Biotechnology
Therapeutics and bioinsecticides: production by spider venom
The venom of a single spider can contain up to 3000 components. The components, mostly peptides, can be used to develop promising active ingredient candidates for the treatment of diseases. Spider venom can also be used in pest control – as a biological pesticide. A team of researchers from the Fraunhofer Institute for Molecular Biology and Applied Ecology IME and the Justus Liebig University Giessen is focusing on native spiders and their venom mix, which have received little attention to date. The research results on the biology of the toxins – especially on the venom of the wasp spider – have been published in scientific journals.
Spiders make many people uncomfortable, and some are even afraid of the eight-legged creatures. At the Fraunhofer Institute for Molecular Biology and Applied Ecology IME in Giessen, however, they are welcome. Here, biochemist Dr. Tim Lüddecke and his team are conducting research on spider toxins.
“Spider toxins are a largely untapped resource, this is partly due to the sheer diversity – some 50,000 species are known. There is a lot of potential in spider venom for medicine, for example in researching disease mechanisms.”
– Dr. Tim Lüddecke, head of the new “Animal Venomics” research group
For example, it is possible to study in the laboratory how individual toxins act on pain receptors of nerve cells. The venom cocktail of the Australian funnel web spider is particularly promising. It is assumed that it can be used to treat neuronal damage after strokes and to make hearts for organ transplants last longer. Other drug candidates are of interest for use as antibiotics or painkillers. “This is a very young field of research. The substances have been discovered and described, but they are not yet in the preclinical stage,” Lüddecke said. Pesticide research is a different story. Spiders stun insects with their venom and then eat them. Because the toxins are very effective against insects, they provide a good basis for biopesticides; they are suitable for crop pest control.
Research to date has focused on the toxins of the very large or potentially dangerous species that live in the tropics. The native, small and harmless spiders have not been in focus. “Most spiders in Central Europe are no more than two centimeters in size, and their venom levels were not sufficient for experiments. But now we have precise analytical methods to study even the small amounts of the previously neglected majority of spiders,” explains Lüddecke. The working group at the Giessen Bioresources branch of the Fraunhofer IME is devoting itself to these species as part of a research project. In the process, they are collaborating with research teams from the Justus Liebig University in Giessen, among others. The work is funded by the LOEWE Center for Translational Biodiversity Genomics (LOEWE-TBG) in Frankfurt am Main.
The scientists are paying particular attention to the wasp spider (Argiope bruennichi), which owes its name to its striking wasp-like coloration. They have succeeded in decoding its venom, identifying numerous novel biomolecules. The research findings were published in the journal Biomolecules.
New biomolecules from wasp spider venom
Spider venoms are highly complex, they can contain up to a maximum of 3000 components. The venom of the wasp spider, on the other hand, contains only about 53 biomolecules. It is heavily dominated by high-molecular-weight components, including so-called CAP proteins and other enzymes. As in other spider venoms, knottins are present – but these make up only a small part of the total mixture.
Knottins represent a group of neurotoxic peptides that are robust to chemical, enzymatic, and thermal degradation due to their nodal structure. One could therefore administer these molecules orally as a component of drugs without digesting them in the gastrointestinal tract. They can therefore exert their effects very well, which is why they offer great potential for medicine. In addition, knottins bind specifically to ion channels. “The more specifically a molecule docks onto its target molecule, attacking only a single type of ion channel, the fewer side effects it triggers,” explains Lüddecke. Moreover, even in small amounts, the knottins affect the activity of the ion channels, i.e., they are effective at low concentrations. As a result, derived drugs can be administered in low doses. The combination of these properties is what makes spider venoms so interesting for science.
The project partners also discovered molecules in the wasp spider’s venom that are similar in structure to neuropeptides, which are responsible for transporting information between nerve cells. “We have found novel families of neuropeptides that we have not previously seen in other spiders. We suspect that the wasp spider uses them to attack the nervous system of insects. It has been known for some time that neuropeptides in the animal kingdom are frequently converted into toxins in the course of evolution,” says the researcher.
Replicating toxins in the lab
Since the toxin yield is low in small spiders, the researchers extract the toxin glands and sequence the mRNA from them. Based on the gene structure, the toxins can be decoded. The venom profile of the wasp spider is now available in its entirety, and the next step is to produce the relevant components. For this, the gene sequence is incorporated into a bacterial cell using biotechnology, which then produces the toxin. “We are building quasi genetically modified bacteria that produce the toxin on a large scale.” Lüddecke and his team have been able to mass produce the main component of the wasp spider toxin, the CAP protein. The first functional studies will start soon.
