Final ReportSummary - SENSBIOSYN (Biosensors and Sensors for the industrial biosynthesis process of widely used commercial antioxidants: nutraceuticals as additives for food and aquaculture promoting
Monitoring industrial whole cell bioprocesses for the production of algal biomass and xanthophyll pigments with antioxidant activity. There is an increasing demand worldwide for low cost, fast, and reliable methods for monitoring chemical species, additives, and xenobiotics in clinical chemistry, environmental sciences and food related processes. Chemical sensors and biosensors offer all these advantages since they can be easily used in both laboratory and field applications, are easy to use without need of a skilled operator, automatic, cheap, with no needs for maintenance and sample pre-treatments. The SENSBIOSYN adopted a multidisciplinary approach in which the cooperation between small private companies and RTD performers was central. The team is composed by two research institutes: the National Research Council of Italy (CNR) and the National Institute for Biological Sciences, Centre of Bioanalysis (NIB, Romania); one university: the Ben-Gurion University (BGU, Israel); three SMEs: Biosensor (BIO, Italy), Nanosens (NANO, the Netherland), Algatechnologies (ALGATECH, Israel). The industrial process selected in this proposal is the cultivation of the green alga Haematococcus pluvialis at Kibbuz Qetura in Israel, in the largest continuously operating industrial facility consisting of over 30 tubular photobioreactors having 10 000 litre capacity, covering an area of over 10 000 m2. Algae are cultivated first under nutrient replete conditions for production of green biomass, in a second stage those algae are transferred to nutrient deficient medium, whereby the nutrient stress leads to induction of astaxanthin biosynthesis and accumulation to about 4 % of the dry biomass. Once this stage is achieved, the cells are harvested by centrifugation and lysed. The asthaxanthin containing oil is then extracted and marketed. Every day at the plant, five different algal parameters are manually determined in order to control the process. In this context, the development of on-site biosensors for monitoring and optimisation of the process and deriving product is really advisable. The project produced four single sensors, two optical and two electrochemical, according to the detection technique used. The first optical biosensor realised allow to determine the H. pluvialis cells turbidity providing information on the cells concentration in the culture, hence on the ideal time for culture harvest. The second optical sensor was build-up in order to measure the fluorescence induction emitted by the chlorophyll a inside the H. pluvialis cells culture. This sensor monitors the cellular changes that occur under high irradiance in terms of changes in the PSII photosynthetic activity inside the cell. These information allow to derive important parameters on the carotenoid and chlorophyll accumulation. The second set of biosensors are electrochemical devices aimed to determine the astaxanthin concentration in the H. pluvialis culture (red phase) and its antioxidant capacity for the control quality assessment of the final product. Electrochemical biosensors were developed based on both screen printed electrodes (SPE) and nanowire-FET (NWFET). The SPE for antioxidant capacity (SPE-Aox) determination was successfully based on the exploitation of magnetic nanoparticles modified with phosphatidylcholine derivatives functioning as lipidic substrates for oxidative stress assessment, providing the overall antioxidant capacity of a sample. It has also been applied for evaluation of antioxidant efficacy against lipoperoxidation. The NWFET biosensor was based on the activity of the photosystem II (PSII) of spinach thylakoids with the aim to measure the astaxanthin concentration in a solution. Spinach thylakoids were immobilised by the highly efficient LIFT procedure on nonowires providing reliable results. An additional new chronoamperometric biosensor has been developed exploiting thylakoids extracted by different mutants of the unicellular green alga C. reinhardtii, obtained by a combined approach of directed evolution strategy and side-directed mutagenesis targeted to the photosynthetic D1 protein. Different protocols have been set-up for thylakoids purification from mutants that were immobilised on SPE after entrapment in calcium alginate matrix. This biosensor provide information on astaxanthin concentration and was realised to fulfill the project requirements regarding the necessity of implementing on large scale industrial line of highly sensitive, low cost and easy to use and fabricate sensors Each prototypes has been tested using different concentrations of astaxanthin providing their applicability to the purpose.
The introduction of such sensors in the antioxidants production line will increase production through online monitoring that will ease decision about time of harvest and culture performance. It is also expected a reduction in the production cost by at least 30 %, by saving work time and manpower, which is a big industrial breakthrough.
The new monitoring approaches provided are targeted to the bioproduction in the industrial green technology sector which has an extremely high potential of offering wide market opportunities while increasing the environmental sustainability and promoting public health and human safety.
Project context and objectives:
There is an increasing demand worldwide for low cost, fast, and reliable methods for monitoring chemical species, additives, and xenobiotics in clinical chemistry, environmental sciences, and food related processes. Chemical sensors and biosensors offer all these advantages since they can be easily used in both laboratory and field applications, are easy to use without need of a skilled operator, automatic, cheap, with no needs for maintenance and sample pre-treatments. A wide range of applications is foreseen in agriculture, veterinary analysis, food and drink industry, fermentation industry, waste water management, environment defence against pollutants and radiations, microbial contamination, clinical diagnosis, drugs monitoring, mining, military and defence, aerospace, mechanical production and many other fields. The proposed project addresses the development of sensors and biosensors useable for monitoring whole cell bioprocesses for the production of algal biomass and xanthophylls pigments with antioxidant activity.
Two of the SMEs applicants, BIO and NANO, are active in the field of analytical instruments based on electrochemical and optical devices and of innovative sensors technologies; they are familiar with the integration and interface of biologic material with electronics, mechanics and fluidics. By subcontracting experienced RTD performers, these SMEs aim at solving main critical issues involved in biosensor based products which are often responsible for the poor technology transfer from research laboratories to the market:
Technical issues, such as stability and sensitivity of the sensors, biomediator's short lifetime, lack of appropriate modern instrumentation and software tools. On the other hand, researchers in their laboratories have already developed technologies or have access to sophisticated expensive technological tools for miniaturisation (flow cells, transduction systems, etc.), electrochemical-optical transduction (potentiostats, electrometers, pH meters, ion meters, multi-channel analysers, spectrophotometers, etc.), chemical and biochemical recognition (ion carrier, enzymes, mutants, proteins etc.) and software to support the instruments. Often, these resources remain isolated and not available to SMEs in need of these technologies in order to complete a specific biosensor product.
Market issues, related to the partial awareness of the customers' needs, to the poor penetration of their advertising, and to the intrinsic difficulty to investigate and attack potentially huge markets at present still emerging, like the ones interested by the bioactive compounds and biomass production. Needs for a multidisciplinary approach that requires a lot of technical different expertises, always rare to find in a SME. Most SMEs have not stable relationships with R&D performers, have not education/training plans and have poor employees' time to dedicate to upgrading knowledge and skills.
The third SME, Algatech, is a producer of Astaxanthin and algal biomass and suffers critical problems which are typical of microalgae companies. Key parameters such as biomass (gr/l), pigment content (mg/l) and accumulation profile during the induction process are now manually determined routinely everyday at commercial production sites to measure overall. Since there is a need for very clean product at high quality, the xanthophylls growth must be carried out in expensive systems like tubular photobioreactors; in this framework monitoring growth parameters and pigment content daily further increases production costs, being critical time consuming and requiring manpower.
This is a major challenge faced by microalgae companies today, especially in the production of natural carotenoids such as beta carotene and astaxanthin in comparison with the relatively cheap synthetic analogues. European farmers are highly price sensitive to the cost of all inputs, and suppliers can only use ingredients that offer a favourable price: performance ratio in comparison with alternatives. So as long as synthetic antioxidant options are effective, low in price and still authorised for use, uptake of naturals, in the present economic climate will be limited. SENSBIOSYN intends to contribute to a faster spread of natural antioxidants by providing a mean for a significant cost reduction and optimisation of the production. The proposed sensors and biosensors will offer a solution to the lack of existing devices able to provide online, rapid, less laborious and reliable information on active compounds accumulation profile and efficacy during their biosynthesis.
The aim of the SENSBIOSYN project is to set up a collaborative framework between private companies and research institutes and universities to deliver new instruments for the benefit of all the involved industries in terms of enhanced competitiveness and market share. SENSBIOSYN will address the above problems through a dual approach, which allows SMEs to focus on their core competencies and concentrate on what they do best, while leaving the research activities to RTD performers:
i) a team of SMEs with close related activities but minimum overlap so that the exchange of technical information and know-how will not be hampered due to conflict of interests;
ii) a well balanced and strong scientific team from research centres and universities with long experience in biological aspects, in biosensor design and optimisation, instrument design and evaluation, industrial bioprocesses, as well as already developed laboratory methods and industrial technology transfer.
Production of bioactive compounds and algal biomass
Microalgae for xantophylls synthesis
In recent decades, some photosynthetic microorganisms, especially some prokaryotic and eukaryotic microalgae, have been identified as efficient biological systems for producing various chemicals and pharmaceuticals: carotenoids, phycobilins, fatty acids, polysaccharides, vitamins and sterols, which actually cover a large part of the market). Microalgae represent a potential source for both biomass production or high-value secondary metabolites preparation and purification. These chemicals usually exist at low concentration in cells growing under favourable conditions, but could be overproduced under unfavourable circumstances like biotic or abiotic environmental stresses. An additional important feature is that many microalgal strains are considered GRAS (generally recognised as safe) organisms, which is a basic requirement for the production of compounds or compounds-enriched microalgae for pharmaceutical, nutraceutical and feed applications.
Among the most modern whole cell processes is the production of the Astaxanthin from the green alga Haematococcus pluvialis. This is the industrial biosynthetic process selected in this proposal for the development of new biosensors to increase the quality control of the final product while reducing production costs.
The microalga H. pluvialis represents the richest natural source of astaxanthin and it is nowadays successfully cultivated at industrial scale. Cultivating the algal culture in closed systems allows an environmentally controlled process. The commercial production process is based on two distinct cultivation stages. The first is called the 'green stage' which starts indoors with a single-cell colony of the microalga, and continues outdoors in solar-powered photobioreactors. The aim of this stage is to produce plenty of viable, unstressed 'green' algal cells by normal cell-division process. The 'green stage' provides optimal growth conditions (carefully controlled pH, temperature and nutrient concentration) in order to achieve maximal biomass production rate. After a sufficient volume of vegetative cell suspension is produced, the second cultivation stage, the 'red stage' starts, in which the algal cells synthesise and accumulate the pigment astaxanthin. In this stage the cells are subjected to severe environmental stress conditions:
(1) deprivation of nitrate and phosphate;
(2) increased temperature and light irradiation; or
(3) addition of sodium chloride to the culture medium.
Within 2-3 days after the culture is stressed, the Haematococcus cells start to form cysts by producing thick cell walls, and to synthesise and accumulate Astaxanthin in its esterified form. 3 - 5 days after formation of haematocysts, the latter contain about 1.5 - 3.0 % astaxanthin and would be ready for harvest. The astaxanthin content of the 'red cells' is correlated to the severity of the stress conditions, mainly to the light flux through the culture, and it may reach up to about 4 % of their dry weight. At this stage, the size of the cells differ between 15 and 50 µm and the astaxanthin content is not uniformly distributed at the same time in the cells but it differs between the different sized cells. In this context, the on-site monitoring of the astaxanthin concentration / activity would be of outstanding interest to determine the time of harvest at a set astaxanthin content. In due time, the 'red' culture is pumped to the down-processing area, where the cells are cracked (to a) b) c) render the pigment bioavailable), dried, and vacuum-packed. Haematococcus oleoresin is produced in an additional step, using the CO2 Supercritical fluid extraction process. The SME Algatech partner in this project produces up to 1000 Kg/year of Astaxanthin.
