Formation of ethyl acetate by Kluyveromyces marxianus on whey during aerobic batch cultivation at specific trace element limitation

Kluyveromyces marxianus is able to transform lactose into ethyl acetate as a bulk product which offers a chance for an economical reuse of whey-borne sugar. Ethyl acetate is highly volatile and allows its process-integrated recovery by stripping from the aerated bioreactor. Extensive formation of ethyl acetate by K. marxianus DSM 5422 required restriction of yeast growth by a lack of trace elements. Several aerobic batch processes were done in a 1-L stirred reactor using whey-borne culture medium supplemented with an individual trace element solution excluding Mn, Mo, Fe, Cu, or Zn for identifying the trace element(s) crucial for the observed ester synthesis. Only a lack of Fe, Cu, or Zn restricted yeast growth while exclusion of Mn and Mo did not exhibit any effect due to a higher amount of the latter in the used whey. Limitation of growth by Fe or Cu caused significant production of ethyl acetate while limitation by Zn resulted in formation of ethanol. A lack of Fe or Cu obviously makes the respiratory chain inefficient resulting in an increased mitochondrial NADH level followed by a reduced metabolic flux of acetyl-SCoA into the citrate cycle. Synthesis of ethyl acetate from acetyl-SCoA and ethanol by alcoholysis is thus interpreted as an overflow metabolism.     

C. elegans Eats Its Own Intestine to Make Yolk Leading to Multiple Senescent Pathologies

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Hydrogen production and anaerobic decolorization of wastewater containing Reactive Blue 4 by a bacterial consortium of Salmonella subterranea and Paenibacillus polymyxa

Anaerobic biodegradability of wastewater (3,000 mg CODcr/l) containing 300 mg/l Reactive Blue 4, with different co-substrates, glucose, butyrate and propionate by a bacterial consortium of Salmonella subterranea and Paenibacillus polymyxa, concomitantly with hydrogen production was investigated at 35°C. The accumulative hydrogen production at 3,067 mg CODcr/l was obtained after 7 days of incubation with glucose, sludge, the bacterial consortium. The volatile fatty acids, residual glucose and the total organic carbon were correlated to hydrogen obtained. Interestingly, the bacterial consortium possess decolorization ability showing approximately 24% dye removal after 24 h incubation using glucose as a co-substrate, which was about two and eight times those of butyrate (10%), propionate (12%) and control (3%), respectively. RB4 decolorization occurred through acidogenesis, as high volatile fatty acids but low methane was detected. The bacterial consortium will be the bacterial strains of interest for further decolorization and hydrogen production of industrial waste water.     

Formation of ethyl acetate by Kluyveromyces marxianus on whey during aerobic batch and chemostat cultivation at iron limitation

The ability of Kluyveromyces marxianus to convert lactose into ethyl acetate offers a chance for an economic reuse of whey. Former experiments with K. marxianus DSM 5422 proved limitation of growth by iron (Fe) or copper as a precondition for significant ester synthesis. Several aerobic batch and chemostat cultivations were done with whey-borne media of a variable Fe content for exploring the effect of Fe on growth, the Fe content of biomass, and metabolite synthesis. At low Fe doses, Fe was the growth-limiting factor, the available Fe was completely absorbed by the yeasts, and the biomass formation linearly depended on the Fe dose governed by a minimum Fe content in the yeasts, x _Fe,min. At batch conditions, x _Fe,min was 8.8???g/g, while during chemostat cultivation at D?=?0.15?h^?1, it was 23???g/g. At high Fe doses, sugar was the growth-limiting factor, Fe was more or less absorbed, and the formed biomass became constant. Significant amounts of ethyl acetate were only formed at Fe limitation while high Fe doses suppressed ester formation. Analysis of formed metabolites such as glycerol, pyruvate, acetate, ethanol, ethyl acetate, isocitrate, 2-oxoglutarate, succinate, and malate during chemostat cultivation allowed some interpretation of the Fe-dependent mechanism of ester synthesis; formation of ethyl acetate from acetyl-SCoA and ethanol is obviously initiated by a diminished metabolic flux of acetyl-SCoA into the citrate cycle and by a limited oxidation of NADH in the respiratory chain since Fe is required for the function of aconitase, succinate dehydrogenase, and the electron-transferring proteins.     

