Influence of Metronidazole, CO, CO2, and Methanogens on the Fermentative Metabolism of the Anaerobic Fungus Neocallimastix sp. Strain L2

The effects of metronidazole, CO, methanogens, and CO₂ on the fermentation of glucose by the anaerobic fungus Neocallimastix sp. strain L2 were investigated. Both metronidazole and CO caused a shift in the fermentation products from predominantly H₂, acetate, and formate to lactate as the major product and caused a lower glucose consumption rate and cell protein yield. An increased lactate dehydrogenase activity and a decreased hydrogenase activity were observed in cells grown under both culture conditions. In metronidazole-grown cells, the amount of hydrogenase protein was decreased compared with the amount in cells grown in the absence of metronidazole. When Neocallimastix sp. strain L2 was cocultured with the methanogenic bacterium Methanobrevibacter smithii, the fermentation pattern changed in the opposite direction: H₂ and acetate production increased at the expense of the electron sink products lactate, succinate, and ethanol. A concomitant decrease in the enzyme activities leading to these electron sink products was observed, as well as an increase in the glucose consumption rate and cell protein yield, compared with those of pure cultures of the fungus. Low levels of CO₂ in the gas phase resulted in increased H₂ and lactate formation and decreased production of formate, acetate, succinate, and ethanol, a decreased glucose consumption rate and cell protein yield, and a decrease in most of the hydrogenosomal enzyme activities. None of the tested culture conditions resulted in changed quantities of hydrogenosomal proteins. The results indicate that manipulation of the pattern of fermentation in Neocallimastix sp. strain L2 results in changes in enzyme activities but not in the proliferation or disappearance of hydrogenosomes.     

Requirement for Ethylene Synthesis and Action during Relief of Thermoinhibition of Lettuce Seed Germination by Combinations of Gibberellic Acid, Kinetin, and Carbon Dioxide

Application of exogenous ethylene in combination with gibberellic acid (GA₃), kinetin (KIN), and/or CO₂ has been reported to induce germination of lettuce seeds at supraoptimal temperatures. However, it is not clear whether endogenous ethylene also plays a mediatory role when germination under these conditions is induced by treatment regimes that do not include ethylene. Therefore, possible involvement of endogenous ethylene during the relief of thermoinhibition of lettuce (Lactuca sativa L. cv Grand Rapids) seed germination at 32°C was investigated. Combinations of GA₃ (0.5 millimolar), KIN (0.05 millimolar), and CO₂ (10%) were used to induce germination. Little germination occurred in controls or upon treatment with ethylene, KIN, or CO₂. Neither KIN nor CO₂ affected the rate of ethylene production by seeds. Both germination and ethylene production were slightly promoted by GA₃. Treatments with GA₃+CO₂, GA₃+KIN, or GA₃+CO₂+KIN resulted in approximately 10-to 40-fold increases in ethylene production and 50 to 100% promotion of germination as compared to controls. Initial ethylene evolution from the treated seeds was greater than from the controls and a major surge in ethylene evolution occurred at the time of visible germination. Application of 1 millimolar 2-aminoethoxyvinyl glycine (AVG), an inhibitor of ethylene synthesis, in combination with any of above three treatments inhibited the ethylene production to below control levels. This was accompanied by a marked decline in germination percentage. Germination was also inhibited by 2,5-norbornadiene (0.25-2 milliliters per liter), a competitive inhibitor of ethylene action. Application of exogenous ethylene (1-100 microliters per liter) overcame the inhibitory effects of AVG and 2,5-norbornadiene on germination. The results demonstrate that endogenous ethylene synthesis and action are essential for the alleviation of thermoinhibition of lettuce seeds by combinations of GA₃, KIN, and CO₂. It also appears that these treatment combinations do not act exclusively via promotion of ethylene evolution as the application of exogenous ethylene alone did not promote germination.     