Biosensors based on photosynthetic biomediators
Due to its unique features and structural constituents, photosynthesis and photosynthetic systems are suitable to the biosensoristic field, due to their ability to operate charge separation and electron transfer, and represent also a super model to be mimicked for developing artificial photochemicalm systems with high yield of energy capture and conversion.
Natural photosynthetis is allowed by the action of two distinct photosystems. This proposal will consider the photosystem II (PSII) for the construction of one of the proposed bio/sensors. PSII is a multisubunit chlorophyll-protein complex embedded into the thylakoid membranes of chloroplast that drives electron transfer from water to plastoquinone using energy derived from light. Its native form, consisting of more than 30 proteins, is surrounded by the light-harvesting complex, which binds six individual proteins and an array of chlorophyll a, chlorophyll b, lutein, violaxanthin, zeaxanthin and antheraxanthin molecules. At the centre of PSII-LHCII complex is the reaction centre (RC), which is composed of the D1 and D2 heterodimer which bind all cofactors involved in PSII mediated electron transport.
The electron flow through PSII begins with the release of the electron from an excited P680 molecule. When the primary donor, P680, is excited by light energy captured by antenna pigments, the primary charge separation takes place between P680 and the intermediate acceptor, Phe; this reaction generates the radical pair (P680+/Phe). Reduced Phe transfers an electron to the primary acceptor, QA, to generate QA-, and subsequently reduces the secondary acceptor, QB. This linear electron transfer reaction in PSII catalyses the light-induced water-plastoquinone oxidation-reduction with a high quantum yield (about 0.85 a little lower than PSI which is approximately 1.0). PSII can be extracted by algae, higher plants and cianobacteria.
Chlamydomonas reinhardtii is a microalga considered in this project as source of the PSII biomediator component required for the realisation of a specific biosensor. Chlamydomonas reinhardtii is a unicellular green alga phylogenetically closely related to H. pluvialis. The vegetative cell averages about 10 µm in diameter and is enclosed within a cell wall consisting primarily of hydroxyproline-rich glycoproteins, which not contain cellulose. It holds several mitochondria, two anterior flagella for motility and mating, and a single chloroplast surrounding the nucleus that houses the photosynthetic apparatus and critical metabolic pathways. Large quantities of cells can be produced quickly with relatively simple and inexpensive media. Chlamydomonas is used to study eukaryotic photosynthesis because, unlike flowering plants, it has the unique ability to grow heterotrophically in the dark by metabolising an exogenous organic carbon source (acetate), and to maintain functional green chloroplast that retains the capacity to perform oxygenic photosynthesis when illuminated following growth in the dark. In spite of these differences, however, the photosynthetic apparatus of Chlamydomonas is very similar to that of land plants, making it a useful comparative system for understanding plant metabolism and photosynthesis. Chlamydomonas possesses three genetic systems located in the nucleus, the chloroplast and the mitochondria, all sequenced and available at the JGI Chlamydomonas Genome Portal (see http://www.chlamy.org online). The newly available information concerning the sequence and organisation of the C. reinhardtii genomes combined with the many types of physiological, genetic, and molecular manipulations, make C. reinhardtii the organism of election for the generation and characterisation of a wealth of mutants with modifications in structural, metabolic and regulatory genes for biotechnological purposes.
Four single sensors will be developed specifically for the addressed Astaxanthin bioprocess in the tubular photobioreactor, divided into two groups according to the detection technique used: optical (2) and electrochemical (2). Their main characteristics and descriptions are summarised below:
1) Optical sensor to measure the fluorescence emitted by chlorophyll inside the Haematococcus pluvialis cells in culture medium
During the induction process of astaxanthin accumulation, the intensity of fluorescence emitted by chlorophyll inside the Haematococcus cells changes, .The proposed online sensor monitors the cellular changes that occur under high irradiance in terms of changes in the PSII photosynthetic activity inside the cell. The miniaturised fluorimeter instrumental set-up will consist of:
1) excitation LED light source with high intensity emission at 660 nm;
2) optical detector;
3) small and optimised flow cell to flux the cell culture sample into the measurement chamber.
The pulse-amplitude-modulation measuring principle is based on selective amplification of a signal generated by short subsaturating light pulses of high intensity.
This biosensor is innovative with respect to the offline Clark-type oxygen electrode previously used and reported for monitoring the photosynthetic activity of Haematococcus pluvialis cells.
2) Optical sensor to measure the density of the H. pluvialis cells culture medium through a light transmission measurement
The online measurement of the density and turbidity of the culture medium is very important because it gives information on the H. pluvialis cells concentration in the solution. This data is linearly correlated to the dry weight (DW), which plays a fundamental role for the final determination of produced astaxanthin, normally measured as DW percentage at the end of the process. A good estimation of DW will be achieved through a standard calibration curve that will be measured offline in the laboratory to extract a reliable conversion factor. The sensor will measure the light transmitted by the culture sample by a photodiode mounted at 180 with respect to the light source, after excitation with a LED at 730 nm. Such excitation wavelength has been chosen to avoid the spectrum range of chlorophyll absorption and fluorescence activation, which would mask and affect the turbidity measurement.
3) Electrochemical biosensor to measure the antioxidant potential of astaxanthin, after disruption of the Haematococcus pluvialis cells, by measuring the amperometric current generated by stabilised Phosphatidylcholine (PC) derivatives bound to magnetic nanoparticles
The antioxidant activity is the ability of a compound (mixture) to inhibit oxidative degradation (like lipid peroxidation). Direct methods to assess antioxidant activity (AoxA) are based on studying the effect of a sample containing presumed antioxidants on the oxidative degradation of a testing system. The substrate of oxidation may be individual lipids, lipid mixtures (oils), proteins, DNA, or lipid containing biologically relevant species, like blood plasma, LDL, biological membranes, etc. Lipid peroxidation appears to be the most convenient for the purpose of the SENSBIOSYN project. According to the solubility of the tested sample, the preference should be given to the oxidation of homogeneous lipids or to aqueous micro-heterogeneous systems, such as micelles and liposomes. Based on these arguments, we propose the use of phosphatidyl choline derivatives as lipid substrate for oxidative stress, the lipo-peroxyd formation being potentially reduced or even completely quenched by the presence of xanthophylls. The third proposed biosensor aims at measuring the antioxidant capacity in terms of the degree of efficacy of the red cells against Phosphatidylcholine (PC) peroxidative damage initiated by radical initiators.
4) Electrochemical biosensor to measure the astaxanthin concentration by measuring the output current of a nanowire field effect transistor (FET) whose nanowire channel is deposited with PSII biomolecules
The fourth proposed biosensor is a nanosensor which uses the immobilised Photosystem II (PSII) complex extracted by the microalga C. reinhardtii as sensing. The nanodevice employed has been already developed by the SME participant Nanosens (NANO) but it has never been coupled to PSII and never been applied in industrial monitoring applications. It will transduce the electrochemical charge and electric field variations induced by the PSII reaction with the antioxidant agent into an output current. It is a nanowire FET, in which, in contrast to conventional FET, the source and drain contacts are ohmic contacts, realised at the ends of a silicon nanowire which acts as the FET channel, basically as a semi-conducting nano-resistor. To function as a biosensor the surface of the nanowire is coated with a functional biologic layer, capable of coupling with targeted species present in the sample solution. When the targeted species activate an electron transfer and a charge unbalance in the biomeadiator layer, a change in the electric field is induced at the surface of the nanowire which causes an enrichment or depletion of the charge carriers in the semiconductor. This broadening or narrowing of the local conducting channel causes a measurable change in the electrical conductance of the whole nanowire, even at a nearly 0 V gate potential, which is measured as a variation of the FET drain-source current.
Project results:
The main objective of the SENSBIOSYN project is design, manufacturing and testing innovative biosensors for supervision of the astaxanthin natural production process. The industrial process involves cultivation of the green alga Haematococcus pluvialis at Kibbuz Qetura in Israel, in the largest continuously operating industrial tubular photobioreactor facility, consisting of over 30, 10 000 litre reactors, covering an area of over 10 000 m2. This process is based on cultivation of the green alga Haematococcus pluvialis in two stages:
- stage 1 in complete medium for rapidly producing large amounts of green biomass; and
- stage 2 where those green cells are transferred to nutrient stress during which they accumulate the red pigment astaxanthin to a concentration of up to 4 % of its dry weight.
Once this stage is achieved, the cells are harvested by centrifugation and lysed. The asthaxanthin containing oil is then extracted and marketed. This is the only commercial facility worldwide successfully in operation for 10 years relying on such a highly controlled cultivation process for production of pigment rich algal biomass and, as such, the only available option for realistic testing of the planned sensors, though experimental and pilot facilities have now put in place at increasing frequencies.
During the first project year, BGU has provided all the necessary background on the production process, control and management of the cultivation process, details of culture parameters measured, and detailed description of the bioreactor facilities with pictures depicting possible attachment points for additional biosensors. Subsequently, during the project meeting in Israel, it has been decided to insert the sensors in the same locations where sensors for pH or temperature detection are already placed. Furthermore, as several spare connections for additional sensors are available, it has been verified by the BIO, CNR project engineer the suitability to place the same sensor in different reactor locations for testing consistency of the readouts and their independence from the sensor locations.
To identify the system specifications for novel biosensors development, BGU provided a list of parameters routinely measured, recorded and controlled at the Qetura facility along the process which include:
- the pH of the culture medium (measured on line and controlled by varying the flow CO2 into the reactor);
- reactor temperature (monitored online and controlled by cooling water applied to the outside of the reactor tubing);
- dry algal biomass concentration (measured manually on a daily bases, both in the green and the red growth stages);
- chlorophyll concentration (measured manually on a daily basis);
- photosynthetic efficiency (fv/fm, determined manually on a daily bases);
- astaxanthin concentration during the red stage (measured daily by a manual procedure);
- turbidity (OD 750, measured daily manually);
- oxygen concentration (monitored online);
- nitrate concentration (monitored online);
- light intensity.
To demonstrate the importance of continuous multi-parameter supervision of the cultivation process BGU determined a number of relevant parameters in two green reactor units and two red reactor units each continuously during June 2010. These measurements revealed a significant unexploited production potential: average reactor performance is far below optimal most of the time. For the green stage daily productivities vary widely, dilution intervals are irregular and as a consequence the biomass produced is not of reproducible quality, leading to differences in subsequent astaxanthin productivity as well. A similar situation was experienced analyzing the accumulation of astaxanthin during the nitrate starvation growth phase. Similar to the green stage, both accumulation of dry weight and astaxanthin in the red stage are hampered seriously by unreliable and erratic growth phenomena. The situation is complicated by the fact that success of the red stage depends strongly on the quality of the green material used to charge the reactor, which in itself is not of consistent and reproducible quality.
