The field of synthetic biology has exploded over the past decade, having a major influence on fields such as metabolic engineering, protein engineering, digital biology, and whole-genome engineering.

Biosensors & Biochips Technologies: Contribution to the Future Sustainable Life

The field of synthetic biology has exploded over the past decade, having a major influence on fields such as metabolic engineering, protein engineering, digital biology, and whole-genome engineering. In the framework of iterative “design-build-test” development cycles, a significant portion of synthetic biology innovation has taken place. In the field of synthetic biology, progress can be associated with innovations in each of the processes of “design”, “build” and “test”. For example, there has been a major push to standardize elements within synthetic biology, with significant attention being paid to modularity and “plug and play” components. This modularization, along with the accelerated progress in systems biology, has allowed the “design” stage to become less time-consuming and less reliant on advanced knowledge. In recent years, the cost of DNA sequencing and synthesis has also decreased dramatically, allowing large constructs to be synthesized cheaply. In the ‘build’ phase, this has facilitated a rapid improvement, helping researchers to investigate a larger percentage of the space of the biological solution. Finally, within the synthetic biology “test” phase, high-throughput screening has also become a focal point. The increased “design” and “build” potential has contributed to an increased demand for success in the assessment of the plethora of new designs. In turn, this was done by incorporating robots and high-throughput analytics into the laboratory setting, in which new models can be evaluated to a level that is not achievable for human researchers.

Biosensors represent a groundbreaking emerging technology for high-throughput screening that can be implemented. Most precisely, they are classified as an analytical tool consisting of biological components used to detect and generate a signal for the presence of a target ligand. Synthetic biology is at the forefront of biosensors, both as a tool for high-throughput screening, but also as the direct result of developments within the field of synthetic biology itself. In addition, because of the unparalleled specificity and sensitivity that biological parts provide relative to conventional analytical methods, biosensors have gained expanded interest as alternatives to traditional analytics.

The design and construction of biosensors is a multidisciplinary endeavour and can include expertise in areas such as protein engineering, molecular biology, affinity chemistry, molecular dynamics of nucleic acid, materials sciences, and nanotechnology. Biosensors interface with a target ligand at their most simple stage, undergo some type of modification, and output a signal. There is a great variety of potential configurations in all the parts of this process. Target ligands range from single atoms such as calcium, to entire proteins such as thrombin, all the way through. Processes as varied as enzymatic activity, fluorescence, electrical current generation, and transcriptional activity include output signals. The mechanisms that transduce ligand recognition into functional signals are just as diverse.

In the field of analytics, biosensors represent a significant step forward. In order to move analytics away from purely physics- or chemistry-based frameworks, the integration of biological components in sensory diagnostics has begun. This has allowed analytical functions that are not well adapted to conventional methods to conduct a vast diversity and specificity of biological components. The theoretical and demonstrated biosensor applications cover a significant range of human society and activity. Biosensor applications are grouped into three broad categories, depending on their measurement scale.

• Group Diagnostics: Environmental, Agricultural, and Industrial Applications
• Point-of-Use Diagnostics: Medical, and Security Applications
• Single-Cell Diagnostics: Metabolic Engineering, and Synthetic Biology Applications

The current food production faces immense challenges from the increasing human population, the maintenance of clean resources and food quality, and the protection of the environment and climate. Food sustainability is mainly a cooperative effort that results in the development of technology funded by both governments and companies. Several attempts have been supported to overcome challenges and improve the drivers in food production. Via their applications, biosensors and biosensing technologies are widely used to solve the major challenges of food production and its sustainability. As a result, there is a rising need for biosensing technology in the area of food sustainability. A technological system combining several technologies is defined by microfluidics. Nanomaterials, with its biosensing technology, is known to be the most innovative tool strongly associated with world populations in dealing with health, energy, and environmental issues. The need for point of care (POC) technology in this area focuses on analytical tools that are fast, simple, precise, compact, and low-cost.