Venom of male and female spiders differs
In another review paper, the biochemist, in cooperation with colleagues at the Justus Liebig University of Giessen and researchers at the Australian University of the Sunshine Coast, was able to deduce that spider venoms are very dynamic and that many influences shape their composition and functionality. “The dynamics of spider venom have been completely underestimated. The biochemical repertoire is critically influenced by life stage, habitat, and especially sex. Even the venom cocktail of juveniles and adults is not necessarily identical. It is rather the interaction of the many components that makes spider venom so effective than the effect of a single toxin. Through their interactions, the components increase their effectiveness,” the researcher sums up.
Biotechnology News Processing Technologies Quality Management Water & Waste Water
Negative influence by CO2 on important plankton group
The most important producers of plant biomass in the ocean are diatoms. Because they rely on silica rather than calcium carbonate to build their shells, they were previously considered winners of ocean acidification – a chemical change in seawater caused by the uptake of CO2 that makes calcification more difficult. In a study published today in the journal Nature, scientists at GEOMAR Helmholtz Centre for Ocean Research Kiel show that diatoms, which belong to the plankton, are also affected. Analysis suggests that increasing acidification could drastically reduce populations of diatoms.
While calcifying organisms in particular struggle to form their shells and skeletons in more acidic seawater, diatoms (diatoms) were previously thought to be less vulnerable to the effects of ocean acidification – a chemical change triggered by the uptake of carbon dioxide (CO2). The globally widespread tiny diatoms use silica, a compound of silicon, oxygen and hydrogen, as a building material for their shells. That diatoms are nevertheless threatened has now been demonstrated for the first time by researchers from GEOMAR Helmholtz Centre for Ocean Research Kiel, the Institute of Geological and Nuclear Sciences New Zealand and the University of Tasmania in a study published in the journal Nature. For their study, they linked an overarching analysis of various data sources with Earth system modeling. The findings provide a new assessment of the global impact of ocean acidification.
As a result of ocean acidification, the silicon shells of diatoms dissolve more slowly. This is not an advantage – because it causes diatoms to sink to deeper water layers than before before they chemically dissolve and are converted back to silica. Consequently, the nutrient is increasingly withdrawn from the global cycle and thus becomes scarcer in the light-flooded surface layer, where it is needed to form new shells. This causes a decline in diatoms, the scientists:in their current publication. Diatoms contribute 40 percent of the production of plant biomass in the ocean and are the basis of many marine food webs. They are also the main driver of the biological carbon pump that transports CO2 to the deep ocean for long-term storage.
“Using an overarching analysis of field experiments and observational data, we wanted to determine how ocean acidification affects diatoms on a global scale. Our current understanding of ecological effects of ocean change is largely based on small-scale experiments, i.e., from a particular place at a particular time. These findings can be deceptive if the complexity of the Earth system is not taken into account. Our study uses diatoms as an example to show how small-scale effects can lead to ocean-wide changes with unforeseen and far-reaching consequences for marine ecosystems and matter cycles. Since diatoms are one of the most important plankton groups in the ocean, their decline could lead to a significant shift in the marine food web or even a change for the ocean as a carbon sink.”
– Dr. Jan Taucher, marine biologist
The meta-analysis examined data from five mesocosm studies from 2010 to 2014, from different ocean regions, from Arctic to subtropical waters. Mesocosms are a type of large-volume, oversized test tube in the ocean, with a capacity of tens of thousands of liters, in which changes in environmental conditions in a closed but otherwise natural ecosystem can be studied. For this purpose, the water enclosed in the mesocosms was enriched in carbon dioxide to correspond to future scenarios with moderate to high increases in atmospheric CO2 levels. For the present study, the chemical composition of organic material from sediment traps was evaluated as it sank through the water contained in the experimental containers over the course of the experiments, which lasted several weeks. Combined with measurements from the water column, an accurate picture of biogeochemical processes within the ecosystem emerged.
The findings obtained from the mesocosm studies could be confirmed using global observational data from the open ocean. They show – in line with the results of the analysis – a lower dissolution of the silicon shells at higher seawater acidity. The resulting data sets were used to run simulations in an Earth system model to assess the ocean-wide consequences of the observed trends.
“Already by the end of this century, we expect a loss of up to ten percent of diatoms. That’s immense considering how important they are to life in the ocean and to the climate system,” Dr. Taucher continued. “However, it is important to think beyond 2100. Climate change will not stop abruptly, and global effects in particular take some time to become clearly visible. Depending on the amount of emissions, our model in the study predicts a loss of up to 27 percent silica in surface waters and an ocean-wide decline in diatoms of up to 26 percent by 2200 – more than a quarter of the current population.”