Whey Fermentation by Anaerobiospirillum succiniciproducens for Production of a Succinate-Based Animal Feed Additive

Anaerobic fermentation processes for the production of a succinate-rich animal feed supplement from raw whey were investigated with batch, continuous, and variable-volume fed-batch cultures with Anaerobiospirillum succiniciproducens. The highest succinate yield, 90%, was obtained in a variable-volume fed-batch process in comparison to 80% yield in a batch cultivation mode. In continuous culture, succinate productivity was 3 g/liter/h, and the yield was 60%. Under conditions of excess CO₂, more than 90% of the whey-lactose was consumed, with an end product ratio of 4 succinate to 1 acetate. Under conditions of limited CO₂, lactose was only partially consumed and lactate was the major end product, with lower levels of ethanol, succinate, and acetate. When the succinic acid in this fermentation product was added to rumen fluid, it was completely consumed by a mixed rumen population and was 90% decarboxylated to propionate on a molar basis. The whey fermentation product formed under excess CO₂, which contained mainly organic acids and cells, could potentially be used as an animal feed supplement.     

Influence of environmental factors on endo-β-1,4-glucanase production by Bacillus HR68, isolated from a Zimbabwean hot spring

The production of endo-β-1,4-glucanase by a Bacillus strain isolated from a hot spring in Zimbabwe was studied in batch culture, chemostat culture, and carbon dioxide-regulated auxostat (CO₂-auxostat). The bacteria produced the enzyme in the presence of excess glucose or sucroso, but not under carbon-limited conditions in a chemostat using mineral medium. There was a specific growth rate dependent linear increase in enzyme production in glucose excess, nitrogen-limited chemostat cultures. A high specific growth rate of 2.2 h^-1 and a high rate of enzyme production of 362 nkat (mg dry mass h)^-1 were attained under nutrient rich conditions in the CO₂-auxostat. The bacteria had the highest specific growth rate and endo-β-1,4-glucanase enzyme production at 50° C. The maximum specific growth rate and the rate of enzyme production increased when yeast extract and tryptone were added in increasing amounts to the mineral medium used for cultivation in separate experiments. Increasing the glucose concentration in the CO₂-auxostat cultures increased the rate of enzyme production but did not affect the specific growth rate.     

Absorbed substrate fermentation for pectinase production with Aspergillus niger

Summary A 130 litre packed-bed bioreactor was used for pectinase production with Aspergillus niger using absorbed substrate fermentation techniques. Pectinolytic enzyme activity and relative CO₂ production were used as indicators of metabolic activity. Absorbed substrate fermentation is an efficient process for pectinase production and is also an interesting model because the culture medium, water, nutrients and specific inducers, can be designed at the desired concentrations.     

Outdoor cultivation of Chlamydomonas reinhardtii for photobiological hydrogen production

Photobiological hydrogen production by the unicellular green alga Chlamydomonas reinhardtii has been studied under laboratory conditions to a vast extend but has not been investigated under outdoor conditions yet. Because the hydrogen-producing hydrogenase is very sensitive to oxygen, the production must be performed in a two-stage process: generation of the required algal biomass under oxygenic photosynthesis, followed by hydrogen biosynthesis under anaerobic conditions. In order to design a sustainable process, cultivation and subsequent hydrogen production under cost-free sunlight was investigated in this work for the first time. First, cells were grown in closed photobioreactors under simulated outdoor conditions according to the light intensities of an idealized summer day (up to 2,000?μmol photons m^?2?s^?1) in order to achieve results independent of varying, and therefore not reproducible, weather conditions. The following outdoor experiments showed comparable growth characteristics and similar cell densities. However, the use of cells grown under outdoor, simulated outdoor, or high light conditions generally resulted in significantly lower hydrogen yields compared to the use of cells cultivated under low and continuous irradiance. In order to lower cultivation costs during the growth phase, the use of 10% CO₂ corresponding to the CO₂ content of flue gas was investigated. By supplying additional CO₂ during growth under the light profile corresponding to an idealized summer day, no significant increase of cell densities could be achieved, but the subsequent hydrogen production increased compared to hydrogen production of cells grown under atmospheric CO₂.     

