Christian Löser

Bioavailability of hydrocarbons during microbial remediation of a sandy soil

Abstract The microbial degradation of hydrocarbons was studied in an artificially contaminated sandy soil, using a pilot-scale percolator system. After a short lag period, an intensive degradation occurred, which diminished in time and completely stopped in the end, despite large residual contaminations (residues of 56% diesel fuel, 20% n-hexadecane and 3.5% phenanthrene at the initial loadings of each 3000 mg/kg). The remaining pollutant content was influenced by the kind of hydrocarbon but was nearly independent of its initial loading. According to a model-aided analysis of the carbon dioxide production during remediation, the observed stagnation of degradation was caused by a limited bioavailability of the pollutants. The degradation in the soil-free aqueous phase was more extensive than in the soil, which suggests that the limited bioavailability in the soil can be attributed mainly to matrix-dependent rather than substrate-dependent influences. Generally, fine particles and organic matter are mainly responsible for the adsorption of pollutants to the soil matrix. Our sandy soil also bound hydrocarbons adsorptively although it contained neither silty material nor significant amounts of organic matter. As shown by Brunauer Emmett Teller (BET) analysis, the soil particles were covered by micropores, which enlarged the soil surface by a factor of 120 in comparison with the macroscopic surface area. The microporosity is the reason for the hydrocarbons being more strongly adsorbed to the sandy soil than expected.

Microbial degradation of hydrocarbons in soil during aerobic/anaerobic changes and under purely aerobic conditions

Abstract Microbial hydrocarbon degradation in soil was studied during periodical aerobic/anaerobic switching and under purely aerobic conditions by using a pilot-scale plant with diesel-fuel-contaminated sand. The system worked according to the percolation principle with controlled circulation of process water and aeration. Periodical switching between 4 h of aerobic and 2 h of anaerobic conditions was achieved by repeated saturation of the soil with water. Whatever the cultivation mode, less than 50% of the diesel was degraded after 650 h because the hydrocarbons were adsorbed. Contrary to expectations, aerobic/anaerobic changes neither accelerated the rate of degradation nor reduced the residual hydrocarbon content of the soil. Obviously the pollutant degradation rate was determined mainly by transport phenomena and less by the efficiency of microbial metabolism. The total mass of oxygen consumed and carbon dioxide produced was greater under aerobic/anaerobic changing than under aerobic conditions, although the mass of hydrocarbons degraded was nearly the same. As shown by an overall balance of microbial growth and by a carbon balance, the growth yield coefficient was smaller during aerobic/anaerobic changes than under aerobic conditions.

Aerobic 4-nitrophenol degradation by microorganisms fixed in a continuously working aerated solid-bed reactor

Abstract Studies of microbial purification of a model waste water containing 4-nitrophenol were carried out in a continuously working aerobic solid-bed reactor. The main emphasis was on the dynamic behaviour of the system after a sudden change in cultivation conditions and on the steady-state performance of the reactor as a function of the pollution load. A change from ammonium-free to ammonium-containing medium hardly influenced the nitrophenol degradation. The reactor responded differently to an increase in pollutant load, which was brought about by increasing either the 4-nitrophenol content or the flow of the waste water. Up to a load of 270 mg l−1 h−1 the pollutant was stably and almost completely degraded. At a higher load, only a partial 4-nitrophenol degradation took place. A mathematical model was derived to describe the processes that occurred in the reactor. By segregation into two compartments – the aqueous phase and the biofilm – account was taken of the fact that the pollutant is carried into the biofilm by diffusion and is degraded there. The observed relations between the pollutant load, the pollutant concentration in the outlet of the reactor and the reactor performance agreed with the simulated process behaviour. As the model simulation showed, the incomplete pollutant degradation at a higher reactor load was caused by oxygen limitation.

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.

Composting of Wood Containing Polycyclic Aromatic Hydrocarbons (PAHs)

The addition of various nitrogen sources, such as liquid hog manure and mineral medium, to pine wood accelerated the composting process in Dewar vessels, which was obvious from the increased decomposition temperature and the more intensive oxygen consumption and carbon dioxide production. During composting in Dewar vessels of artificially PAH-contaminated pine wood soaked with liquid manure, the PAH degradation was influenced by the inoculum used. The fastest PAH degradation was achieved by compost addition, but the most intensive carbon dioxide evolution was measured with hydrocarbon-polluted soil as an additive. After 61 days, the PAH content of the wood was reduced from each 1000 mg/kg to 26 mg/kg of phenanthrene and 83 mg/kg of pyrene. The relation between the microbial wood decay and PAH degradation shows that the detoxification at least of artificially PAH-polluted wood demands only a partial wood decay.A pilot scale percolator was applied to composting of artificially contaminated pine wood and rea...

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.