This finding of the study is in sharp contrast to the current state of ocean research, which sees calcifying organisms as losers and diatoms as profiteers from ocean acidification. Professor Ulf Riebesell, marine biologist at GEOMAR and head of the mesocosm experiments adds: “This study once again highlights the complexity of the Earth system and the associated difficulty in predicting the consequences of man-made climate change in its entirety. Surprises of this kind remind us again and again of the incalculable risks we run if we do not counteract climate change swiftly and decisively.”
Biotechnology News Processing Technologies Water & Waste Water
Solutions for energy recovery from wastewater and organic waste
From May 30 to June 3, the sales team of biogas specialist Weltec Biopower will be available in Hall A4, Stand 217, to answer all questions relating to the construction and retrofitting of anaerobic energy plants: The range includes proven processes from the field of biogas technology. The high savings potential of these processes is demonstrated by the modernization of the municipal wastewater treatment plant in Bückeburg, Germany, which serves 33,000 inhabitants. Since Weltec Biopower switched to anaerobic sludge stabilization in 2021, operation of the plant at full load has become significantly more economical.
As general contractor, the company was responsible for the construction of the wastewater treatment system at the municipal sewage treatment plant. In addition to the earthworks, the construction of the foundation and the electrical cabling, the work included the construction of a new static sludge thickener, a machine room for the combined heat and power plant, the control and pumping station, and a stainless steel digester with a gas storage roof. Thanks to anaerobic wastewater treatment, the sludge volume has dropped by 35 percent, resulting in a significant reduction in transport and disposal costs. In addition, the digester gas produced can now generate around 465,000 kWh of electricity at full load. This allows the operator to meet about 40 percent of its electricity needs and save two-thirds of its electricity costs.
“In view of the new greenhouse gas reduction targets and the sharp rise in energy prices, an anaerobic stage is an economically attractive solution for wastewater treatment plant operators, which also benefits from public subsidies. Ultimately, the combination of wastewater treatment, power and heat generation, and climate protection enables more efficient operation, especially for small and medium-sized wastewater treatment plants.”
– Jens Albartus, Managing Director
How these goals can be achieved with organic waste is demonstrated by a WELTEC plant in Piddlehinton, southwest England. Here, a mix of food waste, expired food from supermarkets and organic waste is fed to the biogas plant. In addition to the substrate mix, the technical approach is also special. Before feeding and shredding, a de-packaging machine separates the food from the packaging.
Another efficiency bonus: The waste heat from the cogeneration plant is sold to a nearby feed producer, which also uses most of the electricity. The biogas plant operator feeds the excess electricity directly into the power grid, generating further revenue. The digestate from the process meets the requirements of the British industry standard PAS-100, so local farmers can use it as fertilizer.
Following a capacity expansion in 2014 from 20,000 t of substrate input per year to 30,000 t, the Group installed an additional digester and storage tank, as well as GasMix blending systems and a separation unit. A plant with this equipment would also support the conversion to biomethane production.
Biotechnology Measurement, Instrumentation, Control & Automation Safety & Security Water & Waste Water
Method for determination of legionella in a water
New method for fully automated determination of the concentration of legionella in a water sample within a few hours
The hygienic necessity to control the concentration of legionella in technical water systems from which aerosols can be discharged leads to the problem that the cultivation method (ISO 11731-2017) used for this purpose only provides reliable results after a delay of 7-12 days. On this basis, necessary measures can only be taken and controlled with a considerable time delay. Rapid tests currently available on the market either do not correlate reliably with the accredited cultivation method or require (time-) consuming preparation steps. Some rapid tests provide highly specific detection for single Legionella species, but not for all Legionella species in a water sample (Legionella spp. = species pluralis). The newly developed measuring device INWATROL L.nella+ from Inwatec is based on the method of measuring the metabolic activity of living cells and reliably determines the parameter Legionella spp. from a water sample within a few hours. The measuring device is directly connected to the technical water system with automatic and self-disinfecting sample feed, including self-disinfection of the water contained in the measuring cell after the measurement is completed. This enables the plant operator to determine the hygienic water quality continuously and safely. In addition to the direct control of the success of the measures carried out, it is also possible to control e.g. biocides according to requirements.