Partitioning pathways of CO2 production in peatlands with stable carbon isotopes

Although methanogenic pathways generally produce equimolar amounts of carbon dioxide and methane, CO₂ concentrations are often reported to be higher than CH₄ concentrations in both field and laboratory incubation studies of peat decomposition. In field settings, higher pore water concentrations of CO₂ may result from the loss of methane by: (1) ebullition due to the low solubility of methane in pore water and (2) vascular-plant transport. Higher CO₂ concentrations may also be caused by: (1) production of additional CO₂ by high-molecular weight (HMW) organic matter (OM) fermentation and/or (2) respiration from non-methanogenic pathways. In this study of a peatland where advection and transverse dispersion were the dominant pore water solute transport mechanisms, an isotope-mass balance approach was used to determine the proportions of CO₂ formed from non-fractionating OM respiration and HMW fermentation relative to CO₂ production from methanogenesis. This approach also allowed us to estimate the loss of CH₄ from the belowground system. The pathways of CO₂ production varied with depth and surface vegetation type. In a Carex-dominated fen, methane production initially produced 40 % of the total CO₂ and then increased to 90–100 % with increasing depth. In a Sphagnum-dominated bog, methanogenesis resulted in 60 % of total CO₂ production which increased to 100 % at depth. Both bogs and fens showed 85–100 % of methane loss from pore waters. Our results indicate that the isotopic composition of dissolved CO₂ is a powerful indicator to allow partitioning of the processes affecting peat remineralization and methane production.     

Effects of Carbon Dioxide on Growth and Maltose Fermentation by Bacteroides amylophilus

The requirement of carbon dioxide for growth of Bacteroides amylophilus is quantitatively similar to that of certain other rumen bacteria. Carbon dioxide could be replaced by bicarbonate, but not by formate or certain amino acids. Label from ¹⁴CO₂ was incorporated into the succinate produced during maltose fermentation by B. amylophilus, and during glucose fermentation by B. ruminicola, and during cellobiose fermentation by B. succinogenes. All of the incorporated label could be associated with the carboxyl function of the molecule. The depression in radioactivity per micromole of carbon in the succinate formed from the fermentation of uniformly labeled ¹⁴C-maltose by B. amylophilus was greater than would be expected if all of the succinate formed was produced via a direct CO₂ fixation pathway(s) involving phosphoenolpyruvate or pyruvate; the radioactivity per micromole of carbon suggests that as much as 60% of the total succinate results from a pathway(s) involving direct CO₂ fixation. Maltose fermentation by B. amylophilus was dependent upon CO₂ concentration, but CO₂ concentration could not be shown to influence either the fermentation end-product ratios or the proportion of total succinate formed attributable to CO₂ fixation.     

Minimization of Glycerol Production during the High-Performance Fed-Batch Ethanolic Fermentation Process in Saccharomyces cerevisiae, Using a Metabolic Model as a Prediction Tool

On the basis of knowledge of the biological role of glycerol in the redox balance of Saccharomyces cerevisiae, a fermentation strategy was defined to reduce the surplus formation of NADH, responsible for glycerol synthesis. A metabolic model was used to predict the operating conditions that would reduce glycerol production during ethanol fermentation. Experimental validation of the simulation results was done by monitoring the inlet substrate feeding during fed-batch S. cerevisiae cultivation in order to maintain the respiratory quotient (RQ) (defined as the CO₂ production to O₂ consumption ratio) value between 4 and 5. Compared to previous fermentations without glucose monitoring, the final glycerol concentration was successfully decreased. Although RQ-controlled fermentation led to a lower maximum specific ethanol production rate, it was possible to reach a high level of ethanol production: 85 g · liter⁻¹ with 1.7 g · liter⁻¹ glycerol in 30 h. We showed here that by using a metabolic model as a tool in prediction, it was possible to reduce glycerol production in a very high-performance ethanolic fermentation process.     

