Parallel use of shake flask and microtiter plate online measuring devices (RAMOS and BioLector) reduces the number of experiments in laboratory-scale stirred tank bioreactors

Background Conventional experiments in small scale are often performed in a Black Box fashion, analyzing only the product concentration in the final sample. Online monitoring of relevant process characteristics and parameters such as substrate limitation, product inhibition and oxygen supply is lacking. Therefore, fully equipped laboratory-scale stirred tank bioreactors are hitherto required for detailed studies of new microbial systems. However, they are too spacious, laborious and expensive to be operated in larger number in parallel. Thus, the aim of this study is to present a new experimental approach to obtain dense quantitative process information by parallel use of two small-scale culture systems with online monitoring capabilities: Respiration Activity MOnitoring System (RAMOS) and the BioLector device. Results The same mastermix (medium plus microorganisms) was distributed to the different small-scale culture systems: 1) RAMOS device; 2) 48-well microtiter plate for BioLector device; and 3) separate shake flasks or microtiter plates for offline sampling. By adjusting the same maximum oxygen transfer capacity (OTRmax), the results from the RAMOS and BioLector online monitoring systems supplemented each other very well for all studied microbial systems (E. coli, G. oxydans, K. lactis) and culture conditions (oxygen limitation, diauxic growth, auto-induction, buffer effects). Conclusions The parallel use of RAMOS and BioLector devices is a suitable and fast approach to gain comprehensive quantitative data about growth and production behavior of the evaluated microorganisms. These acquired data largely reduce the necessary number of experiments in laboratory-scale stirred tank bioreactors for basic process development. Thus, much more quantitative information is obtained in parallel in shorter time.Cluster of Excellence “Tailor-Made Fuels from Biomass”, which is funded by the Excellence Initiative by the German federal and state governments to promote science and research at German universities

Utilizing high-throughput experimentation to enhance specific productivity of an E.coli T7 expression system by phosphate limitation

<p>Abstract</p> <p>Background</p> <p>The specific productivity of cultivation processes can be optimized, amongst others, by using genetic engineering of strains, choice of suitable host/vector systems or process optimization (e.g. choosing the right induction time). A further possibility is to reduce biomass buildup in favor of an enhanced product formation, e.g. by limiting secondary substrates in the medium, such as phosphate. However, with conventional techniques (e.g. small scale cultivations in shake flasks), it is very tedious to establish optimal conditions for cell growth and protein expression, as the start of protein expression (induction time) and the degree of phosphate limitation have to be determined in numerous concerted, manually conducted experiments.</p> <p>Results</p> <p>We investigated the effect of different induction times and a concurrent phosphate limitation on the specific productivity of the T7 expression system <it>E.coli </it>BL21(DE3) pRhotHi-2-EcFbFP, which produces the model fluorescence protein EcFbFP upon induction. Therefore, specific online-monitoring tools for small scale cultivations (RAMOS, BioLector) as well as a novel cultivation platform (Robo-Lector) were used for rapid process optimization. The RAMOS system monitored the oxygen transfer rate in shake flasks, whereas the BioLector device allowed to monitor microbial growth and the production of EcFbFP in microtiter plates. The Robo-Lector is a combination of a BioLector and a pipetting robot and can conduct high-throughput experiments fully automated. By using these tools, it was possible to determine the optimal induction time and to increase the specific productivity for EcFbFP from 22% (for unlimited conditions) to 31% of total protein content of the <it>E.coli </it>cells via a phosphate limitation.</p> <p>Conclusions</p> <p>The results revealed that a phosphate limitation at the right induction time was suitable to redirect the available cellular resources during cultivation to protein expression rather than in biomass production. To our knowledge, such an effect was shown for the first time for an IPTG-inducible expression system. Finally, this finding and the utilization of the introduced high-throughput experimentation approach could help to find new targets to further enhance the production capacity of recombinant <it>E.coli</it>-strains.</p

Cisplatin-induced ototoxicity: the current state of ototoxicity monitoring in New Zealand.

Background: Many well-known pharmacologic agents have been shown to have toxic effects to the cochleo-vestibular system. Examples of such ototoxic agents include cisplatin and aminoglycoside antibiotics. Ototoxicity monitoring consists of a comprehensive pattern of audiological assessments designed to detect the onset of any hearing loss. Three main methods have emerged over the past decade, and include the basic audiological assessment, extended high frequency (EHF) audiometry, and otoacoustic emission (OAE) measurement. These measures can be used separately or in combination, depending on clinical purpose and patient considerations. It is suggested by the American Academy of Audiology Position Statement and Clinical Practice Guidelines: Ototoxicity Monitoring, that baseline testing be done in a fairly comprehensive manner, including pure-tone thresholds in both the conventional- and extended high frequency ranges, tympanometry, speech audiometry, and the testing of OAEs (AAA, 2009). Anecdotal evidence suggests that New Zealand Audiologists do not currently follow a national ototoxicity monitoring protocol. Therefore the main aim of this study was to explore the current status of ototoxicity monitoring within New Zealand. Hypothesis: It was hypothesized that hospital based Audiology departments across New Zealand each followed their own internal ototoxicity monitoring protocol based, to a large extent, on the guidelines proposed by the American Academy of Audiology and by the American Speech-Language-Hearing Association. Method: Through the use of a Telephone Interview Questionnaire, 16 charge Audiologists were interviewed to establish their current state of knowledge regarding ototoxicity monitoring at 16 out of 20 district health boards in New Zealand. Enquiries about the current systems and procedures in place at their departments together with any suggestions and recommendations to improve on these systems were made. Results: This study found that only 9 of the 16 DHBs interviewed currently follow an ototoxicity monitoring protocol. Furthermore, other than initially hypothesized the origin of the protocols followed by the remaining 7 departments were reported to have ranged from independently developed protocols to historically adopted protocols. One department implemented an adapted version of a protocol by Fausti et al. (Ear and Hearing 1999; 20(6):497-505). This diversity in origin however, does confirm our initial suspicion that no universal and standardized monitoring protocol is currently being followed by Audiologists working in the public health sector of New Zealand

تأثير الاكسجين والتفريغ الجزئي ودرجة الحرارة والرطوبة النسبية مع أشعة جاما على بعوضة الكيولكس ببينز 1- تأثير درجة الحرارة والرطوبة النسبية وحدهما (بدون إشعاع )

The results revealed that a temperature of 10°C was the most effective temperature on pupal mortality of Culex pipiens complex L. followed by 32°C then 20 and 26°C. There was a gradual increase in pupal mortality with increasing the time of exposure to temperatures. The pupal mortality increased with decreasing the relative humidity levels at the same time of exposure. Exposure for short time periods did not affect significantly the pupal mortality. Increasing the exposure time increased mortality.أظهرت النتائج أن درجة 10 م هي أنسب درجات الحرارة المؤثرة عل نسبة الوفيات في العذارى المعاملة ، تليها درجات 32 ، 20 ثم 26 م . كما أظهرت النتائج زيادة تدريجية في نسبه الوفيات مع زيادة مدة التعريض لهذه الدرجات (2/1-48 ساعة ) . وعند تعريض العذارى ولفترات زمنية مختلفة (2/1-48 ساعة) لمستويات رطوبة نسبية مماثلة ازدادت الوفيات مع نقص هذه المستويات . وقد أظهرت النتائج تأثير غير معنوي على نسبة الوفيات في حالة التعريض لفترات زمنية قصيرة بينما زادت نسبة الوفيات بزيادة فترة التعريض