1. Introduction
The hygienic relevance of the spread of pathogenic Legionella via aerosols from technical water systems such as evaporative cooling systems and cooling towers has led to the creation of technical hygiene guidelines in many countries. In Germany, VDI 2047 part 2 and 3, generally accepted technical rules for ensuring the hygienic operation of evaporative cooling systems and cooling towers came into force for the first time in 2015. In addition, in many countries the tolerable concentration of legionella in the circulation water of the respective plants is limited by the legislator. In Germany, the forty-second ordinance for the implementation of the Federal Immission Control Act (Ordinance on Evaporative Cooling Systems, Cooling Towers and Wet Separators – 42nd BImSchV) came into force on 19.08.2017, which also includes wet separators. So far, the basis for hygiene control has always been the determination of the concentration of legionella in the water by cultivation according to ISO 11731:2017 with system-dependent threshold values. In this cultivation method, cell division produces visible and therefore countable colonies. In comparison to other bacterial species, Legionella bacteria divide relatively slowly, so that the results of the measurement are only available after 7-12 days, whereby in some cases further investigations to confirm suspicious colonies follow.
For the operator of a plant with monitoring obligation, this means a strongly delayed control of the hygiene status. Furthermore, the efficiency of any necessary measures can only be determined with a long delay. Additional rapid tests for estimating the contamination of water with Legionella are available, e.g. based on immunological reactions (antibody reaction), detection of genetic material (PCR) or by means of color fluorescence microscopy. The limitations of these rapid tests are the live/dead quantification, the comparability to the culture method or the complex sample preparation.
The newly developed and patented automatic measuring device INWATROL L.nella+ allows the reliable and continuous determination of the parameter Legionella species pluralis with high correlation to the cultivation method according to ISO 11731:2017 within a few hours without further preparation steps by the user.
2. Rapid test for the fully automated determination of Legionella species pluralis
2.1 Measuring principle
The detection of metabolically active Legionella bacteria is based on a non-specific enzymatic conversion of a non-polar fluorescein acid ester, which only passes through the cell membrane of living cells into the cell interior where it is converted into color-active fluorescein. The increase in fluorescence as a function of time is directly proportional to the number of living cells and is converted into colony forming units per 100 ml. Due to a combined heat and pH pretreatment and the high measuring temperature compared to the cultivation method, the accompanying flora is killed. The measurement is performed undiluted in a sample volume of approx. 350 ml. In comparison to the cultivation method, the measurement is not significantly influenced by accompanying flora and a high measuring inaccuracy due to a high dilution.
2.2 Continuous, automated measurement
For continuous measurements, the measuring device is directly connected to a water system. A thermally self-disinfecting sampling line ensures that no reproduction of legionella in the supply line affects the measurement result. Ideally, the sampling tap is in continuous operation to exclude stagnation of water between two measurements. The measuring cell in the device is rinsed several times during filling. After the rinsing process is completed, the combined heat and pH pretreatment starts. When the pre-treatment is completed, the measuring cell cools down to the measuring temperature and the measurement begins. The measuring cell is thermally disinfected before the device is filled again for the follow-up examination. The measuring cell is ready for the next measurement. Usually, a sampling tap is installed directly at the sampling point before the sampling line. This tap can be used to take microbiological samples at the time of filling the measuring cell or at any other times, e.g. for further validation measurements.
2.3 Automated measurement of manually loaded samples
The continuous measuring operation can be interrupted for manual feeding of further water samples via the filling funnel. For cleaning, rinsing and filling the measuring cell, only the valve position on the device has to be changed. When the filling is completed, the valve position is returned to its original position and the measuring device switches back to automatic mode when the measurement is completed. The measuring procedure itself does not differ from the automatic mode.
2.4 Cultivation according to ISO 11731:2017/ UBA1
The cultivation method uses several approaches with different dilution and pretreatment stages (heat or acid). The aim is to obtain evaluable results for both low and high levels of Legionella. For the result, the preparation with the highest number of confirmed Legionella colonies is used (if the measurement accuracy/number of colonies is sufficiently high). The limits of the accuracy of the cultivation method are mainly due to the possible influence of the accompanying flora, i.e. other microorganisms which can suppress the growth of the legionella or overgrow their colonies. Furthermore, bacteria are particles in a water sample and are not homogeneously distributed. Therefore, when taking small volumes from the sample bottle, inaccuracies may occur due to the sometimes high dilution factors. During cultivation, living but non-cultivable cells in the so-called VBNC2 status are not detected. Many Legionella from a coherent agglomerate, e.g. by propagation within an amoeba, are only visible and evaluated as one colony during cultivation (see Lindner, Hahn: Microbiological analyses of the cooling water according to the 42nd BImSchV, p. 74, VGB PowerTech 9, 2018).