Development of a system for the on‐line measurement of carbon dioxide production in microbioreactors: Application to aerobic batch cultivations of Candida utilis

We developed and applied a conductometric method for the quantitative online measurement of the carbon dioxide (CO₂) production during batch cultivations of Candida utilis on a 100‐μL scale. The applied method for the CO₂ measurement consisted of absorption of the produced CO₂ from the exhaust gas of the microbioreactor in an alkali solution, of which the conductivity was measured on‐line. The measured conductivity change of the alkali solution showed a linear relation with the total amount of CO₂ absorbed. After calibration of the CO₂ measurement system, it was connected to a well of a 96‐well microtiter plate. The mixing in the well was achieved by a magnetic stirrer. Using online measurement of the CO₂ production during the cultivation, we show reproducible exponential batch growth of C. utilis on a 100‐μL scale. The CO₂ production measurements obtained from the microcultivation were compared with the CO₂ production measurement in a 4‐L bioreactor equipped with a conventional off‐gas analyzer. The measurements showed that on‐line measurement of the CO₂ production rate in microbioreactors can provide essential data for quantitative physiological studies and provide better understanding of microscale cultivations. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Valorization of CO2 through lithoautotrophic production of sustainable chemicals in Cupriavidus necator

Coupling recent advancements in genetic engineering of diverse microbes and gas-driven fermentation provides a path towards sustainable commodity chemical production. Cupriavidus necator H16 is a suitable species for this task because it effectively utilizes H₂ and CO₂ and is genetically tractable. Here, we demonstrate the versatility of C. necator for chemical production by engineering it to produce three products from CO₂ under lithotrophic conditions: sucrose, polyhydroxyalkanoates (PHAs), and lipochitooligosaccharides (LCOs). We engineered sucrose production in a co-culture system with heterotrophic growth 30 times that of WT C. necator. We engineered PHA production (20–60% DCW) and selectively altered product composition by combining different thioesterases and phaCs to produce copolymers directly from CO₂. And, we engineered C. necator to convert CO₂ into the LCO, a plant growth enhancer, with titers of ~1.4 mg/L—equivalent to yields in its native source, Bradyrhizobium. We applied the LCOs to germinating seeds as well as corn plants and observed increases in a variety of growth parameters. Taken together, these results expand our understanding of how a gas-utilizing bacteria can promote sustainable production.     

Mevalonate production by engineered acetogen biocatalyst during continuous fermentation of syngas or CO2/H2 blend

Naturally mevalonate-resistant acetogen Clostridium sp. MT1243 produced only 425 mM acetate during syngas fermentation. Using Clostridium sp. MT1243 we engineered biocatalyst selectively producing mevalonate from synthesis gas or CO₂/H₂ blend. Acetate production and spore formation were eliminated from Clostridium sp. MT1243 using Cre-lox66/lox71-system. Cell energy released via elimination of phosphotransacetylase, acetate kinase and early stage sporulation genes powered mevalonate accumulation in fermentation broth due to expression of synthetic thiolase, HMG-synthase, and HMG-reductase, three copies of each, integrated using Tn7-approach. Recombinants produced 145 mM mevalonate in five independent single-step fermentation runs 25 days each in five repeats using syngas blend 60 % CO and 40 % H₂ (v/v) (p < 0.005). Mevalonate production was 97 mM if only CO₂/H₂ blend was fed instead of syngas (p < 0.005). Mevalonate from CO₂/H₂ blend might serve as a commercial route to mitigate global warming in proportion to CO₂ fermentation scale worldwide.     

Mutual influence of light and CO2 on carbon sequestration via cultivating mixotrophic alga Auxenochlorella protothecoides UMN280 in an organic carbon-rich wastewater

This study focusses on the assimilation of carbon in concentrated municipal wastewater rich in organic carbon using the mixotrophic microalga Auxenochlorella protothecoides UMN280 with the addition of supplemental CO₂. The entire growth period of A. protothecoides UMN280 can be characterized by three phases: first, a phase where algae grew in a mixotrophic-dominated mode; second, a transition phase; and last, a phase where algae grew in a photoautotrophic-dominated mode. In this study, it was found that light intensity had a strong effect on algal biomass production; the culture system would transfer from a mixotrophic-dominated mode to a photoautotrophic-dominated mode quicker under higher light intensities. The addition of CO₂ exhibited an important role in the photoautotrophic-dominated cultivation stage. At certain level of irradiance and certain range of CO₂ injection rate, higher CO₂ injection rate would result in a higher level of carbon fixation. It is clearly beneficial to inject exogenous CO₂ in the mixotrophic wastewater algae production system when a light source is available, such as during daylight hours.     