For our existence and lives, food with its production industry is essential; and its sustainability is essential in continuous human growth on the planet. Current food production is facing immense difficulties from increasing human population, maintaining clean resources and food quality, and protecting environment and climate. Some of these issues stem from food production itself; others stem from other food production-related industries. Food recalls, for example, trigger major damage to food brands’ credibility and prestige, with an estimate of $15 million per incident over the last few years. 48 million sick cases are responsible for 3000 fatalities annually due to foodborne illnesses.

Food safety is largely a cooperative effort arising from both governments and companies in technology development. In order to pose new challenges in food safety issues, information technologies such as blockchain technology can accelerate communication between food quality, media and consumers. Five challenges can be summarized as the main challenges in the sustainability of food production: the production challenge of food safety and security; the quality challenge of food diversity and quality; the economic challenge in the leading food system, including its packaging and supply chain; the environmental challenge, including the processing of food waste; and the engineering challenge in the creation and generation of novel food.

Basically, a biosensor is an analytical instrument used to measure a sample’s molecule of interest (target). In general, a bio-recognition factor (aptamer, antibody, enzyme, etc.) that is unique to the target is used. A physiochemical or biological signal is elicited by molecular recognition events between the recognition element and the target compound, which is transformed into a measurable quantity by the transducer. Signals are shown in either optical (colorimetric, fluorescence, chemiluminescence and plasmon surface resonance) or electrical (voltammetry, impedance and capacitance) or any other chosen format (Figure 3).

Figure 3. Classification of biosensors based on transducer and bio-recognition elements used in food analysis

As one of the primary objectives of food analysis, food safety is a major health issue in both animal and human lives. The advancement of food safety analytical technology means that it thrives in line with the rising interest in and emphasis on food supply safety issues. In food safety analysis, traditional approaches are labour-intensive, time-consuming, and need trained technicians. The application of microfluidics in food safety analysis provides fresh insight about how to detect foodborne toxins, allergens, pathogens, hazardous substances, heavy metals, and other contaminants effectively and rapidly. Microfluidics’ features, such as it miniaturize-capability, compact and reducible quantities of samples and reagents, make it a perfect technology for the development of food sustainability. Complex food matrix preparation and difficult manufacturing steps are the current challenges in the application of microfluidics to food sustainability. These challenges can be addressed by leveraging physical properties dependent on specific test targets, designing complex real food analysis microfluidic platforms, and incorporating into microfluidic systems biomolecules such as food proteins and DNA.

Nanomaterials in biosensing technology

With its biosensing technology, nanomaterials are the most promising tool in dealing with health, energy and environmental problems associated with population in the world. Particles smaller than 100 nm in at least one size dimension are known as nanomaterials. These nanomaterials are biocomposite polymers based on metal, metal oxide and carbon, and different types of nanoparticles have been established, such as magnetic iron, aluminum, gold, silver, copper, silica, zinc, zinc oxide, cerium oxide and titanium dioxide nanoparticles, and single/multiple walled carbon nanotubes (CNTs). Nanotechnology and its agricultural development have been greatly extended in different fields. These fields include food production, crop protection, detection of pathogens and toxins, purification of water, food packaging, disposal of wastewater, and environmental remediation. Improving the productivity and performance of applications is the priority of these agricultural fields.

In the field of food safety and protection, biosensing technologies have been developed for nutrient and quality detection, detection of pathogens and detection of toxins, as listed below.

Nutrient and quality detection

Food protection measures can be split down into two categories: post-harvest loss and food biosecurity. Food biosecurity means food contamination and degradation, which is addressed in the later sections, by environmental, political, unfair economic gain, warfare, or exacting revenge. Post-harvest loss, on the other hand, suggests the nutrients and edible conditions in food that need to be maintained between the harvest period and the moment of consumption by technologies. Since time differs from minutes to years, in maintaining and reducing losses, technologies focusing on reducing post-harvest losses are important.