These data gave a strong indication on the urgency to move to sensor based culture control and management. All process stages, methods and culture parameters have been listed and used to recommend sensor specifications and design. Those recommendations were supported by a detailed literature review on optical and fluorescence properties of Haematococcus cultures helpful in designing suitable biosensors. BGU has performed further experiments for further refinement and improvement both the planned fluorescence optical sensors. The most appropriate sensor dimensions and wavelengths for an optical flow through sensor as well as excitation and emission wavelengths for the fluorescence detector were identified and provided to the sensor manufacturer. Main specifications are reported below. The flow through cell for measuring absorption at different wavelengths is the most important tool for controlling and managing cultures. Such a cell should measure simultaneously turbidity by OD 750 nm and chlorophyll peak by OD at 665 nm. Since suspended cells of various sizes will be measured, the geometry and hydrodynamics of the flow through cell is of crucial importance, to pass a homogenous sample stream without bubbles and turbulences through the sample cell. This detector should be linked on line to a control centre where raw data would be directly transformed into parameters such as dry-weight, chlorophyll content, astaxanthin content, cell number and chlorophyll / astaxanthin ratio, by application of suitable methods of calibration and empirical formula.
Regarding the sensor for the photosynthetic efficiency measurements, it has been determined that Fv/Fm cannot be measured continuously. This sensor can perform periodic measurements whereby a sample is drawn into a mixed dark chamber and dark adapted for about 20 min, and Fo can be measured. A light flash then induces very shortly maximal PSII fluorescence Fm that is then relaxing. Fv (Fm - Fo) divided by Fm is then a measure for photosynthetic efficiency. This sensor is thus fundamentally different from the first one determining absorption in flow through mode. However it too should feed its data directly to the central control unit where adequate algorithms will supervise the culture status. Fv/Fm changes with increasing astaxanthin content. Chlorophyll fluorescence allows to study the different functional levels of photosynthesis indirectly. Final technical specification of sensor design and testing were defined by BIO, BGU and CNR.
Furthermore, BGU carried out associated experiments with other prominent microalgae species such as Nannochloropsis and Parietochloris. Obtained data confirmed that under stress conditions, those species display the same optical changes (strongly reduced chlorophyll per biomass) we intend to exploit for quantifying Haematococcus. Thus our sensors can be applied to cultivation of other algal species based on identical principles, though those species are not commercially produced in large scale yet.
For verification of sensors developed in Rome and Bucharest, Haematococcus cells from different growth stages grown both in the Qetura bioreactors or in the lab at BGU, representing green to fully red cells were sent repeatedly to the partners for testing the antioxidant and flow through sensors respectively, and further protocols and details on maintaining the algal cultures were provided to both partners. The samples were successfully tested for antioxidant capacity and for fluorescence and OD determination using the respective sensors.
From the beginning of the project, biologists at the CNR worked in order to screen, identify, characterise and purify wild-type and molecular engineered Chlamydomonas reinhardtii photosystem II complexes to be used as bio-recognition elements for biosensor development. The screening is based on several crucial points:
i) PSII functional characterisation of the biomediators;
ii) stability of biomediator performance in immobilised conditions;
iii) response of the biomediator's PSII activity to unfavourable conditions.
Research activities were carried out on chlamydomonas strains mutated in the chloroplast psbA gene encoding the D1 reaction centre protein, which hosts several crucial components of the photosynthetic electron transfer. Mutations in this site can lead to modification of electron transport rate and overall PSII performance. Random mutagenesis was used for generating D1 proteins with novel properties by in vitro psbA gene evolution technique. This has been achieved by 'error-prone' PCR mutagenesis through an iterative process consisting of recombinant generation to create combinatorial libraries. The result was a pool of D1 variants that was delivered in algae chloroplast by particle gun bombardments. Mutants were further selected for their capability to overcome ionising radiation exposures represented by proton and neutron sources in order to get biological photosynthetic material with improved capability to tolerate radical stress. One of the milestone using biological materials as a sensing element in the biosensor devices is their capability to maintain their long-term activity often under unfavourable environment.
About 2000 transformed cells were subjected to the ionising radiation treatments and 32 colonies overcoming the radiation-induced stress were analyzed by psbA sequencing. Among them, twenty strains were identified as different mutants hosting both single and double mutations, indicating that in the unicellular green alga C. reinhardtii even single amino acidic substitutions in the reaction centre D1 protein enable the cells to survive in the presence of free radicals produced by both high light fluencies and neutron and proton radiation. The mutations, located in regions of the D1 protein responsible for the electron transport and oxygen evolution processes, involved replacement of polar amino acids, more prone to oxidative damage, with less sensitive ones. To exclude the possibility that additional random mutations induced by the radiation exposure could confer the observed tolerance, a set of the identified amino acid substitutions were introduced by site-directed mutagenesis in untransformed strains. The choice of the residue substitutions was made to include mutations located in two different structural regions of the D1 protein, near Tyr161 and the oxygen evolving complex (OEC) (I163T, P162S, M172L) and near the QB binding pocket (G207S, L200I, I281T). Physiological characterisation of the obtained mutants was carried out by estimating the growth parameters and the photosynthetic efficiency. Functional characterisation of the produced Chlamydomonas D1 mutants in physiological conditions revealed very similar trends in growth rates, but showed a reduction in their chlorophyll content and photosynthetic performance. The mutants generally showed a lower maximal quantum yield of PSII photochemistry (Fv/Fm) and a reduced efficiency of the electron transport through PSII primary and secondary electron acceptors (1-VJ) than the control strain. Despite the lower electron transfer efficiency, a higher oxygen evolution capacity under high photon fluency conditions was demonstrated in all the mutants compared to the reference strain, IL. The D1 mutants were able to achieve and maintain more than two-fold higher O2 evolution rate even under the very high light intensity of 900 µmol m-2 s-1. The long-term resistance to stressful conditions, such as low temperature (13 °C) and light intensity (20 µmol/m2/s), of the chlamydomonas strains produced by site-directed mutagenesis and selected, as biomediators was tested for a period longer than two months. On one hand, low temperature stress slow down all the catabolic reactions, which reduced the demand of absorbed energy, and on the other hand hold up the photosynthetic electron transport, leading to accumulation of long-lasting reduced forms of the PSII primary quinine (QA). As a result, the probability for oxygen radical species generation and oxidative damage increase considerably.
In these conditions, some of the D1 mutants were able to preserve between 70 % and 80 % of their initial photosynthetic activity (Fv/Fm) in contrast to the 54 % maintained by the reference strain. Moreover, the I281T mutant resulted very stable since even after 100 days under low temperature the reduction of PSII performance did not exceed 30 %, in comparison to 57 % reduction observed in IL strain. Few other mutants showed lower decrease in Fv/Fm than IL, such as M172L maintained 66 % of the initial value, L159M 56 %.
Additional mutant were produced by site-directed mutagenesis and protein engineering following a rational design accomplished by molecular modelling and binding energy calculation procedures to produce mutants having novel binding niche for the sensing event. These activities led to the production of a lot of mutants. Among these mutants at least five strains resulted suitable for our purposed. The step forward was the purification of these mutant's photosynthetic active sub-components, such as thylakoid membrane. Due to specific ultrastructural features, C. reinhardtii requires care in cell disruption to preserve thylakoids integrity and PSII sovramolecular organisation and functionality for biosensoristic purposes. Different physico-chemical procedures, including the use of Yeda press, French press and sonication in the presence of specific solvents, has been applied and tested for the best results. This task required a lot of time, as different mutants had a different migration pattern of the PSII-enriched thylakoid fraction during the particle preparation. Finally, a protocol for each mutant was set-up. Measurements of the photosynthetic activity by fluorescence methods has been used to test and compare the most suitable thylakoid preparations. The developed biorecognition elements were realised in order to build up electrochemical biosensors based on nanowire transducers to detect astaxanthin. However, taking into account the project requirements to implement large scale industrial line with highly sensitive, low cost and easy to use and fabricate sensors, mutated algal strains and their active sub-components were exploited to build-up a novel chronoamperometric sensor, as alternative solution to NWFET sensor.
The proof-of-concept for PSII-based biosensors functioning relies on their capability to harness light and convert it in an electrical or optical signals. The excitation of the PSII by red light induces charge separation and electron transfer processes resulting in a measurable intensity current. In the presence of an inhibitor of the PSII photosynthetic activity (such as exposure to high intensity of white light, or herbicides), a decrease of the charge transfer occurs, consequently a decrease of the measured current occurs. It has been assumed that the presence of Astaxanthin can preserve the photosynthetic activity protecting the PSII. The degree of restored activity is proportional to the amount of the astaxanthin.
The experimental conditions for PSII-chronoamperometric biosensor were optimised in terms of:
1) material of the working electrode;
2) immobilisation procedure of the phototosynthetic material;
3) applied potential to the working electrode during amperometric measurements;
4) photoperiod, namely the duration of dark/light cycles, during electrochemical measurements;
5) flow rate;
6) cell density and chlorophyll content;
7) composition and ionic strength of the buffer solution flowing through the measuring cell (referred as measurement buffer solution),
in order to maximise the biomediator viability (half-time, see below) and response stability to the analyte of interest. Flow rate, cell density and chlorophyll content and even characteristics of buffer solution were optimised in order to ensure the highest signal to noise (S/N) ratio in the working conditions, as all these parameters affect the S/N ratio, the optimum values for all these parameters were chosen. The choice of the appropriate working electrode material was performed among different and commercially available screen-printed electrochemical cell electrodes, SPEs (Dropsens, Oviedo, Spain) made of gold (Au), graphite (C) or carbon nanotubes (CNT). All measurements were performed in a lab-made biosensor instrument, settled specifically for this project, which is composed by:
(i) a software-controlled PG 581 potentiostat (Uniscan Ltd. UK);
(ii) a peristaltic pump joined to a sample cell box equipped with inlet and outlet flow holes; and
(iii) SPE modified electrode with algae.
SPE cell fits into the measurements cell, while a suitable buffer solution flows through the measuring cell the instrument provides amperometric signals subsequent to biological material illumination at a suitable wavelength and intensity (650 nm and 325 µmol respectively, from two red LEDs at the bottom of the sample cell; the whole device has been specifically designed for dynamic flow chronoamperometry measurements. In order to establish the more suitable material for the SPE working electrode and the appropriate applied potential values for chronoamperometric measurements, first experiments performed were of cyclic voltammetry (CV), the experimental conclusions leading to the choice of CNT-SPE as optimal working electrodes, with a wider available potential window to work on. Concerning the immobilisation procedure, the most reliable providing low S/N ration was based on physical gelation with calcium alginate polysaccharide and was applied throughout.
Exploiting Chlamydomonas reinhardtii whole cells as biorecognition element, the value of applied potential for chronoamperometric experiments is around -0.7 V vs reference electrode Ag/AgCl. At this value, the oxygen reduction current signal originated from the algal photosynthetic activity following illumination is registered. The optimum applied potential in these chronoamperometric measurements was chosen according to a 'change and trial' procedure, the values of the applied potential ranging between -0.7 V and -0.1 V and the obtained current intensities (which are proportional to oxygen evolution) are registered.
