranking on the photo-hydrogen production performance was white > yellow > green > blue > red without stirring and white > yellow > blue > red > green under stirring. The best light source for hydrogen production was tungsten filament lamp. The optimum temperature was 35 °C. The maximal hydrogen production rate and cumulative hydrogen production reached 78.7 ± 2.3 ml/l/h and 1728.1 ± 92.7 mol H2/l culture, respectively, under 35 °C, 240 rpm, and illumination of 4000 lux. Pyruvate was one of the main sources of CO2 and has a great impact on the gas composition.

Bioprocess-Technological Potential of Irradiation-Based Fungal Pretreatment Platform Relevant to Lignocellulolytic Biocascade.

Lignocellulose-decaying fungal bioplatforms available are not commercially accessible and are limited to short-term use. In this study, those limitations were overcome by developing a platform using water-soaked rice straw (RS) biodegraded by irradiation-based fungal pretreatment (IBFP). This eco-friendly system increased the ability of RS to biodegrade and ferment without the generation of inhibitory compounds. When processed RS (i.e., with a water-soaking ratio of 81 % and irradiation dose of 80 kGy at 1 MeV and 0.12 mA) was pretreated with Dichomitus squalens for 9 days, the sugar yield was 58.5 % of the theoretical maximum. This sugar yield was comparable to that obtained with unirradiated RS for 15 days, which was 57.9 %. Furthermore, the ethanol concentration of 9.7 g L(-1) provided a yield of 58.1 %; the theoretical maximum and productivity at 0.40 g L(-1) h(-1) were determined after simultaneous saccharification and fermentation for 24 h. In addition, microscopic images revealed that IBFP induced predominant ultrastructural modifications to the surface of cell wall fibers. The peroxidative profiles for different biosystems were analyzed in order to understand substrate-specific biocascades based on the differences in biomass components. The activation level of core lignocellulolysis-related factors was analogous under the optimized conditions of each system.

The role of pH control on biohydrogen production by single stage hybrid dark- and photo-fermentation.

The role of pH control on biohydrogen production by co-culture of dark-fermentative Clostridium acetobutylicum and photofermentative Rhodobacter sphaeroides was studied. Single stage dark fermentation, photofermentation and hybrid co-culture systems were studied at different values of controlled and uncontrolled pH. Increasing pH during dark fermentation resulted in lower hydrogen production rate (HPR) and longer lag time for both controlled and uncontrolled conditions. However, it only slightly affected cumulative H2 volume. Results have shown that pH control at pH 7.5 increased photofermentative hydrogen production from 0.966 to 2.502 L H2/L(medium) when compared to uncontrolled process. Fixed pH value has proven to be an important control strategy also for the hybrid process and resulted in obtaining balanced co-culture of dark and photofermentative bacteria. Control of pH at 7.0 was found optimum for bacteria cooperation in the co-culture what resulted in obtaining 2.533 L H2/L(medium) and H2 yield of 6.22 mol H2/mol glucose.

Influence of Altered NADH Metabolic Pathway on the Respiratory-deficient Mutant of Rhizopus oryzae and its L-lactate Production.

Respiratory-deficient mutants of Rhizopus oryzae (R. oryzae) AS 3.3461 were acquired by ultraviolet (UV) irradiation to investigate changes in intracellular NADH metabolic pathway and its influence on the fermentation characteristics of the strain. Compared with R. oryzae AS 3.3461, the intracellular ATP level of the respiratory-deficient strain UV-1 decreased by 52.7 % and the glucose utilization rate rose by 8.9 %; When incubated for 36 h, the activities of phosphofructokinase (PFK), hexokinase (HK), and pyruvate kinase (PK) in the mutant rose by 74.2, 7.2, and 12.0 %, respectively; when incubated for 48 h, the intracellular NADH/NAD(+) ratio of the mutant rose by 14.6 %; when a mixed carbon source with a glucose/gluconic acid ratio of 1:1 was substituted to culture the mutant, the NADH/NAD(+) ratio decreased by 4.6 %; the ATP content dropped by 27.6 %; the lactate dehydrogenase (LDH) activity rose by 22.7 %; and the lactate yield rose by 11.6 %. These results indicated that changes to the NADH metabolic pathway under a low-energy charge level can effectively increase the glycolytic rate and further improve the yield of L-lactate of R. oryzae.