To maintain food quality and to avoid post-harvest losses, new technology such as biosensing can be used. Biosensors have been developed, for example, to detect and analyse quantities of sweeteners in foods that can be used to detect both natural and artificial sweeteners. Sweeteners are widely used in food production and processing, but they have recently been identified in humans as causing health problems. A multi-channel biosensor has been developed to use electro-physiological sensing from taste epithelia to detect and analyse both natural and artificial sweeteners. To detect long-term signals from sucrose, glucose, cyclamate, and saccharin, respectively, the signals are studied through spatiotemporal techniques. The biosensor can distinguish between different concentrations with dose-dependent increased responses of the taste epithelium from different sweeteners. It can also distinguish between two natural sweeteners: sucrose and glucose, with two signal patterns. For glucose, the detection range is 50-150 mM, and for saccharin, 5-15 mM.

Detection of pathogens

Due to their reduced format, biosensors targeting pathogen detection such as bacteria (Table 1) and fungi (Table 2) started more than two decades ago; one device to address multiple problems, and a multi-panel signal detection. The ligand motif is a crucial element in the biosensor design for pathogen detection since it determines the sensitivity and efficiency of the device. The aim is to establish a fast, specific, and sensitive platform to detect in food samples the presence or absence of pathogens. It has been discovered that there is no ideal ligand, and various ligands have different advantages. The combination of bioreceptors to detect a large variety of microbes in different samples poses current challenges in pathogen biosensor detection; new synthetic ligand designs such as aptamers, small molecules, and peptides; and the incorporation of different ligands into a portable device to achieve rapid, effective, and low-cost detection.

Detection of toxins

The mainstream of development in food safety is electrochemical biosensors for rapid detection and assessment of food toxins. Numerous platforms have been developed to allow customized and individualized devices to meet particular environmental and organizational requirements and to reach the nM to fM detection limit levels. For example, to encourage unique binding profiles, bioreceptor arrays address individual electrodes functionalized with different bioreceptors with binding targets. In addition to electrochemical biosensing, toxin and chemical detection in food production have been applied to other biosensors such as optic and piezoelectric sensing (Figure 4). In order to sense toxins, fluorescent nanoparticles have been produced in foods and bodies, including on-surface, inter- and intra-cellular foods.

Figure 4. Predominant food contaminants and the target analytes in the food manufacturing industries

Toxin extraction from complicated food samples is one of the main obstacles in creating a fully automated toxin detector. To automatically assess their harmful levels from food and water samples, potential systems are expected to extract, process, and measure toxins. In identifying, discriminating, and quantifying chemical toxins in food matrices, sophisticated separation strategies have been coupled with SERS. In addition, even though they are typically in lower amounts, chemical contaminants from food processing can be a challenge. Lower stability, selectivity and sensitivity are another challenge in food toxin detection, where MIPs can be a solution to provide stable and low-cost alternatives.

Heavy metals like Ag⁺, As³⁺, Cd²⁺, Hg²⁺, Pb²⁺, and Zn²⁺ are known as chemical pollutants that form stable states of oxidation and interfere with metabolic pathways, resulting in health problems. Aptamer and DNA-based biosensors can detect heavy metals at both nanoscale and very large-scale levels, which are appropriate for food safety screening and monitoring. In order to detect arsenate in food, a heavy metal detecting biosensor is based on genetically modified bacterial cells and a green, fluorescent signal amplifier. With a detection range of 5-140 μg/L of arsenic, its arsenic detection lasts just one hour and can be integrated with optical power output for its future biosensing optical fibre. Other biosensing technologies like aptamers, nanoparticles and graphene electrodes have been successfully applied to the identification and evaluation of arsenic, with the potential to be produced as fast, simple, easy-to-use, and low-cost devices.

