Thanet Urit

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.

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.

Screening of Kluyveromyces strains for the production of ethyl acetate: Design and evaluation of a cultivation system

Some members of the yeast Kluyveromyces marxianus are known to convert sugars and ethanol into ethyl acetate. This metabolic activity offers a chance for an economically favorable use of whey. An exemplary batch experiment is presented to demonstrate the formation of ethyl acetate by K. marxianus DSM 5422 on whey‐based medium in an aerated 70‐L stirred reactor. A screening system was developed to check Kluyveromyces strains regarding their capability to form ethyl acetate. The designed screening system takes the volatility of ethyl acetate and the requirement of oxygen for sugar conversion into account and allows the analysis of ester formation kinetics. Modeling and measurement of the ester stripping from screening bottles guaranteed reliable data evaluation. Twelve of 23 tested strains produced noteworthy amounts of ethyl acetate with a yield between 0.13 and 0.23 g ester per gram sugar consumed. The productive strains showed individual kinetics of ester formation; some strains had a short interval of intense production while others exhibited a longer period of stable formation. Another factor of interest was the selectivity of product formation expressed as the mass ratio of formed ester and ethanol, which ranged from 0.9 to 3.5 g/g.

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.

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.

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.