Formation of ethyl acetate by Kluyveromyces marxianus on whey: Influence of aeration and inhibition of yeast growth by ethyl acetate

The ability of the yeast Kluyveromyces marxianus to convert lactose into ethyl acetate offers good opportunities for the economical reuse of whey. The formation of ethyl acetate as a bulk product depends on aerobic conditions. Aeration of the bioreactor results in discharge of the volatile ester with the exhaust gas that allows its process‐integrated recovery. The influence of aeration (varied from 10 to 50 L/h) was investigated during batch cultivation of K. marxianus DSM 5422 in 0.6 L whey‐borne medium using a stirred reactor. With lower aeration rates, the ester accumulated in the bioreactor and reached higher concentrations in the culture medium and the off gas. A high ester concentration in the gas phase is considered beneficial for ester recovery from the gas, while a high ester concentration in the medium inhibited yeast growth and slowed down the process. To further investigate this effect, the inhibition of growth by ethyl acetate was studied in a sealed cultivation system. Here, increasing ester concentrations caused a nearly linear decrease of the growth rate with complete inhibition at concentrations greater than 17 g/L ethyl acetate. Both the cultivation process and the growth rate depending on ethyl acetate were described by mathematical models. The simulated processes agreed well with the measured data.     

Growth of Kluyveromyces marxianus and formation of ethyl acetate depending on temperature

Conversion of lactose into ethyl acetate by Kluyveromyces marxianus allows economic reuse of whey-borne sugar. The high volatility of ethyl acetate enables its process-integrated recovery by stripping. This stripping is governed by both the aeration rate and the partition coefficient, K _EA,L/G. Cultivation at elevated temperatures should decrease the K _EA,L/G value and thus favor stripping. K. marxianus DSM 5422 as a potent producer of ethyl acetate was cultivated aerobically in whey-borne media for studying temperature-dependent growth and ester formation. Shake flask cultivation proved thermal tolerance of this yeast growing from 7 to 47 °C with a maximum rate of 0.75 h^?1 at 40 °C. The biomass yield was 0.41 g/g at moderate temperatures while low and high temperatures caused distinct drops. The observed μ-T and Y _X/S-T dependencies were described by mathematical models. Further cultivations were done in an 1-L stirred reactor for exploring the effect of temperature on ester synthesis. Cultivation at 32 °C caused significant ester formation (Y _EA/S?=?0.197 g/g) while cultivation at 42 °C suppressed ester synthesis (Y _EA/S?=?0.002 g/g). The high temperature affected metal dissolution from the bioreactor delivering iron for yeast growth and preventing ester synthesis. Cultivation at 32 °C with a switch to 42 °C at the onset of ester synthesis allowed quick and efficient ester production (Y _EA/S?=?0.289 g/g). The high temperature lowered the K _EA,L/G value from 78 to 44 L/L which heightened the gas-phase ester concentration (favoring ester recovery) without increasing the liquid-phase concentration (avoiding product inhibition).     

Formation of ethyl acetate by Kluyveromyces marxianus on whey: studies of the ester stripping

Kluyveromyces marxianus is capable of converting lactose into ethyl acetate offering a chance for an economical reuse of whey. The microbial formation of ethyl acetate as a bulk product calls for an aerobic process and, thus, the highly volatile ethyl acetate is discharged from the aerated bioreactor. This stripping process was modeled and investigated experimentally. The stripping rate was proportional to the gas flow and nearly independent of the stirring rate since the stripping was governed by the absorption capacity of the exhaust gas rather than the phase transfer. Cooling the exhaust gas did not noticeably influence the stripping. One batch experiment is presented in detail to demonstrate the formation of ethyl acetate by K. maxianus DSM 5422 on whey. Further batch experiments showed that a substantial formation of ethyl acetate only occurred when the yeast growth was limited by a lack of trace elements. The highest product yield observed was 0.25 g ethyl acetate per g lactose which is nearly 50% of the theoretical maximum.