Exploiting thylakoids extracted by both spinach plants and Chlamydomonas reinhardtii mutants strains, the applied potential was +0.2 V versus reference electrode Ag/AgCl. At this value, the DCPIP current signal originated from the PSII photosynthetic activity following illumination is registered. Photosynthetic activity in both whole cells or thylakoids was stimulated or inhibited by red (652 nm, 540 µmol photons cm-2s-1) and white light (1350 µmol photons cm-2s-1), respectively. The algal cell density and chlorophyll content on the working electrode were carefully determined. Generally, a specific relationship exists among algal cell density and the chlorophyll content for each algal strain. Indeed the current signal (and the S/N ratio too) depends on many factors such as cell density, surface properties of the immobilised cells / SPE interface, chlorophyll content, etc. Some tests were performed, aiming to optimise the biosensor response as function of the algae / alginate mixture volume dropped onto the SPE surface using optimal immobilisation procedure. The volume of algae / alginate mixture providing the highest signal was somewhere around 6 µl. When the signal to noise (S/N) ratios are considered, it was noticed that the best volume to drop over the SPE for optimising the S/N response of the biosensor is 5 µl. As consequence, this volume was further used by us in biosensor fabrication. Illumination time was set-up at a sequence of 30 s light/5 min dark and flow rate at 0.1 ml/min. Amperometric measurements performed with both algal strains and extracted thylakoids proved the developed PSII-CNT SPE electrochemical sensors to be suitable to the purpose, meaning, to the determination of astaxanthin amount by assessing its capability to reduce the PSII inhibition generated by light stress. Data supporting this conclusion are: no matter the employed mutant in fabrication of PSII-CNT, amperometric sensors, the sensor responses correlates to astaxanthin amount, the stress inhibition being higher with increasing the astaxanthin amount; the developed sensors provide accurate and precise responses (SD ranges between 0.3 and 0.5); the developed method proved a good sensor sensitivity of 1.15 µA/mmolL-1.
The project aimed to produce four single sensors, two optical and two electrochemical, according to the detection technique used. Optical biosensors aimed to determine the H. pluvialis cells turbidity and photosynthetic performance providing information on the cells concentration in the culture, hence on the ideal time for culture harvest (mainly involved SMEs and RTD performer: BIO, Algatech and CNR). Electrochemical biosensors aimed to determine the astaxanthin concentration in the H. pluvialis culture (red phase) and its antioxidant capacity for the control quality assessment of the final product (mainly involved SMEs and RTD performer: BIO, Algatech, NIB, NANO and CNR).
Development of two optical (chlorophyll fluorescence, culture turbidity) and two electrochemical (PSII-nanowireFET, magnetic beads stabilised PC-SPE) biosensors able to assess the content and the antioxidant efficacy of accumulated xanthophylls was achieved during the two-years activity in tight collaboration with all parties.
Two optical biosensor for fluorescence and density measurement has been developed taking into carefully consideration the previous reported specification provided by BGU and the technical specification of BIO engineers. The fluorescence system has been equipped with a LED excitation source operated in continuous mode, generating high intensity red light to excite the fluorescence emission without altering astaxanthin induction and formation, making the measured results reliable. In order to efficiently collect the emitted fluorescence, different photodetectors and configurations has been investigated according to the S/N ratio achievable. In addition, an interferential band-pass filter centred on the set fluorescence spectral range has been used. As the cell culture are kept in constant flow in the photobioreactor tubes, a direct interface of the fluorescence sensor with the solution under test through the tube glass wall is not reliable, so the fluorescence measurement has be planned in static condition to provide the necessary time interval elapsing between the excitation and the successive collection of the emitted fluorescence.
The optical biosensor for the determination of the culture turbidity has also been delivered by BIO in tight collaboration with CNR and BGU. The device is equipped with red emission wavelength for the turbidity measurement that consider the chlorophyll absorption peak at 660 nm and consequently shift to 730 nm to avoid mask effects.
Electrochemical biosensors were developed based on both screen printed electrodes (SPE) and nanowire-FET (NWFET) transducers by CNR and NIB.
The SPE for antioxidant capacity determination was successfully set-up. The electrochemical biosensor was based on magnetic nanoparticles modified with phosphatidylcholine derivatives as substrate for oxidative stress. Phosphatidylcholine (PC) derivatives were bound to magnetic nanoparticles, characterised and stabilised. The optimal recommended storage conditions for composite PC-nanobeads were finally defined as: room temperature, no light contact storage in inert atmosphere. Subsequently, the PC peroxidation assessment following ROS attack was evaluated as well as the lipophilic antioxidant efficacy against PC Peroxidation. The obtained results proved that the developed model is applicable even to assessment of antioxidant efficacy against lipoperoxidation. After the flow system design, the effect of magnetic field on registered chronoamperometric response was evaluated.
Procedure of measurements using the assembled biosensors was drawn and, as further detailed, laboratory and industrial testing were performed. SPE electrochemical sensor for astaxanthin antioxidant capacity determination was developed, tested and validated in terms of linearity, limit of quantitation (LQ), limit of detection (LoD), repeatability; accuracy; selectivity with real samples. The developed SPE-Aox sensors are applicable to various samples (oil or H. pluviallis cell cultures), proving their versatility. Responses provided by SPE-Aox are given as overall antioxidant capacity of the sample (or extract). Three new protocols / methods were established and validated:
- protocols of SPE-Aox use to assess ASTAXATHIN from oils;
- protocols of SPE-Aox use to assess astaxathin from H. pluviallis cell cultures.
Finally, the sensors response statistical validation was carried out.
The SPE biosensors for astaxanthin determination were also set-up, exploting Chlamydomonas reinhardtii algae cells as photosynthetic bio-sensing materials. The experimental conditions were optimised in terms of:
1) material of the working electrode;
2) applied potential to the working electrode during amperometric measurements;
3) immobilisation procedure of the phototosynthetic material;
4) duration of dark / light cycles during electrochemical measurements;
5) flow rate;
6) cell density and chlorophyll content;
7) composition and ionic strength of the buffer solution flowing through the measuring cell, in order to maximise the biomediator viability and response stability to the analyte of interest.
Flow, rate, cell density and chlorophyll content and even characteristics of buffer solution were chosen as those ensuring the highest S/N ratio in the working conditions. Two main immobilisation procedures were tested in order to achieve the best results:
(a) coating of algae cells with a Nafion membrane;
(b) physical gelation of alginate for algae cells entrapment, the second one providing the best outcome.
The amperometric set-up was exploited to determine algal photosynthetic performance as preliminary approach for the development of biosensor for astaxanthin concentration determination. Subsequently, the analytical device developed was set-up in order to determine the astaxanthin concentration necessary to revert a photoinhibitory event. The latter was induced by including a saturating white-light led in the instrument that in active operation mode decrease the photosynthetic current produced. Astaxanthin inhibits this phenomenon in a concentration manner. Dose response curves were carried out leading to the production of a reliable titration curve. A similar approach was followed in order to set-up functionality of biosensor hosting thylakoid membranes extracted by whole algal cell.
The photosystem-based Nanowire FET electrochemical biosensor to measure the xanthophyll concentration in a solution was successfully developed by CNR and NIB. Spinach thylakoids were immobilised by the highly efficient LIFT procedure on nanowires (provided by NANO) producing the PSII-NWFET. The performance of this assembly was assessed determining crucial parameters, such as the excitation time, the range of potential appropriate for modified PSII-NWFET, registered intensity current range, useful domain of inhibitor concentration, useful range of antioxidant / preservative compound concentration, PSII-NWFET operational stability. Furthermore, astaxanthin preservative effects were assessed.
As required by task 3.4 protocols of H. pluviallis cell disruption and carotenoids extraction were set-up.
Continuation of experiments on PSII-NWFET is expected to be improved exploiting additional thylakoids extracted by different strains of Chlamydomonas on new nanowires. NANO is currently looking at ways to optimise the nanowire fabrication process (design, surface smoothness of the NWs) and also team with larger industrial foundry partners to obtain 'ready to measure reliable devices' on a larger scale. In addition, beyond the scope of the SENSBIOSYN project, Nanosens is currently focused on improving the electrical device characterisation setup, the microfluidics delivery, and the readout electronics a compact instrumentation setup so that our research partners could focus on sample measurements together.
The introduction of such sensors in the antioxidants production line will increase production through online monitoring that will ease decision about time of harvest and culture performance. It is also expected a reduction in the production cost by at least 30 %, by saving work time and manpower, which is a big industrial breakthrough.
The new monitoring approaches provided are targeted to the bioproduction in the industrial green technology sector which has an extremely high potential of offering wide market opportunities while increasing the environmental sustainability and promoting public health and human safety.
Potential impact:
The potential impact of the project is the result of three converging parts, emerging from the project specificity.
First, the results obtained lead us to supporting the project impact on the participating SMEs in terms of advantages gained from the project:
Technical impact:
SMEs will have the opportunity to push the production of new products toward real problems solution with the help of the directly involved end user, ALgateh.
Biosensor, based on fabricated and tested prototypes, SMA and ASPE have the opportunity of producing new instrumentation; Biosensor is be able to further incorporate in their automatic fabricated and commercialised systems these new developed devices. Biosensor access specific technical and scientific knowledge on industrial biosynthesis of algae, which is very difficult and uncommon to find, but fundamental to attack a promising emerging market for sensors and biosensors.
The technical advantages gained by Algatech directly impact their astaxanthin production process: they have the possibility of employing control devices online and to be the first industry to test and use them on board their photobioreactor, as long as registered drawback during in field testing is solved.
Commercial impact:
Biosensor, Nanosens and Algatech are characterised by a good quality standard production and a high innovation degree. As long as Nanosens is solving the issue in nanowires production, all three SMEs will be able to offer on the market a wider range of high performance. Algatech will reduce the production cost by improving the manpower costs and will offer a more qualified and controlled product.
Educational and training impact:
Biosensor offered internships to young researchers. Based on developed protocols for new biosensors fabrication, as included even in the dissemination plan, CNR, NIB and BGU are transferring the know-how by organising educational courses for the study and the application of the implemented methodologies.
Secondly, based on the fact that in the last decade the direction of biosensor R&D significantly changed in response to new biotechnology innovations in biocomplementary chemistry, surface characterisation, molecular markers, and nanotechnology a market impact is expected.
Based on obtained and tested prototypes, considering the data obtained when devices were validated, it is estimated that in the next years the following sensors will be on the market as presented in tables below, were even the associated business plan is given.
The business plan was developed considering the estimated production costs, sales volume other costs related to product introduction to the market (prices in thousands of Euro). The business plan was drastically adjusted with respect to initial one, considering the economic crisis waves.
This project is significantly relevant to the industrial and applied research and development. The prototypes developed, intended for field trials, feature a high potential for commercialisation, if supported by a well-developed marketing strategy in order to increase the stakeholders and public awareness on the biosensors use in processes control and monitoring.
The need for on-line monitoring of growth parameters of Xanthophyll metabolites in cultivation systems and process streams is a main issue faced today by producer companies.
Biomass (gr/l) and pigment content (mg/l) are nowadays determined offline routinely everyday at commercial production sites by means of complex manual analyses carried on by skilled operators.
The cost of these operations further increase the production cost that today is a major challenge, especially for small companies in the business of biosynthesis of Xanthophylls that are recently starting up in Europe.
The innovative offer of the biosensors developed in this project is about the possibility of doing online not only the classical measurements but also specific analyses without time consumption and manpower.
Biomass, pigment concentration, accumulation profile during the induction process, photosynthetic activity (through the chlorophyll fluorescence measurement) and antioxidant capacity level will be monitored online, automatically and rapidly. This will allow to increase production and reduce production cost by at least 20 %, which is an important industrial breakthrough.
It is envisaged, also, that SENSBIOSYN will even contribute to increase the applicant SMEs' market share being attractive worlwide to international companies, public / private research institutes and laboratories, operating in the above mentioned fields and in the more recent field of algae-based bioenergy.
In addition, the flexibility and wide applicability of the SENSBIOSYN products allow to target other potential end-user companies producing natural food additives, functional food and nutraceutics, not exclusively extracted by algae.
