Application of A. adeninivorans as a biosensor

Due to its physiology and the ease of genetic manipulation of A. adeninivorans collaborations especially with small and medium-sized enterprises (KMUs) have been developed. Among others a cooperation with the Dr. Bruno Lange GmbH in the late 1990's was established to test A. adeninivorans cells for the first time as microbial component of biosensors for the measurement of the biochemical oxygen demand (BOD). The tests revealed that Arxula based biosensors can determine the so-called sensorBOD in waste water, tap water, brackish water and sea water within 70 seconds and demonstrated the possibility to analyse the pollution degree of aqueous samples. These biosensors were marketed by the Dr. Bruno Lange GmbH in combination with the ARAS device system. At the same time the biosensors were used in Hong Kong for the analysis of waste water, brackish water and sea water. A comparison of the measured sensorBOD data (gained within only 70 seconds) with the results of the conventional BSB5-method (measurement period of 5 days) emphasised the correctness of the biosensors which are still in use by the Hong Kong University Of Science and Technology (HKUSC) for waste water analysis.

 

During the last years we have focused on Arxula based biosensors for application to environmental survey, in the agriculture and for medical purposes In collaboration with industrial partners (quo data GmbH - Dresden, PROLATEK GmbH - Dresden, Gesellschaft für Silizium und Mikrosysteme mbH (GeSiM) – Dresden), research facilities (Forschungszentrum Rossendorf – Dresden) and federal offices (Bundesamt für Verbraucherschutz und Lebensmittelsicherheit [BVL – Referenzlabor], Bundesamt für Materialforschung und –prüfung [BAM]) a set of microbial biosensors for the determination of hormones, dioxins and pharmaceuticals in tap water, soda water, waste water, urine and blood serum was established. The used transgenic A. adeninivorans cells contain the genetic information for the constitutive expression of the particular human receptor which interacts with the appropriate ligand (estrogen, androgen, progesteron, glucocorticoids, dioxin, pharmaceuticals) resulting in expression of a reporter gene which encodes a recombinant enzyme detectable by simple biochemical and/or amperometrical measurements. Since the activity of the enzyme correlates directly with the concentration of the hormone and/or pharmaceutical, all substances interacting with the appropriate receptor can be measured as so-called sum parameters.

 

The following sensors have been established on basis of transgenic A. adeninivorans cells:

A-YES assay – Measurement of estrogenic activities in tap water, soda water, waste water, urine and blood serum

The assay was developed at the IPK and validated by the quo DATA GmbH/Dresden. It is already commercialised for the measurement of tap water, soda water and waste water (sold by the quo DATA GmbH) and since 2018 an internationally approved biological test system for monitoring surface waters according to DIN / ISO.

 

Microbial biosensor for the determination of estrogenic substances - measurement principle: The microbial component of the biosensor consists of transgenic A. adeninivorans yeast cells. Integrated in the genome these cells carry a receptor gene cassette with TEF1-promotor - hERα gene - PHO5-terminator and a reporter gene cassette with GAA-ER6-promotor - reporter gene - PHO5-terminator. The TEF1-promotor provides constitutive expression of the receptor gene which leads to the synthesis of the recombinant estrogen receptor α (hERα). While incubation with estrogenic substances hERα is expressed and forms dimers which act as transcription factors that bind to the ERE region of the GAA-ERE-promoters. Consequently the expression of the reporter gene (phyK) is activated leading to synthesis and secretion of the reporter protein (Phytase) whose activity can be measured subsequently.

A-YAS assay – Measurement of androgenic activities in waste water, urine and blood serum

The Assay was developed at the IPK and validated by the quo DATA GmbH/Dresden. It is already commercialised for the measurement of tap water, soda water and waste water (sold by the quo DATA GmbH) and the validation of the A-YAS assays for urine and blood serum is in progress.

A-YPRS assay – Measurement of progesterone in waste water, urine and blood serum

The assay was established in the last years and tested for road capability. At present it is validated by the quo DATA GmbH/Dresden for waste water, urine and blood serum. Commercialisation of the A-YPRS assays by the quo DATA GmbH was done in 2017.

A-YGS assay - Measurement of glucocorticoid activities in urine and blood serum

This assay was also established in the last years and tested for road capability. A validation for urine and blood serum and its commercialisation by the quo DATA GmbH is intended.

A-YDS-assay - Measurement of dioxins in urine, blood serum and animal feeds

This assay is currently established and tested for road capability. It is to be validated for animal feeds, urine and blood serum. Its commercialisation is intended by the quo DATA GmbH.

A-YPS assay - Measurement of pharmaceuticals (e.g. Diclofenac, Chlozapine, Diazepam)

This assay was established for the measurement of pharmaceuticals excreted by humans and animals into the environment. It is currently tested for road capability and it's a subsequent commercialisation for aqueous samples is in discussion.

In environmental analytics not only the monitoring of hormones and pharmaceuticals is important but also the complete removal of these substances from waste water plants. At present those molecules are eliminated by ozonation but this procedure is prone to insufficient removal with the risk of even higher hormonal and pharmaceutical activities after the treatment. In order to make the substance removal procedure more effective a survey of the ozonation process using Arxula based biosensors is planned. For this purpose the A-YES, A-YAS and A-YPS assays will be designed for amperometric measurement. Thus, the yeast cells are immobilised in a temperature controlled, electrode containing measuring unit as part of the measuring system “EstraMonitor”. This system, designed by the the KMUs quo DATA GmbH/Dresden and PROLATEK GmbH/Dresden is suitable for on-line measurement of hormones and pharmaceuticals by means of amperometric measuring method. In order to fulfil all requirements of the genetic engineering law performing on-site measurements in waste water plants an inactivating unit for killing all living material was integrated. 

 

The “EstraMonitor” has been used already for amperometric measurements of estrogenic substances at the IPK and by the cooperation partners quo DATA GmbH in Dresden as well as the Institut für Energie und Umwelttechnik e.V. (UITA) in Duisburg. The data showed that the immobilised cells can be used for up to 20 measurements with periods of 4 hours per measurement. The next step will be an on-site test in a waste water plant with ozonisation unit in Duisburg. Our aim is to provide an on-line measuring system for the semi-continuously detection of hormones and pharmaceuticals which automatically activates ozonisation at distinct pollutant concentration thresholds. Therefore, measuring devices as well as the microbial components are subject to further improvement throughout the next years.

 

 

Another ongoing project is the in vitro testing of pharmaceutical substances and their degradation products in terms of functional binding to human receptors. The aim is to develop a rapid screening procedure, which is part of the characterization of different substances (hormones, inhibitors, activators) in the development of potential pharmaceuticals. For this purpose, human receptors, which dimerize in the natural system after binding of a ligand, are fused with split fluorescent proteins or enzymes. Upon dimerization by ligand binding, complementation of the reporter occurs and consequently a measurement signal to be detected.