Aerobic batch cultivation in micro bioreactor with integrated electrochemical sensor array

Aerobic batch cultivations of Candida utilis were carried out in two micro bioreactors with a working volume of 100 μL operated in parallel. The dimensions of the micro bioreactors were similar as the wells in a 96‐well microtiter plate, to preserve compatibility with the current high‐throughput cultivation systems. Each micro bioreactor was equipped with an electrochemical sensor array for the online measurement of temperature, pH, dissolved oxygen, and viable biomass concentration. Furthermore, the CO₂ production rate was obtained from the online measurement of cumulative CO₂ production during the cultivation. The online data obtained by the sensor array and the CO₂ production measurements appeared to be very reproducible for all batch cultivations performed and were highly comparable to measurement results obtained during a similar aerobic batch cultivation carried out in a conventional 4L bench‐scale bioreactor. Although the sensor chip certainly needs further improvement on some points, this work clearly shows the applicability of electrochemical sensor arrays for the monitoring of parallel micro‐scale fermentations, e.g. using the 96‐well microtiterplate format. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

On-line recognition of CO2 exit gas profiles for operational harvesting time of intracellular enzyme products from S. cerevisiae

This paper concerns the recognition of significant events in the trends from CO₂ exit gas profiles of a S.cerevisiae fermentation. Distinct changes in the on-line monitored variable, CO₂ exit gas concentration, are used to recognise identifiable phases of glucose depletion, biomass formation and the onset of intracellular enzyme production. Process automation for the harvesting of some intracellular enzyme products from S. cerevisiae is considered using on-line recognition of CO₂ exit gas peaks.     

The Possibility of Meeting Greenhouse Energy and CO<Subscript>2</Subscript> Demands Through Utilisation of Cucumber and Tomato Residues

The article examines the possibility of using residues from greenhouse cucumber and tomato cultivation as biomass for energy and CO₂ production in order to meet greenhouse needs. Methane fermentation and combustion were compared. Moreover, the legitimacy of ensiling as a storage method for biogas plant was evaluated. The tested waste was found to be an unsuitable feedstock for the production of silage due to low sugar and high protein content. Fresh waste had a higher biogas yield than silage; however, its fermentation lasted longer. Furthermore, the results showed that, in the case of fresh residues, the methane fermentation proved to be a more energy-efficient process, while air-dry biomass is a more sustainable feedstock for combustion. The energy and CO₂ balance showed that, regardless of the method used, the available quantity of waste is too small to meet the greenhouse needs.     

Metabolic engineering of Propionibacterium freudenreichii: effect of expressing phosphoenolpyruvate carboxylase on propionic acid production

Propionic acid is currently produced mainly via petrochemicals, but there is increasing interest in its fermentative production from renewable biomass. However, the current propionic acid fermentation process suffers from low product yield and productivity. In this work, the gene encoding phosphoenolpyruvate carboxylase (PPC) was cloned from Escherichia coli and expressed in Propionibacterium freudenreichii. PPC catalyzes the conversion of phosphoenolpyruvate to oxaloacetate with the fixation of one CO₂. Its expression in P. freudenreichii showed profound effects on propionic acid fermentation. Compared to the wild type, the mutant expressing the ppc gene grew significantly faster, consumed more glycerol, and produced propionate to a higher final titer at a faster rate. The mutant also produced significantly more propionate from glucose under elevated CO₂ partial pressure. These effects could be attributed to increased CO₂ fixation and resulting changes in the flux distributions in the dicarboxylic acid pathway.     

Fermentative products and dark CO2 fixation during germination of seeds of Cicer arietinum

During the germination of Cicer arietinum L. the amounts of ethanol, lactate and malate reached their highest values at 24 hr, the concentration of ethanol being about 4 times that of lactate and twice that of malate. The activities of phosphoenolpyruvate carboxylase and malic enzyme seem to be correlated with the ability of cotyledons to fix CO₂ from NaH¹⁴CO₃ into malate and with the further decrease in this metabolise from 36 hr onwards.