The choice of the distribution channels is fundamental because the product is highly innovative and target specific markets.
The distribution politics take into account various channels we intend to take advantage of to commercialise the products. 20 % of our effort to distribute the new product will use the same distributors already employed by each SME for their existing instrumentations.
Ongoing dissemination activities will be as follow:
Specific dealers distribution (40 %) are the most relevant channels offered by dealers that are well-known in the microalgae and fish industry. Through the internet (20 % effort), in addition to the project and partners' websites, it is possible to advertise the products on web magazines and journals. The products can be displayed at conferences and exhibitions (10 %). Direct contacts (10 % effort) through the experience and acquaintances of the main partners in the project.
The experimental results will be presented at international conferences and, where possible, be made available to an extended audience. We will make use of an updated SESNBIOSYN webpage and the various European networks and work groups to disseminate the project results. Also, the services of the European Innovation Relay Centres, where applicable, will be used to disseminate the results and work towards a commercialisation of the biosensors.
There will be an international symposium during the next year within the International Conference on Photosynthesis.
The partners will continue to attend the European Biosensor Symposia organised in 2012 for the exchange of knowledge and technology.
A brochure of the developed biosensors was electronically issued, printed out upon request of farms and firms that will be updated next 4 - 6 months also after the project end.
The group will organise 'hangar-stands' for technological exhibitions in EU countries. A first stand was organised in the area of research and presentations to companies took place. A stand was already organised for the biosensor meeting expected in Mexico on 2012 and for the biosensing meeting in Edimburgh expected in March 2012.
Biosensors prototypes were previously presented on TV programs and this aroused great interest; they were presented again by the CNR press office and in the near future. Several meetings will include participation of interested industries beside the extended consortium.
Concerning the first results of dissemination nine articles have been published on impact factor journals and other five articles under review and in preparation.
SENSBIOSYN partners participated to eleven Conference/exhibitions presenting stands of the results and prototypes.
Project website: http://www.sensbisyn.com
The introduction of such sensors in the antioxidants production line will increase production through online monitoring that will ease decision about time of harvest and culture performance. It is also expected a reduction in the production cost by at least 30 %, by saving work time and manpower, which is a big industrial breakthrough.
The new monitoring approaches provided are targeted to the bioproduction in the industrial green technology sector which has an extremely high potential of offering wide market opportunities while increasing the environmental sustainability and promoting public health and human safety.
Project context and objectives:
There is an increasing demand worldwide for low cost, fast, and reliable methods for monitoring chemical species, additives, and xenobiotics in clinical chemistry, environmental sciences, and food related processes. Chemical sensors and biosensors offer all these advantages since they can be easily used in both laboratory and field applications, are easy to use without need of a skilled operator, automatic, cheap, with no needs for maintenance and sample pre-treatments. A wide range of applications is foreseen in agriculture, veterinary analysis, food and drink industry, fermentation industry, waste water management, environment defence against pollutants and radiations, microbial contamination, clinical diagnosis, drugs monitoring, mining, military and defence, aerospace, mechanical production and many other fields. The proposed project addresses the development of sensors and biosensors useable for monitoring whole cell bioprocesses for the production of algal biomass and xanthophylls pigments with antioxidant activity.
Two of the SMEs applicants, BIO and NANO, are active in the field of analytical instruments based on electrochemical and optical devices and of innovative sensors technologies; they are familiar with the integration and interface of biologic material with electronics, mechanics and fluidics. By subcontracting experienced RTD performers, these SMEs aim at solving main critical issues involved in biosensor based products which are often responsible for the poor technology transfer from research laboratories to the market:
Technical issues, such as stability and sensitivity of the sensors, biomediator's short lifetime, lack of appropriate modern instrumentation and software tools. On the other hand, researchers in their laboratories have already developed technologies or have access to sophisticated expensive technological tools for miniaturisation (flow cells, transduction systems, etc.), electrochemical-optical transduction (potentiostats, electrometers, pH meters, ion meters, multi-channel analysers, spectrophotometers, etc.), chemical and biochemical recognition (ion carrier, enzymes, mutants, proteins etc.) and software to support the instruments. Often, these resources remain isolated and not available to SMEs in need of these technologies in order to complete a specific biosensor product.
Market issues, related to the partial awareness of the customers' needs, to the poor penetration of their advertising, and to the intrinsic difficulty to investigate and attack potentially huge markets at present still emerging, like the ones interested by the bioactive compounds and biomass production. Needs for a multidisciplinary approach that requires a lot of technical different expertises, always rare to find in a SME. Most SMEs have not stable relationships with R&D performers, have not education/training plans and have poor employees' time to dedicate to upgrading knowledge and skills.
The third SME, Algatech, is a producer of Astaxanthin and algal biomass and suffers critical problems which are typical of microalgae companies. Key parameters such as biomass (gr/l), pigment content (mg/l) and accumulation profile during the induction process are now manually determined routinely everyday at commercial production sites to measure overall. Since there is a need for very clean product at high quality, the xanthophylls growth must be carried out in expensive systems like tubular photobioreactors; in this framework monitoring growth parameters and pigment content daily further increases production costs, being critical time consuming and requiring manpower.
This is a major challenge faced by microalgae companies today, especially in the production of natural carotenoids such as beta carotene and astaxanthin in comparison with the relatively cheap synthetic analogues. European farmers are highly price sensitive to the cost of all inputs, and suppliers can only use ingredients that offer a favourable price: performance ratio in comparison with alternatives. So as long as synthetic antioxidant options are effective, low in price and still authorised for use, uptake of naturals, in the present economic climate will be limited. SENSBIOSYN intends to contribute to a faster spread of natural antioxidants by providing a mean for a significant cost reduction and optimisation of the production. The proposed sensors and biosensors will offer a solution to the lack of existing devices able to provide online, rapid, less laborious and reliable information on active compounds accumulation profile and efficacy during their biosynthesis.
The aim of the SENSBIOSYN project is to set up a collaborative framework between private companies and research institutes and universities to deliver new instruments for the benefit of all the involved industries in terms of enhanced competitiveness and market share. SENSBIOSYN will address the above problems through a dual approach, which allows SMEs to focus on their core competencies and concentrate on what they do best, while leaving the research activities to RTD performers:
i) a team of SMEs with close related activities but minimum overlap so that the exchange of technical information and know-how will not be hampered due to conflict of interests;
ii) a well balanced and strong scientific team from research centres and universities with long experience in biological aspects, in biosensor design and optimisation, instrument design and evaluation, industrial bioprocesses, as well as already developed laboratory methods and industrial technology transfer.
Production of bioactive compounds and algal biomass
Microalgae for xantophylls synthesis
In recent decades, some photosynthetic microorganisms, especially some prokaryotic and eukaryotic microalgae, have been identified as efficient biological systems for producing various chemicals and pharmaceuticals: carotenoids, phycobilins, fatty acids, polysaccharides, vitamins and sterols, which actually cover a large part of the market). Microalgae represent a potential source for both biomass production or high-value secondary metabolites preparation and purification. These chemicals usually exist at low concentration in cells growing under favourable conditions, but could be overproduced under unfavourable circumstances like biotic or abiotic environmental stresses. An additional important feature is that many microalgal strains are considered GRAS (generally recognised as safe) organisms, which is a basic requirement for the production of compounds or compounds-enriched microalgae for pharmaceutical, nutraceutical and feed applications.
Among the most modern whole cell processes is the production of the Astaxanthin from the green alga Haematococcus pluvialis. This is the industrial biosynthetic process selected in this proposal for the development of new biosensors to increase the quality control of the final product while reducing production costs.
The microalga H. pluvialis represents the richest natural source of astaxanthin and it is nowadays successfully cultivated at industrial scale. Cultivating the algal culture in closed systems allows an environmentally controlled process. The commercial production process is based on two distinct cultivation stages. The first is called the 'green stage' which starts indoors with a single-cell colony of the microalga, and continues outdoors in solar-powered photobioreactors. The aim of this stage is to produce plenty of viable, unstressed 'green' algal cells by normal cell-division process. The 'green stage' provides optimal growth conditions (carefully controlled pH, temperature and nutrient concentration) in order to achieve maximal biomass production rate. After a sufficient volume of vegetative cell suspension is produced, the second cultivation stage, the 'red stage' starts, in which the algal cells synthesise and accumulate the pigment astaxanthin. In this stage the cells are subjected to severe environmental stress conditions:
(1) deprivation of nitrate and phosphate;
(2) increased temperature and light irradiation; or
(3) addition of sodium chloride to the culture medium.
Within 2-3 days after the culture is stressed, the Haematococcus cells start to form cysts by producing thick cell walls, and to synthesise and accumulate Astaxanthin in its esterified form. 3 - 5 days after formation of haematocysts, the latter contain about 1.5 - 3.0 % astaxanthin and would be ready for harvest. The astaxanthin content of the 'red cells' is correlated to the severity of the stress conditions, mainly to the light flux through the culture, and it may reach up to about 4 % of their dry weight. At this stage, the size of the cells differ between 15 and 50 µm and the astaxanthin content is not uniformly distributed at the same time in the cells but it differs between the different sized cells. In this context, the on-site monitoring of the astaxanthin concentration / activity would be of outstanding interest to determine the time of harvest at a set astaxanthin content. In due time, the 'red' culture is pumped to the down-processing area, where the cells are cracked (to a) b) c) render the pigment bioavailable), dried, and vacuum-packed. Haematococcus oleoresin is produced in an additional step, using the CO2 Supercritical fluid extraction process. The SME Algatech partner in this project produces up to 1000 Kg/year of Astaxanthin.
Biosensors based on photosynthetic biomediators
Due to its unique features and structural constituents, photosynthesis and photosynthetic systems are suitable to the biosensoristic field, due to their ability to operate charge separation and electron transfer, and represent also a super model to be mimicked for developing artificial photochemicalm systems with high yield of energy capture and conversion.
Natural photosynthetis is allowed by the action of two distinct photosystems. This proposal will consider the photosystem II (PSII) for the construction of one of the proposed bio/sensors. PSII is a multisubunit chlorophyll-protein complex embedded into the thylakoid membranes of chloroplast that drives electron transfer from water to plastoquinone using energy derived from light. Its native form, consisting of more than 30 proteins, is surrounded by the light-harvesting complex, which binds six individual proteins and an array of chlorophyll a, chlorophyll b, lutein, violaxanthin, zeaxanthin and antheraxanthin molecules. At the centre of PSII-LHCII complex is the reaction centre (RC), which is composed of the D1 and D2 heterodimer which bind all cofactors involved in PSII mediated electron transport.
The electron flow through PSII begins with the release of the electron from an excited P680 molecule. When the primary donor, P680, is excited by light energy captured by antenna pigments, the primary charge separation takes place between P680 and the intermediate acceptor, Phe; this reaction generates the radical pair (P680+/Phe). Reduced Phe transfers an electron to the primary acceptor, QA, to generate QA-, and subsequently reduces the secondary acceptor, QB. This linear electron transfer reaction in PSII catalyses the light-induced water-plastoquinone oxidation-reduction with a high quantum yield (about 0.85 a little lower than PSI which is approximately 1.0). PSII can be extracted by algae, higher plants and cianobacteria.
