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.

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.

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.

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.

UVC-mutagenesis in acetogens: resistance to methanol, ethanol, acetone, or n-butanol in recombinants with tailored genomes as the step in engineering of commercial biocatalysts for continuous CO2/H2 blend fermentations.

Time- and cost-efficient six-step UVC-mutagenesis was developed and validated to generate acetogen mutants with preliminary reduced genomes to prevent product inhibition in the to-be-engineered commercial biocatalysts. Genome reduction was performed via elimination of pta, ack, spo0A, spo0J and some pro-phage genes. UVC-mutants such as Clostridium sp. MT1784RG, Clostridium sp. MT653RG, Clostridium sp. MT896RG, and Clostridium sp. MT1962RG (all 4 share 97 % DNA homology with Clostridium ljungdahlii ATCC 55383) were selected based on resistance to methanol (3 M), ethanol (3.6 M), acetone (2.5 M), or n-butanol (0.688 M), respectively. As a part of the biocatalyst engineering algorithm, genome reduction step was associated with integration of attTn7 recognition sequence to the chromosomes of each of the above strains to prepare the defined integration sites for future integration of multi-copy synthetic operons encoding biosynthesis of methanol, ethanol, acetone or n-butanol. Reduced genome mutants had cell duplication times decreased compared to the same for the respective parental strains. All groups of mutants had decreased share of palmitic (C16:0) and increased share of oleic (C18:1) acids along with detection of isopropylstearate (C20) compared to the parental strains. Mutants resistant to acetone and n-butanol also had monounsaturated fatty acid (C20:1) not found in parental strains. Cyclopropane fatty acid (C21) was identified only in n-butanol resistant mutants.

Overexpression of plastidial thioredoxin f leads to enhanced starch accumulation in tobacco leaves.

Starch, the most abundant storage carbohydrate in plants, has been a major feedstock for first-generation biofuels. Growing fuel demands require, however, that the starch yields of energy crops be improved. Leaf starch is synthesised during the day and degraded at night to power nonphotosynthetic metabolism. Redox regulation has been associated with the coordination of the enzymes involved in starch metabolism, but neither the signals nor mechanisms that regulate this metabolism are entirely clear. In this work, the thioredoxin (Trx) f and m genes, which code for key enzymes in plastid redox regulation, were overexpressed from the plastid genome. Tobacco plants overexpressing Trx f, but not Trx m, showed an increase of up to 700% in leaf starch accumulation, accompanied by an increase in leaf sugars, specific leaf weight (SLW), and leaf biomass yield. To test the potential of these plants as a nonfood energy crop, tobacco leaves overexpressing Trx f were subjected to enzymatic hydrolysis, and around a 500% increase in the release of fermentable sugars was recorded. The results show that Trx f is a more effective regulator of photosynthetic carbon metabolism in planta than Trx m. The overexpression of Trx f might therefore provide a means of increasing the carbohydrate content of plants destined for use in biofuel production. It might also provide a means of improving the nutritional properties of staple food crops.

Influence of anaerobiosis and low temperature on Bacillus cereus growth, metabolism, and membrane properties.

The impact of simultaneous anaerobiosis and low temperature on growth parameters, metabolism, and membrane properties of Bacillus cereus ATCC 14579 was studied. No growth was observed under anaerobiosis at 12°C. In bioreactors, growth rates and biomass production were drastically reduced by simultaneous anaerobiosis and low temperature (15°C). The two conditions had a synergistic effect on biomass reduction. In anaerobic cultures, fermentative metabolism was modified by low temperature, with a marked reduction in ethanol production leading to a lower ability to produce NAD(+). Anaerobiosis reduced unsaturated fatty acids at both low optimal temperatures. In addition, simultaneous anaerobiosis and low temperatures markedly reduced levels of branched-chain fatty acids compared to all other conditions (accounting for 33% of total fatty acids against more 71% for low-temperature aerobiosis, optimal-temperature aerobiosis, and optimal-temperature anaerobiosis). This corresponded to high-melting-temperature lipids and to low-fluidity membranes, as indicated by differential scanning calorimetry, 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence anisotropy, and infrared spectroscopy. This is in contrast to requirements for cold adaptation. A link between modification in the synthesis of metabolites of fermentative metabolism and the reduction of branched-chain fatty acids at low temperature under anaerobiosis, through a modification of the oxidizing capacity, is assumed. This link may partly explain the impact of low temperature and anaerobiosis on membrane properties and growth performance.