3. Examples of application and correlation to the culture method
The INWATROL L.nella+ is being used in various practical applications. Case studies include the operation in the following plants:
3.1 Monitoring of the circulation water in the cooling tower of a coal-fired power plant
Challenges for the measuring mode:
Changing operating conditions due to load changes between full load, partial load and operation without load at varying flow rates (automatic sampling directly from the line behind the main cooling water pump) and circuit water temperatures.
Increased influence of VBNC cells especially at low circuit water temperatures.
A stable measuring operation has been achieved over several months. The interim influence of VBNC cells can be successfully suppressed in the instrument by changing the automated pretreatment adapted to the main cooling water.
3.2 Monitoring of the circulating water in the evaporative cooling system of a starch factory
Challenges for the measuring operation:
Outdoor location of the instrument (wall mounting) with strongly changing ambient temperatures
Partially strong solid matter input into the circulation water with high organic load
A stable measuring operation over several months was achieved. In particular, the influence of the biocide treatment on the concentration of legionella could be proven directly. When changing from a non-oxidizing to an oxidizing biocide, a directly measurable effect on both the concentration of the legionella and the reaction speed could be observed.
3.3 Monitoring the circulation water of a metal cast house
Challenge for the measuring operation:
Heavy contamination of the water with inorganic and organic impurities (casting oil)
With strong fluctuations in the water quality, reliable measurements have been achieved over a period of several months. Casting plants are often equipped with a hot water storage tank. Depending on the requirements of the casting plant(s), the temperature and hydraulic retention time (stagnation), as well as the load of organic and inorganic contamination fluctuates strongly with a significant influence on the reproduction rate of legionella.
3.4 Monitoring the drinking water network of a beverage manufacturer
Challenge for the measuring operation:
Reliable detection of low and increasing concentration of legionella at changing drinking water temperature in the pipeline network
Suppression of the influence of VBNC cells on measurement results, especially at low water temperatures
With this characteristically low-nutrient and solid-free water, fluctuating Legionella contamination could be reliably detected over several months, depending on the consumption structure and temperatures in the pipeline network.
3.5 Hygienic monitoring of different cooling systems of a food producing company using a laboratory device
Challenge for the measuring operation:
Manual sample application of cooling water samples different in quality
Disinfection of the feed funnel before sample preparation
Guarantee of low work effort for manual samples including result evaluation
A reliable, automated adjustment of the parameters for the pre-treatment in the device could be ensured over several months even with differently buffered and preloaded water samples. Both drinking water samples (monitoring of the make-up water for the cooling systems) and the cooling water samples showed a good correlation to the cultivation method according to UBA with clearly different results.
3.6 Correlation of the rapid test INWATROL L.nella+ with the cultivation method
The correlation of the rapid test was carried out over a high number of measurements with the cultivation method according to ISO 11731:2017. Sampling, sample transport as well as preparation and evaluation of the measurement results were carried out in accordance with the current recommendation of the Federal Environmental Agency for sampling and detection of Legionella in evaporative cooling systems, cooling towers and wet separators (UBA). Validation measurements were made with different accredited laboratories. In order to obtain a reliable qualitative comparison between the rapid test and the cultivation method, the following measurements were carried out in only one accredited laboratory (IWW Rheinisch-Westfälisches Institut für Wasser Beratungs- und Entwicklungsgesellschaft mbH, D-45476 Mülheim an der Ruhr).
4. Discussion
The correlation to the cultural preparations carried out in the laboratory can be rated as very high overall. Two devices showed significant short-term deviations from the laboratory results in the form of additional findings. Here the influence of VBNC cells on the measurement result of the INWATROL L.nella+ rapid test was investigated. Metabolic activity measurements using fluorescein diacetate are used in microbiological tests in addition to other methods (membrane integrity, protein synthesis (FISH), intact polar membrane lipid analysis, cell extension (“direct viable count”)) for the detection of VBNC bacteria. This can be an additional benefit for the operator, because recontamination of water systems with Legionella can also be a “revival” of VBNC organisms (see Hans-Curt Flemming, Jost Wingender – IWW Zentrum Wasser, Biofilm Centre, University Duisburg-Essen). Often, however, the aim of the operator is to achieve the highest possible correlation to the legally required examination by means of cultivation in the laboratory. By adjusting the pre-treatment conditions (mainly by increasing the temperature and lengthening the pre-treatment time), the correlation to the cultivation method can be successfully restored in case of multiple findings with VBNC cells.
Authors
Holger Ohme, Jennifer Becker, Pascal Jahn, Dirk Heinecke