Chlamydomonas reinhardtii is a microalga considered in this project as source of the PSII biomediator component required for the realisation of a specific biosensor. Chlamydomonas reinhardtii is a unicellular green alga phylogenetically closely related to H. pluvialis. The vegetative cell averages about 10 µm in diameter and is enclosed within a cell wall consisting primarily of hydroxyproline-rich glycoproteins, which not contain cellulose. It holds several mitochondria, two anterior flagella for motility and mating, and a single chloroplast surrounding the nucleus that houses the photosynthetic apparatus and critical metabolic pathways. Large quantities of cells can be produced quickly with relatively simple and inexpensive media. Chlamydomonas is used to study eukaryotic photosynthesis because, unlike flowering plants, it has the unique ability to grow heterotrophically in the dark by metabolising an exogenous organic carbon source (acetate), and to maintain functional green chloroplast that retains the capacity to perform oxygenic photosynthesis when illuminated following growth in the dark. In spite of these differences, however, the photosynthetic apparatus of Chlamydomonas is very similar to that of land plants, making it a useful comparative system for understanding plant metabolism and photosynthesis. Chlamydomonas possesses three genetic systems located in the nucleus, the chloroplast and the mitochondria, all sequenced and available at the JGI Chlamydomonas Genome Portal (see http://www.chlamy.org online). The newly available information concerning the sequence and organisation of the C. reinhardtii genomes combined with the many types of physiological, genetic, and molecular manipulations, make C. reinhardtii the organism of election for the generation and characterisation of a wealth of mutants with modifications in structural, metabolic and regulatory genes for biotechnological purposes.
Four single sensors will be developed specifically for the addressed Astaxanthin bioprocess in the tubular photobioreactor, divided into two groups according to the detection technique used: optical (2) and electrochemical (2). Their main characteristics and descriptions are summarised below:
1) Optical sensor to measure the fluorescence emitted by chlorophyll inside the Haematococcus pluvialis cells in culture medium
During the induction process of astaxanthin accumulation, the intensity of fluorescence emitted by chlorophyll inside the Haematococcus cells changes, .The proposed online sensor monitors the cellular changes that occur under high irradiance in terms of changes in the PSII photosynthetic activity inside the cell. The miniaturised fluorimeter instrumental set-up will consist of:
1) excitation LED light source with high intensity emission at 660 nm;
2) optical detector;
3) small and optimised flow cell to flux the cell culture sample into the measurement chamber.
The pulse-amplitude-modulation measuring principle is based on selective amplification of a signal generated by short subsaturating light pulses of high intensity.
This biosensor is innovative with respect to the offline Clark-type oxygen electrode previously used and reported for monitoring the photosynthetic activity of Haematococcus pluvialis cells.
2) Optical sensor to measure the density of the H. pluvialis cells culture medium through a light transmission measurement
The online measurement of the density and turbidity of the culture medium is very important because it gives information on the H. pluvialis cells concentration in the solution. This data is linearly correlated to the dry weight (DW), which plays a fundamental role for the final determination of produced astaxanthin, normally measured as DW percentage at the end of the process. A good estimation of DW will be achieved through a standard calibration curve that will be measured offline in the laboratory to extract a reliable conversion factor. The sensor will measure the light transmitted by the culture sample by a photodiode mounted at 180 with respect to the light source, after excitation with a LED at 730 nm. Such excitation wavelength has been chosen to avoid the spectrum range of chlorophyll absorption and fluorescence activation, which would mask and affect the turbidity measurement.
3) Electrochemical biosensor to measure the antioxidant potential of astaxanthin, after disruption of the Haematococcus pluvialis cells, by measuring the amperometric current generated by stabilised Phosphatidylcholine (PC) derivatives bound to magnetic nanoparticles
The antioxidant activity is the ability of a compound (mixture) to inhibit oxidative degradation (like lipid peroxidation). Direct methods to assess antioxidant activity (AoxA) are based on studying the effect of a sample containing presumed antioxidants on the oxidative degradation of a testing system. The substrate of oxidation may be individual lipids, lipid mixtures (oils), proteins, DNA, or lipid containing biologically relevant species, like blood plasma, LDL, biological membranes, etc. Lipid peroxidation appears to be the most convenient for the purpose of the SENSBIOSYN project. According to the solubility of the tested sample, the preference should be given to the oxidation of homogeneous lipids or to aqueous micro-heterogeneous systems, such as micelles and liposomes. Based on these arguments, we propose the use of phosphatidyl choline derivatives as lipid substrate for oxidative stress, the lipo-peroxyd formation being potentially reduced or even completely quenched by the presence of xanthophylls. The third proposed biosensor aims at measuring the antioxidant capacity in terms of the degree of efficacy of the red cells against Phosphatidylcholine (PC) peroxidative damage initiated by radical initiators.
4) Electrochemical biosensor to measure the astaxanthin concentration by measuring the output current of a nanowire field effect transistor (FET) whose nanowire channel is deposited with PSII biomolecules
The fourth proposed biosensor is a nanosensor which uses the immobilised Photosystem II (PSII) complex extracted by the microalga C. reinhardtii as sensing. The nanodevice employed has been already developed by the SME participant Nanosens (NANO) but it has never been coupled to PSII and never been applied in industrial monitoring applications. It will transduce the electrochemical charge and electric field variations induced by the PSII reaction with the antioxidant agent into an output current. It is a nanowire FET, in which, in contrast to conventional FET, the source and drain contacts are ohmic contacts, realised at the ends of a silicon nanowire which acts as the FET channel, basically as a semi-conducting nano-resistor. To function as a biosensor the surface of the nanowire is coated with a functional biologic layer, capable of coupling with targeted species present in the sample solution. When the targeted species activate an electron transfer and a charge unbalance in the biomeadiator layer, a change in the electric field is induced at the surface of the nanowire which causes an enrichment or depletion of the charge carriers in the semiconductor. This broadening or narrowing of the local conducting channel causes a measurable change in the electrical conductance of the whole nanowire, even at a nearly 0 V gate potential, which is measured as a variation of the FET drain-source current.
Project results:
The main objective of the SENSBIOSYN project is design, manufacturing and testing innovative biosensors for supervision of the astaxanthin natural production process. The industrial process involves cultivation of the green alga Haematococcus pluvialis at Kibbuz Qetura in Israel, in the largest continuously operating industrial tubular photobioreactor facility, consisting of over 30, 10 000 litre reactors, covering an area of over 10 000 m2. This process is based on cultivation of the green alga Haematococcus pluvialis in two stages:
- stage 1 in complete medium for rapidly producing large amounts of green biomass; and
- stage 2 where those green cells are transferred to nutrient stress during which they accumulate the red pigment astaxanthin to a concentration of up to 4 % of its dry weight.
Once this stage is achieved, the cells are harvested by centrifugation and lysed. The asthaxanthin containing oil is then extracted and marketed. This is the only commercial facility worldwide successfully in operation for 10 years relying on such a highly controlled cultivation process for production of pigment rich algal biomass and, as such, the only available option for realistic testing of the planned sensors, though experimental and pilot facilities have now put in place at increasing frequencies.
During the first project year, BGU has provided all the necessary background on the production process, control and management of the cultivation process, details of culture parameters measured, and detailed description of the bioreactor facilities with pictures depicting possible attachment points for additional biosensors. Subsequently, during the project meeting in Israel, it has been decided to insert the sensors in the same locations where sensors for pH or temperature detection are already placed. Furthermore, as several spare connections for additional sensors are available, it has been verified by the BIO, CNR project engineer the suitability to place the same sensor in different reactor locations for testing consistency of the readouts and their independence from the sensor locations.
To identify the system specifications for novel biosensors development, BGU provided a list of parameters routinely measured, recorded and controlled at the Qetura facility along the process which include:
- the pH of the culture medium (measured on line and controlled by varying the flow CO2 into the reactor);
- reactor temperature (monitored online and controlled by cooling water applied to the outside of the reactor tubing);
- dry algal biomass concentration (measured manually on a daily bases, both in the green and the red growth stages);
- chlorophyll concentration (measured manually on a daily basis);
- photosynthetic efficiency (fv/fm, determined manually on a daily bases);
- astaxanthin concentration during the red stage (measured daily by a manual procedure);
- turbidity (OD 750, measured daily manually);
- oxygen concentration (monitored online);
- nitrate concentration (monitored online);
- light intensity.
To demonstrate the importance of continuous multi-parameter supervision of the cultivation process BGU determined a number of relevant parameters in two green reactor units and two red reactor units each continuously during June 2010. These measurements revealed a significant unexploited production potential: average reactor performance is far below optimal most of the time. For the green stage daily productivities vary widely, dilution intervals are irregular and as a consequence the biomass produced is not of reproducible quality, leading to differences in subsequent astaxanthin productivity as well. A similar situation was experienced analyzing the accumulation of astaxanthin during the nitrate starvation growth phase. Similar to the green stage, both accumulation of dry weight and astaxanthin in the red stage are hampered seriously by unreliable and erratic growth phenomena. The situation is complicated by the fact that success of the red stage depends strongly on the quality of the green material used to charge the reactor, which in itself is not of consistent and reproducible quality.
These data gave a strong indication on the urgency to move to sensor based culture control and management. All process stages, methods and culture parameters have been listed and used to recommend sensor specifications and design. Those recommendations were supported by a detailed literature review on optical and fluorescence properties of Haematococcus cultures helpful in designing suitable biosensors. BGU has performed further experiments for further refinement and improvement both the planned fluorescence optical sensors. The most appropriate sensor dimensions and wavelengths for an optical flow through sensor as well as excitation and emission wavelengths for the fluorescence detector were identified and provided to the sensor manufacturer. Main specifications are reported below. The flow through cell for measuring absorption at different wavelengths is the most important tool for controlling and managing cultures. Such a cell should measure simultaneously turbidity by OD 750 nm and chlorophyll peak by OD at 665 nm. Since suspended cells of various sizes will be measured, the geometry and hydrodynamics of the flow through cell is of crucial importance, to pass a homogenous sample stream without bubbles and turbulences through the sample cell. This detector should be linked on line to a control centre where raw data would be directly transformed into parameters such as dry-weight, chlorophyll content, astaxanthin content, cell number and chlorophyll / astaxanthin ratio, by application of suitable methods of calibration and empirical formula.
Regarding the sensor for the photosynthetic efficiency measurements, it has been determined that Fv/Fm cannot be measured continuously. This sensor can perform periodic measurements whereby a sample is drawn into a mixed dark chamber and dark adapted for about 20 min, and Fo can be measured. A light flash then induces very shortly maximal PSII fluorescence Fm that is then relaxing. Fv (Fm - Fo) divided by Fm is then a measure for photosynthetic efficiency. This sensor is thus fundamentally different from the first one determining absorption in flow through mode. However it too should feed its data directly to the central control unit where adequate algorithms will supervise the culture status. Fv/Fm changes with increasing astaxanthin content. Chlorophyll fluorescence allows to study the different functional levels of photosynthesis indirectly. Final technical specification of sensor design and testing were defined by BIO, BGU and CNR.
Furthermore, BGU carried out associated experiments with other prominent microalgae species such as Nannochloropsis and Parietochloris. Obtained data confirmed that under stress conditions, those species display the same optical changes (strongly reduced chlorophyll per biomass) we intend to exploit for quantifying Haematococcus. Thus our sensors can be applied to cultivation of other algal species based on identical principles, though those species are not commercially produced in large scale yet.