Bioethanol production from Scenedesmus obliquus sugars: the influence of photobioreactors and culture conditions on biomass production.

A closed-loop vertical tubular photobioreactor (PBR), specially designed to operate under conditions of scarce flat land availability and irregular solar irradiance conditions, was used to study the potential of Scenedesmus obliquus biomass/sugar production. The results obtained were compared to those from an open-raceway pond and a closed-bubble column. The influence of the type of light source and the regime (natural vs artificial and continuous vs light/dark cycles) on the growth of the microalga and the extent of the sugar accumulation was studied in both PBRs. The best type of reactor studied was a closed-loop PBR illuminated with natural light/dark cycles. In all the cases, the relationship between the nitrate depletion and the sugar accumulation was observed. The microalga Scenedesmus was cultivated for 53 days in a raceway pond (4,500 L) and accumulated a maximum sugar content of 29 % g/g. It was pre-treated for carrying out ethanol fermentation assays, and the highest ethanol concentration obtained in the hydrolysate fermented by Kluyveromyces marxianus was 11.7 g/L.

Microwave radiation and reactor design influence microbial communities during methane fermentation.

The effect of reactor design and method of heating on the efficiency of methane fermentation and composition of microbial communities, especially methanogenic Archaea, were determined. The research was carried out using submerge- and trickling-bed reactors fed with wastewater and the heat supply into the reactors included a convection heating method and microwave radiation. The polymerase chain reaction-denaturing gradient gel electrophoresis and relative real-time PCR were used in order to assess the biofilm communities. The best fermentation results and the highest abundance of methanogenic Archaea in biomass were observed in microwave heated trickling-bed reactors. The research proved that in reactors of identical design, the application of microwaves enabled a higher fermentation efficiency to be obtained and simultaneously increased the diversity of methanogenic Archaea communities that favors process stability. All the identified sequences of Archaea belonged to Methanosarcina sp., suggesting that species from this genera are susceptible to non-thermal effects of microwaves. There were no effects from microwaves on the bacterial communities in both types of reactors, however, the bacterial species composition varied in the reactors of different design.

Application of low magnetic field on inulinase production by Geotrichum candidum under solid state fermentation using leek as substrate.

This study evaluates the application of low magnetic field (LMF) on inulinase enzyme production by Geotrichum candidum under solid state fermentation (SSF) using leek as potential carbon source. First, the fermentation conditions were optimized using normal magnetic field grown microorganism. Among eight G. candidum isolates, the most effective strain called G. candidum OC-7 was selected to use in further experiments. In the second part of the study, SSF was carried out under different LMFs (4 and 7 mT). The results showed that inulinase activity was strongly affected by LMF application. The highest enzyme activity was obtained as 535.2 U/g of dry substrate (gds) by 7 mT magnetic field grown G. candidum OC-7. On the contrary, the control had only 412.1 U/gds. Consequently, the use of leek presents a great potential as an alternative carbon source for inulinase production and magnetic field treatment could effectively be used in order to enhance the enzyme production.

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.

Nano-TiO2 enhanced photofermentative hydrogen produced from the dark fermentation liquid of waste activated sludge.

