Studies on the mechanism of synthesis of ethyl acetate in Kluyveromyces marxianus DSM 5422.

Kluyveromyces marxianus converts whey-borne sugar into ethyl acetate, an environmentally friendly solvent with many applications. K. marxianus DSM 5422 presumably synthesizes ethyl acetate from acetyl-SCoA. Iron limitation as a trigger for this synthesis is explained by a diminished aconitase and succinate dehydrogenase activity (both enzymes depend on iron) causing diversion of acetyl-SCoA from the tricarboxic acid cycle to ester synthesis. Copper limitation as another trigger for ester synthesis in this yeast refers to involvement of the electron transport chain (all ETC complexes depend on iron and complex IV requires copper). This hypothesis was checked by using several ETC inhibitors. Malonate was ineffective but carboxin partially inhibited complex II and initiated ester synthesis. Antimycin A and cyanide as complexes III and IV inhibitors initiated ester synthesis only at moderate levels while higher concentrations disrupted all respiration and caused ethanol formation. A restricted supply of oxygen (the terminal electron acceptor) also initiated some ester synthesis but primarily forced ethanol production. A switch from aerobic to anaerobic conditions nearly stopped ester synthesis and induced ethanol formation. Iron-limited ester formation was compared with anaerobic ethanol production; the ester yield was lower than the ethanol yield but a higher market price, a reduced number of process stages, a faster process, and decreased expenses for product recovery by stripping favor biotechnological ester production.

Perspectives for the biotechnological production of ethyl acetate by yeasts.

Ethyl acetate is an environmentally friendly solvent with many industrial applications. The production of ethyl acetate currently proceeds by energy-intensive petrochemical processes which are based on natural gas and crude oil without exception. Microbial synthesis of ethyl acetate could become an interesting alternative. The formation of esters as aroma compounds in food has been repeatedly reviewed, but a survey which deals with microbial synthesis of ethyl acetate as a bulk product is missing. The ability of yeasts for producing larger amounts of this ester is known for a long time. In the past, this potential was mainly of scientific interest, but in the future, it could be applied to large-scale ester production from renewable raw materials. Pichia anomala, Candida utilis, and Kluyveromyces marxianus are yeasts which convert sugar into ethyl acetate with a high yield where the latter is the most promising one. Special attention was paid to the mechanism of ester synthesis including regulatory aspects and to the maximum and expectable yield. Synthesis of much ethyl acetate requires oxygen which is usually supplied by aeration. Ethyl acetate is highly volatile so that aeration results in its phase transfer and stripping. This stripping process cannot be avoided but requires adequate handling during experimentation and offers a chance for a cost-efficient process-integrated recovery of the synthesized ester.

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 from whey by Kluyveromyces marxianus on a pilot scale.

Whey arising in huge amounts during milk processing is a valuable renewable resource in the field of White Biotechnology. Kluyveromyces marxianus is able to convert whey-borne lactose into ethyl acetate, an environmentally friendly solvent. Formation of ethyl acetate as a bulk product is triggered by iron (Fe). K. marxianus DSM 5422 was cultivated aerobically in whey-borne medium originally containing 40 µg/L Fe, supplemented with 1, 3 or 10 mg/L Fe in the pre-culture, using an 1 L or 70 L stirred reactor. The highest Fe content in the pre-culture promoted yeast growth in the main culture causing a high sugar consumption for growth and dissatisfactory formation of ethyl acetate, while the lowest Fe content limited yeast growth and promoted ester synthesis but slowed down the process. An intermediate Fe dose (ca. 0.5 µg Fe/g sugar) lastly represented a compromise between some yeast growth, a quite high yield of ethyl acetate and an acceptable duration of the process. The mass of ethyl acetate related to the sugar consumed amounted to 0.113, 0.265 and 0.239 g/g in the three processes corresponding to 21.9%, 51.4% and 46.3% of the theoretically maximum yield. The performance on a pilot scale was somewhat higher than on lab scale.

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 µg/g, while during chemostat cultivation at D = 0.15 h(-1), it was 23 µg/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 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.

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.