For verification of sensors developed in Rome and Bucharest, Haematococcus cells from different growth stages grown both in the Qetura bioreactors or in the lab at BGU, representing green to fully red cells were sent repeatedly to the partners for testing the antioxidant and flow through sensors respectively, and further protocols and details on maintaining the algal cultures were provided to both partners. The samples were successfully tested for antioxidant capacity and for fluorescence and OD determination using the respective sensors.
From the beginning of the project, biologists at the CNR worked in order to screen, identify, characterise and purify wild-type and molecular engineered Chlamydomonas reinhardtii photosystem II complexes to be used as bio-recognition elements for biosensor development. The screening is based on several crucial points:
i) PSII functional characterisation of the biomediators;
ii) stability of biomediator performance in immobilised conditions;
iii) response of the biomediator's PSII activity to unfavourable conditions.
Research activities were carried out on chlamydomonas strains mutated in the chloroplast psbA gene encoding the D1 reaction centre protein, which hosts several crucial components of the photosynthetic electron transfer. Mutations in this site can lead to modification of electron transport rate and overall PSII performance. Random mutagenesis was used for generating D1 proteins with novel properties by in vitro psbA gene evolution technique. This has been achieved by 'error-prone' PCR mutagenesis through an iterative process consisting of recombinant generation to create combinatorial libraries. The result was a pool of D1 variants that was delivered in algae chloroplast by particle gun bombardments. Mutants were further selected for their capability to overcome ionising radiation exposures represented by proton and neutron sources in order to get biological photosynthetic material with improved capability to tolerate radical stress. One of the milestone using biological materials as a sensing element in the biosensor devices is their capability to maintain their long-term activity often under unfavourable environment.
About 2000 transformed cells were subjected to the ionising radiation treatments and 32 colonies overcoming the radiation-induced stress were analyzed by psbA sequencing. Among them, twenty strains were identified as different mutants hosting both single and double mutations, indicating that in the unicellular green alga C. reinhardtii even single amino acidic substitutions in the reaction centre D1 protein enable the cells to survive in the presence of free radicals produced by both high light fluencies and neutron and proton radiation. The mutations, located in regions of the D1 protein responsible for the electron transport and oxygen evolution processes, involved replacement of polar amino acids, more prone to oxidative damage, with less sensitive ones. To exclude the possibility that additional random mutations induced by the radiation exposure could confer the observed tolerance, a set of the identified amino acid substitutions were introduced by site-directed mutagenesis in untransformed strains. The choice of the residue substitutions was made to include mutations located in two different structural regions of the D1 protein, near Tyr161 and the oxygen evolving complex (OEC) (I163T, P162S, M172L) and near the QB binding pocket (G207S, L200I, I281T). Physiological characterisation of the obtained mutants was carried out by estimating the growth parameters and the photosynthetic efficiency. Functional characterisation of the produced Chlamydomonas D1 mutants in physiological conditions revealed very similar trends in growth rates, but showed a reduction in their chlorophyll content and photosynthetic performance. The mutants generally showed a lower maximal quantum yield of PSII photochemistry (Fv/Fm) and a reduced efficiency of the electron transport through PSII primary and secondary electron acceptors (1-VJ) than the control strain. Despite the lower electron transfer efficiency, a higher oxygen evolution capacity under high photon fluency conditions was demonstrated in all the mutants compared to the reference strain, IL. The D1 mutants were able to achieve and maintain more than two-fold higher O2 evolution rate even under the very high light intensity of 900 µmol m-2 s-1. The long-term resistance to stressful conditions, such as low temperature (13 °C) and light intensity (20 µmol/m2/s), of the chlamydomonas strains produced by site-directed mutagenesis and selected, as biomediators was tested for a period longer than two months. On one hand, low temperature stress slow down all the catabolic reactions, which reduced the demand of absorbed energy, and on the other hand hold up the photosynthetic electron transport, leading to accumulation of long-lasting reduced forms of the PSII primary quinine (QA). As a result, the probability for oxygen radical species generation and oxidative damage increase considerably.
In these conditions, some of the D1 mutants were able to preserve between 70 % and 80 % of their initial photosynthetic activity (Fv/Fm) in contrast to the 54 % maintained by the reference strain. Moreover, the I281T mutant resulted very stable since even after 100 days under low temperature the reduction of PSII performance did not exceed 30 %, in comparison to 57 % reduction observed in IL strain. Few other mutants showed lower decrease in Fv/Fm than IL, such as M172L maintained 66 % of the initial value, L159M 56 %.
Additional mutant were produced by site-directed mutagenesis and protein engineering following a rational design accomplished by molecular modelling and binding energy calculation procedures to produce mutants having novel binding niche for the sensing event. These activities led to the production of a lot of mutants. Among these mutants at least five strains resulted suitable for our purposed. The step forward was the purification of these mutant's photosynthetic active sub-components, such as thylakoid membrane. Due to specific ultrastructural features, C. reinhardtii requires care in cell disruption to preserve thylakoids integrity and PSII sovramolecular organisation and functionality for biosensoristic purposes. Different physico-chemical procedures, including the use of Yeda press, French press and sonication in the presence of specific solvents, has been applied and tested for the best results. This task required a lot of time, as different mutants had a different migration pattern of the PSII-enriched thylakoid fraction during the particle preparation. Finally, a protocol for each mutant was set-up. Measurements of the photosynthetic activity by fluorescence methods has been used to test and compare the most suitable thylakoid preparations. The developed biorecognition elements were realised in order to build up electrochemical biosensors based on nanowire transducers to detect astaxanthin. However, taking into account the project requirements to implement large scale industrial line with highly sensitive, low cost and easy to use and fabricate sensors, mutated algal strains and their active sub-components were exploited to build-up a novel chronoamperometric sensor, as alternative solution to NWFET sensor.
The proof-of-concept for PSII-based biosensors functioning relies on their capability to harness light and convert it in an electrical or optical signals. The excitation of the PSII by red light induces charge separation and electron transfer processes resulting in a measurable intensity current. In the presence of an inhibitor of the PSII photosynthetic activity (such as exposure to high intensity of white light, or herbicides), a decrease of the charge transfer occurs, consequently a decrease of the measured current occurs. It has been assumed that the presence of Astaxanthin can preserve the photosynthetic activity protecting the PSII. The degree of restored activity is proportional to the amount of the astaxanthin.
The experimental conditions for PSII-chronoamperometric biosensor were optimised in terms of:
1) material of the working electrode;
2) immobilisation procedure of the phototosynthetic material;
3) applied potential to the working electrode during amperometric measurements;
4) photoperiod, namely the duration of dark/light cycles, during electrochemical measurements;
5) flow rate;
6) cell density and chlorophyll content;
7) composition and ionic strength of the buffer solution flowing through the measuring cell (referred as measurement buffer solution),
in order to maximise the biomediator viability (half-time, see below) and response stability to the analyte of interest. Flow rate, cell density and chlorophyll content and even characteristics of buffer solution were optimised in order to ensure the highest signal to noise (S/N) ratio in the working conditions, as all these parameters affect the S/N ratio, the optimum values for all these parameters were chosen. The choice of the appropriate working electrode material was performed among different and commercially available screen-printed electrochemical cell electrodes, SPEs (Dropsens, Oviedo, Spain) made of gold (Au), graphite (C) or carbon nanotubes (CNT). All measurements were performed in a lab-made biosensor instrument, settled specifically for this project, which is composed by:
(i) a software-controlled PG 581 potentiostat (Uniscan Ltd. UK);
(ii) a peristaltic pump joined to a sample cell box equipped with inlet and outlet flow holes; and
(iii) SPE modified electrode with algae.
SPE cell fits into the measurements cell, while a suitable buffer solution flows through the measuring cell the instrument provides amperometric signals subsequent to biological material illumination at a suitable wavelength and intensity (650 nm and 325 µmol respectively, from two red LEDs at the bottom of the sample cell; the whole device has been specifically designed for dynamic flow chronoamperometry measurements. In order to establish the more suitable material for the SPE working electrode and the appropriate applied potential values for chronoamperometric measurements, first experiments performed were of cyclic voltammetry (CV), the experimental conclusions leading to the choice of CNT-SPE as optimal working electrodes, with a wider available potential window to work on. Concerning the immobilisation procedure, the most reliable providing low S/N ration was based on physical gelation with calcium alginate polysaccharide and was applied throughout.
Exploiting Chlamydomonas reinhardtii whole cells as biorecognition element, the value of applied potential for chronoamperometric experiments is around -0.7 V vs reference electrode Ag/AgCl. At this value, the oxygen reduction current signal originated from the algal photosynthetic activity following illumination is registered. The optimum applied potential in these chronoamperometric measurements was chosen according to a 'change and trial' procedure, the values of the applied potential ranging between -0.7 V and -0.1 V and the obtained current intensities (which are proportional to oxygen evolution) are registered.
Exploiting thylakoids extracted by both spinach plants and Chlamydomonas reinhardtii mutants strains, the applied potential was +0.2 V versus reference electrode Ag/AgCl. At this value, the DCPIP current signal originated from the PSII photosynthetic activity following illumination is registered. Photosynthetic activity in both whole cells or thylakoids was stimulated or inhibited by red (652 nm, 540 µmol photons cm-2s-1) and white light (1350 µmol photons cm-2s-1), respectively. The algal cell density and chlorophyll content on the working electrode were carefully determined. Generally, a specific relationship exists among algal cell density and the chlorophyll content for each algal strain. Indeed the current signal (and the S/N ratio too) depends on many factors such as cell density, surface properties of the immobilised cells / SPE interface, chlorophyll content, etc. Some tests were performed, aiming to optimise the biosensor response as function of the algae / alginate mixture volume dropped onto the SPE surface using optimal immobilisation procedure. The volume of algae / alginate mixture providing the highest signal was somewhere around 6 µl. When the signal to noise (S/N) ratios are considered, it was noticed that the best volume to drop over the SPE for optimising the S/N response of the biosensor is 5 µl. As consequence, this volume was further used by us in biosensor fabrication. Illumination time was set-up at a sequence of 30 s light/5 min dark and flow rate at 0.1 ml/min. Amperometric measurements performed with both algal strains and extracted thylakoids proved the developed PSII-CNT SPE electrochemical sensors to be suitable to the purpose, meaning, to the determination of astaxanthin amount by assessing its capability to reduce the PSII inhibition generated by light stress. Data supporting this conclusion are: no matter the employed mutant in fabrication of PSII-CNT, amperometric sensors, the sensor responses correlates to astaxanthin amount, the stress inhibition being higher with increasing the astaxanthin amount; the developed sensors provide accurate and precise responses (SD ranges between 0.3 and 0.5); the developed method proved a good sensor sensitivity of 1.15 µA/mmolL-1.
The project aimed to produce four single sensors, two optical and two electrochemical, according to the detection technique used. Optical biosensors aimed to determine the H. pluvialis cells turbidity and photosynthetic performance providing information on the cells concentration in the culture, hence on the ideal time for culture harvest (mainly involved SMEs and RTD performer: BIO, Algatech and CNR). Electrochemical biosensors aimed to determine the astaxanthin concentration in the H. pluvialis culture (red phase) and its antioxidant capacity for the control quality assessment of the final product (mainly involved SMEs and RTD performer: BIO, Algatech, NIB, NANO and CNR).
Development of two optical (chlorophyll fluorescence, culture turbidity) and two electrochemical (PSII-nanowireFET, magnetic beads stabilised PC-SPE) biosensors able to assess the content and the antioxidant efficacy of accumulated xanthophylls was achieved during the two-years activity in tight collaboration with all parties.