Influence of Light Intensity on Growth and Pigment Production by Monascus ruber in Submerged Fermentation.

To reduce environmental problems caused by glycerine accumulation and to make the production of biodiesel more profitable, crude glycerin without treatment was used as substrate for obtaining higher value-added bioproducts. Monascus ruber is a filamentous fungus that produces pigments, particularly red ones, which are used for coloring foods (rice wine and meat products). The interest in developing pigments from natural sources is increasing due to the restriction of using synthetic dyes. The effects of temperature, pH, microorganism morphology, aeration, nitrogen source, and substrates have been studied in the cultivation of M. ruber. In this work, it was observed that light intensity is also an important factor that should be considered for understanding the metabolism of the fungus. In M. ruber cultivation, inhibition of growth and pigment production was observed in Petri dishes and blaffed flasks exposed to direct illumination. Growth and pigment production were higher in Petri dishes and flasks exposed to red light and in the absence of light. Radial growth rate of M. ruber in plates in darkness was 1.50 mm day(-1) and in plates exposed to direct illumination was 0.59 mm day(-1). Maximum production of red pigments (8.32 UA) and biomass (8.82 g L(-1)) were obtained in baffled flasks covered with red film and 7.17 UA of red pigments, and 7.40 g L(-1) of biomass was obtained in flasks incubated in darkness. Under conditions of 1248 lux of luminance, the maximum pigment production was 4.48 UA, with production of 6.94 g L(-1) of biomass, indicating that the fungus has photoreceptors which influence the physiological responses.

The impact of gamma radiation on sediment microbial processes.

Microbial communities have the potential to control the biogeochemical fate of some radionuclides in contaminated land scenarios or in the vicinity of a geological repository for radioactive waste. However, there have been few studies of ionizing radiation effects on microbial communities in sediment systems. Here, acetate and lactate amended sediment microcosms irradiated with gamma radiation at 0.5 or 30 Gy h(-1) for 8 weeks all displayed NO3 (-) and Fe(III) reduction, although the rate of Fe(III) reduction was decreased in 30-Gy h(-1) treatments. These systems were dominated by fermentation processes. Pyrosequencing indicated that the 30-Gy h(-1) treatment resulted in a community dominated by two Clostridial species. In systems containing no added electron donor, irradiation at either dose rate did not restrict NO3 (-), Fe(III), or SO4 (2-) reduction. Rather, Fe(III) reduction was stimulated in the 0.5-Gy h(-1)-treated systems. In irradiated systems, there was a relative increase in the proportion of bacteria capable of Fe(III) reduction, with Geothrix fermentans and Geobacter sp. identified in the 0.5-Gy h(-1) and 30-Gy h(-1) treatments, respectively. These results indicate that biogeochemical processes will likely not be restricted by dose rates in such environments, and electron accepting processes may even be stimulated by radiation.

Prospective technology on bioethanol production from photofermentation.

The most important global demand is the energy supply from alternative source. Ethanol may be considered an environmental friendly fuel that has been produced by feedstock. The production of ethanol by microalgae represent a process with reduced environmental impact with efficient CO2 fixation and requiring less arable land. This work studied the production of ethanol from green alga Chlamydomonas reinhardtii through the cellular metabolism in a light/dark cycle at 25 °C in a TAP medium with sulfur depletion. The parameters evaluated were inoculum concentration and the medium supplementation with mixotrophic carbon sources. The combination of C.reinhardtii and Rhodobacter capsulatus through a hybrid or co-culture systems was also investigated as well. C.reinhardtii maintained in TAP-S produced 19.25±4.16 g/L (ethanol). In addition, in a hybrid system, with medium initially supplemented with milk whey permeated and the algal effluent used by R. capsulatus, the ethanol production achieved 19.94±2.67 g/L.