After anaerobic dark fermentation of waste activated sludge (WAS) for hydrogen production, there are a large number of organic compounds including protein, polysaccharide, and volatile fatty acids left in the dark fermentation liquid, which can be further bioconverted to hydrogen by photofermentation techniquea. In this study, the enhancement of photofermentative hydrogen produced from WAS dark fermentation liquid by using nano-TiO2 is reported. First, high concentration of NH(4)(+)-N in the dark fermentation liquid was observed to inhibit the photofermentative hydrogen production, and its removal was essential. Then the effect of nano-TiO2 on photofermentative hydrogen generation was investigated, and the addition of 100 mg/L nano-TiO2 increased hydrogen by 46.1%. Finally, the mechanisms for nano-TiO2 improving hydrogen production were investigated. It was found that nano-TiO2 improved the decomposition of protein and polysaccharide to small-molecule organic compounds and promoted the growth of photosynthetic bacteria and the activity of nitrogenase but decreased the H2-uptake hydrogenase activity.

Extremely low frequency magnetic field effects on metabolite of Aspergillus niger.

The effect of the extremely low frequency (ELF) magnetic field on citric acid and cellulase production by Aspergillus niger using liquid Charles culture medium was studied during shake flask culture. The cellular suspension was exposed to a magnetic field (t = 4 h, B = 1 mT, and f = 50 Hz). The dependence of yield of citric acid and activity of cellulase on time of exposure and on the value of the magnetic field induction B was measured. Both yield of citric acid and activity of cellulase increased with increasing exposure time and/or induction B, but the quantity of the effect was dependent on the chemical structure of metabolites. The metabolism of citric acid was more sensitive to the magnetic field than that of cellulase. From the measurement of the metabolism dynamics we concluded that the increase in the citric acid and activity of cellulase started immediately after the magnetic field was switched on.

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.

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.

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.

The temperature dependence of maltose transport in ale and lager strains of brewer's yeast.

Lager beers are traditionally made at lower temperatures (6-14 degrees C) than ales (15-25 degrees C). At low temperatures, lager strains (Saccharomyces pastorianus) ferment faster than ale strains (Saccharomyces cerevisiae). Two lager and two ale strains had similar maltose transport activities at 20 degrees C, but at 0 degrees C the lager strains had fivefold greater activity. AGT1, MTT1 and MALx1 are major maltose transporter genes. In nine tested lager strains, the AGT1 genes contained premature stop codons. None of five tested ale strains had this defect. All tested lager strains, but no ale strain, contained MTT1 genes. When functional AGT1 from an ale strain was expressed in a lager strain, the resultant maltose transport activity had the high temperature dependence characteristic of ale yeasts. Lager yeast MTT1 and MALx1 genes were expressed in a maltose-negative laboratory strain of S. cerevisiae. The resultant Mtt1 transport activity had low temperature dependence and the Malx1 activity had high temperature dependence. Faster fermentation at low temperature by lager strains than ale strains may result from their different maltose transporters. The loss of Agt1 transporters during the evolution of lager strains may have provided plasma membrane space for the Mtt1 transporters that perform better at a low temperature.

Effect of moderate electric field frequency on growth kinetics and metabolic activity of Lactobacillus acidophilus.

Moderate electric fields (MEF) have been previously shown to alter the metabolic activity of microbial cells; thus, the effect of frequency and electric field would be of considerable interest. We investigated herein the effects of MEF frequency on microbial growth kinetics and bacteriocin (Lacidin A) production of Lactobacillus acidophilus OSU 133 during fermentation. The following fermentation treatments were compared: conventional (for 40 h), MEF (1 V cm(-1), for 40 h), combination of MEF (1 V cm(-1), for the first 5 h) and conventional (for 35 h) at various frequency levels (45, 60, and 90 Hz) all at 30 degrees C, and control (conventional) fermentation at 37 degrees C. MEF treatments with purely sinusoidal waveforms at all frequencies at 30 degrees C produced a shorter lag phase than conventional fermentation. However, no lag phase reduction was found for a 60 Hz waveform that contained high-frequency harmonics. There was, however, a significant increase in the bacteriocin production under early MEF treatment at 60 Hz with high-frequency harmonics. On the basis of these observations, the fermentation process is accelerated by applying pure sinusoidal MEF at the early stage of growth while a significant increase in the bacteriocin production occurs when sinusoidal field at 60 Hz with harmonics is applied at the early stage of the growth.