Two optical biosensor for fluorescence and density measurement has been developed taking into carefully consideration the previous reported specification provided by BGU and the technical specification of BIO engineers. The fluorescence system has been equipped with a LED excitation source operated in continuous mode, generating high intensity red light to excite the fluorescence emission without altering astaxanthin induction and formation, making the measured results reliable. In order to efficiently collect the emitted fluorescence, different photodetectors and configurations has been investigated according to the S/N ratio achievable. In addition, an interferential band-pass filter centred on the set fluorescence spectral range has been used. As the cell culture are kept in constant flow in the photobioreactor tubes, a direct interface of the fluorescence sensor with the solution under test through the tube glass wall is not reliable, so the fluorescence measurement has be planned in static condition to provide the necessary time interval elapsing between the excitation and the successive collection of the emitted fluorescence.
The optical biosensor for the determination of the culture turbidity has also been delivered by BIO in tight collaboration with CNR and BGU. The device is equipped with red emission wavelength for the turbidity measurement that consider the chlorophyll absorption peak at 660 nm and consequently shift to 730 nm to avoid mask effects.
Electrochemical biosensors were developed based on both screen printed electrodes (SPE) and nanowire-FET (NWFET) transducers by CNR and NIB.
The SPE for antioxidant capacity determination was successfully set-up. The electrochemical biosensor was based on magnetic nanoparticles modified with phosphatidylcholine derivatives as substrate for oxidative stress. Phosphatidylcholine (PC) derivatives were bound to magnetic nanoparticles, characterised and stabilised. The optimal recommended storage conditions for composite PC-nanobeads were finally defined as: room temperature, no light contact storage in inert atmosphere. Subsequently, the PC peroxidation assessment following ROS attack was evaluated as well as the lipophilic antioxidant efficacy against PC Peroxidation. The obtained results proved that the developed model is applicable even to assessment of antioxidant efficacy against lipoperoxidation. After the flow system design, the effect of magnetic field on registered chronoamperometric response was evaluated.
Procedure of measurements using the assembled biosensors was drawn and, as further detailed, laboratory and industrial testing were performed. SPE electrochemical sensor for astaxanthin antioxidant capacity determination was developed, tested and validated in terms of linearity, limit of quantitation (LQ), limit of detection (LoD), repeatability; accuracy; selectivity with real samples. The developed SPE-Aox sensors are applicable to various samples (oil or H. pluviallis cell cultures), proving their versatility. Responses provided by SPE-Aox are given as overall antioxidant capacity of the sample (or extract). Three new protocols / methods were established and validated:
- protocols of SPE-Aox use to assess ASTAXATHIN from oils;
- protocols of SPE-Aox use to assess astaxathin from H. pluviallis cell cultures.
Finally, the sensors response statistical validation was carried out.
The SPE biosensors for astaxanthin determination were also set-up, exploting Chlamydomonas reinhardtii algae cells as photosynthetic bio-sensing materials. The experimental conditions were optimised in terms of:
1) material of the working electrode;
2) applied potential to the working electrode during amperometric measurements;
3) immobilisation procedure of the phototosynthetic material;
4) duration of dark / light cycles during electrochemical measurements;
5) flow rate;
6) cell density and chlorophyll content;
7) composition and ionic strength of the buffer solution flowing through the measuring cell, in order to maximise the biomediator viability and response stability to the analyte of interest.
Flow, rate, cell density and chlorophyll content and even characteristics of buffer solution were chosen as those ensuring the highest S/N ratio in the working conditions. Two main immobilisation procedures were tested in order to achieve the best results:
(a) coating of algae cells with a Nafion membrane;
(b) physical gelation of alginate for algae cells entrapment, the second one providing the best outcome.
The amperometric set-up was exploited to determine algal photosynthetic performance as preliminary approach for the development of biosensor for astaxanthin concentration determination. Subsequently, the analytical device developed was set-up in order to determine the astaxanthin concentration necessary to revert a photoinhibitory event. The latter was induced by including a saturating white-light led in the instrument that in active operation mode decrease the photosynthetic current produced. Astaxanthin inhibits this phenomenon in a concentration manner. Dose response curves were carried out leading to the production of a reliable titration curve. A similar approach was followed in order to set-up functionality of biosensor hosting thylakoid membranes extracted by whole algal cell.
The photosystem-based Nanowire FET electrochemical biosensor to measure the xanthophyll concentration in a solution was successfully developed by CNR and NIB. Spinach thylakoids were immobilised by the highly efficient LIFT procedure on nanowires (provided by NANO) producing the PSII-NWFET. The performance of this assembly was assessed determining crucial parameters, such as the excitation time, the range of potential appropriate for modified PSII-NWFET, registered intensity current range, useful domain of inhibitor concentration, useful range of antioxidant / preservative compound concentration, PSII-NWFET operational stability. Furthermore, astaxanthin preservative effects were assessed.
As required by task 3.4 protocols of H. pluviallis cell disruption and carotenoids extraction were set-up.
Continuation of experiments on PSII-NWFET is expected to be improved exploiting additional thylakoids extracted by different strains of Chlamydomonas on new nanowires. NANO is currently looking at ways to optimise the nanowire fabrication process (design, surface smoothness of the NWs) and also team with larger industrial foundry partners to obtain 'ready to measure reliable devices' on a larger scale. In addition, beyond the scope of the SENSBIOSYN project, Nanosens is currently focused on improving the electrical device characterisation setup, the microfluidics delivery, and the readout electronics a compact instrumentation setup so that our research partners could focus on sample measurements together.
The introduction of such sensors in the antioxidants production line will increase production through online monitoring that will ease decision about time of harvest and culture performance. It is also expected a reduction in the production cost by at least 30 %, by saving work time and manpower, which is a big industrial breakthrough.
The new monitoring approaches provided are targeted to the bioproduction in the industrial green technology sector which has an extremely high potential of offering wide market opportunities while increasing the environmental sustainability and promoting public health and human safety.
Potential impact:
The potential impact of the project is the result of three converging parts, emerging from the project specificity.
First, the results obtained lead us to supporting the project impact on the participating SMEs in terms of advantages gained from the project:
Technical impact:
SMEs will have the opportunity to push the production of new products toward real problems solution with the help of the directly involved end user, ALgateh.
Biosensor, based on fabricated and tested prototypes, SMA and ASPE have the opportunity of producing new instrumentation; Biosensor is be able to further incorporate in their automatic fabricated and commercialised systems these new developed devices. Biosensor access specific technical and scientific knowledge on industrial biosynthesis of algae, which is very difficult and uncommon to find, but fundamental to attack a promising emerging market for sensors and biosensors.
The technical advantages gained by Algatech directly impact their astaxanthin production process: they have the possibility of employing control devices online and to be the first industry to test and use them on board their photobioreactor, as long as registered drawback during in field testing is solved.
Commercial impact:
Biosensor, Nanosens and Algatech are characterised by a good quality standard production and a high innovation degree. As long as Nanosens is solving the issue in nanowires production, all three SMEs will be able to offer on the market a wider range of high performance. Algatech will reduce the production cost by improving the manpower costs and will offer a more qualified and controlled product.
Educational and training impact:
Biosensor offered internships to young researchers. Based on developed protocols for new biosensors fabrication, as included even in the dissemination plan, CNR, NIB and BGU are transferring the know-how by organising educational courses for the study and the application of the implemented methodologies.
Secondly, based on the fact that in the last decade the direction of biosensor R&D significantly changed in response to new biotechnology innovations in biocomplementary chemistry, surface characterisation, molecular markers, and nanotechnology a market impact is expected.
Based on obtained and tested prototypes, considering the data obtained when devices were validated, it is estimated that in the next years the following sensors will be on the market as presented in tables below, were even the associated business plan is given.
The business plan was developed considering the estimated production costs, sales volume other costs related to product introduction to the market (prices in thousands of Euro). The business plan was drastically adjusted with respect to initial one, considering the economic crisis waves.
This project is significantly relevant to the industrial and applied research and development. The prototypes developed, intended for field trials, feature a high potential for commercialisation, if supported by a well-developed marketing strategy in order to increase the stakeholders and public awareness on the biosensors use in processes control and monitoring.
The need for on-line monitoring of growth parameters of Xanthophyll metabolites in cultivation systems and process streams is a main issue faced today by producer companies.
Biomass (gr/l) and pigment content (mg/l) are nowadays determined offline routinely everyday at commercial production sites by means of complex manual analyses carried on by skilled operators.
The cost of these operations further increase the production cost that today is a major challenge, especially for small companies in the business of biosynthesis of Xanthophylls that are recently starting up in Europe.
The innovative offer of the biosensors developed in this project is about the possibility of doing online not only the classical measurements but also specific analyses without time consumption and manpower.
Biomass, pigment concentration, accumulation profile during the induction process, photosynthetic activity (through the chlorophyll fluorescence measurement) and antioxidant capacity level will be monitored online, automatically and rapidly. This will allow to increase production and reduce production cost by at least 20 %, which is an important industrial breakthrough.
It is envisaged, also, that SENSBIOSYN will even contribute to increase the applicant SMEs' market share being attractive worlwide to international companies, public / private research institutes and laboratories, operating in the above mentioned fields and in the more recent field of algae-based bioenergy.
In addition, the flexibility and wide applicability of the SENSBIOSYN products allow to target other potential end-user companies producing natural food additives, functional food and nutraceutics, not exclusively extracted by algae.
The choice of the distribution channels is fundamental because the product is highly innovative and target specific markets.
The distribution politics take into account various channels we intend to take advantage of to commercialise the products. 20 % of our effort to distribute the new product will use the same distributors already employed by each SME for their existing instrumentations.
Ongoing dissemination activities will be as follow:
Specific dealers distribution (40 %) are the most relevant channels offered by dealers that are well-known in the microalgae and fish industry. Through the internet (20 % effort), in addition to the project and partners' websites, it is possible to advertise the products on web magazines and journals. The products can be displayed at conferences and exhibitions (10 %). Direct contacts (10 % effort) through the experience and acquaintances of the main partners in the project.
The experimental results will be presented at international conferences and, where possible, be made available to an extended audience. We will make use of an updated SESNBIOSYN webpage and the various European networks and work groups to disseminate the project results. Also, the services of the European Innovation Relay Centres, where applicable, will be used to disseminate the results and work towards a commercialisation of the biosensors.
There will be an international symposium during the next year within the International Conference on Photosynthesis.
The partners will continue to attend the European Biosensor Symposia organised in 2012 for the exchange of knowledge and technology.
A brochure of the developed biosensors was electronically issued, printed out upon request of farms and firms that will be updated next 4 - 6 months also after the project end.
The group will organise 'hangar-stands' for technological exhibitions in EU countries. A first stand was organised in the area of research and presentations to companies took place. A stand was already organised for the biosensor meeting expected in Mexico on 2012 and for the biosensing meeting in Edimburgh expected in March 2012.
Biosensors prototypes were previously presented on TV programs and this aroused great interest; they were presented again by the CNR press office and in the near future. Several meetings will include participation of interested industries beside the extended consortium.
Concerning the first results of dissemination nine articles have been published on impact factor journals and other five articles under review and in preparation.
SENSBIOSYN partners participated to eleven Conference/exhibitions presenting stands of the results and prototypes.
Project website: http://www.sensbisyn.com