Nanotechnology has been adapted to two separate fields of agri-food pesticides: as a pesticide delivery vector for pesticide management and as a trace-amount detector for pesticides. In the first field, nanoparticles are able to slowly modify pesticides to target insect pests, which helps prevent groundwater and topsoil pollution, reduce pesticide levels and improve efficiency. In the second field, bio- or biomimetic-based nanotechnology, like antibodies, enzymes, aptamers, and MIP-like macromolecules, improves stability, selectivity, sensitivity, and speed of detection. Furthermore, bacterial, fungal, algal, and mammalian cells are all cell-based biosensors used in pesticide and herbicide detection, helping to establish fast, reliable, real-time, and cost-effective tools for decontamination procedures and preventive casualty damage.

Carcinogens, odorants, and marine contaminants are other toxins that are significant in food production. Carcinogens are a complex group of trace amount of toxins, like pesticides, heavy metals, mycotoxins, and acrylamide, in which the difficulty of identifying trace-amounts is a challenge; and imprinted aptamers, nanotechnology, and biosensing are optimistic for promising future use. Sensitive and soluble molecules effective in odour detection for olfactory animal systems are odorant binding proteins. A nanosensor combining localized SPR and small odorant binding proteins from honeybees has been established in which the detection range is 10 nM – 1 mM using a quantitative array of nanocups. To monitor and preserve a stable environment for marine food systems, marine contaminant detection is used. Finally, through their sensitive detection capabilities, miniaturized devices, wireless communication, and small-scale networks, biosensors can be applied to marine food safety to be established as advanced analytical and monitoring tools.

Another development kit for food safety biosensors focuses on the detection of genetically modified organisms (GMOs) in food products. Since the 1990s, GMOs in all fields of agricultural products have been considered a biotechnology revolution. To present, more than 45 percent of the world’s soybeans, 40 percent of corn, and 50 percent of cotton are GM products; and GM is also used in livestock. Recent research, however, indicates that GMO products can affect human and animal bodies through gastrointestinal problems, antibiotic resistance, allergenicity, diversity of farm products degradation, and undesired gene flow to other species. Biosensors are designed to measure GMOs in foods and feeds using isothermal DNA amplification and fast detection signal detection to identify GM genes. Detecting unidentified DNA genes that can be resolved by high-throughput technology like the combination of biosensing and arrays, and the development of databases of GMO genes are the key challenges in GMO detection.

Biosensors & Biochips for environmental monitoring

Due to the strong connection between environmental pollution and human health/ socioeconomic progress, environmental monitoring has become one of the priorities on a European and global scale. Biosensors have been commonly used as cost-effective, rapid, in situ, and real-time analytical techniques in this field. The recent development of biosensors with new transduction materials obtained from nanotechnology and for multiplexed pollutant detection, involving multidisciplinary experts, explains the need for compact, fast, and smart biosensing devices. Several recent developments exist in the monitoring of air, water, and soil contaminants by biosensors under real conditions, like pesticides, highly toxic components and small organic molecules, including toxins and endocrine disrupting chemicals.

Biosensors used in environmental monitoring can be categorized as optical (including optical fibre and surface plasmon resonance biosensors), electrochemical (including amperometric and impedance biosensors) and piezoelectric (including quartz crystal microbalance biosensors) based on their transduction or as immunosensors, aptasensors, genosensors and enzymatic biosensors based on their recognition elements, respectively when are used antibodies, aptamers, nucleic acids, and enzymes. The majority of biosensors in environmental monitoring are recognized as immunosensors and enzymatic biosensors, but the development of aptasensors has recently increased due to the beneficial characteristics of aptamers, like ease of modification, thermal stability, in vitro synthesis and the ability to design their structure, to differentiate targets with different functional groups and to rehybridize.

Study on the design of biosensors for the monitoring of organic pollutants, potentially toxic elements and pathogens in the environment has led to the sustainable development of civilization due to the environmental pollution issues confronting human health. Various chromatographic techniques (such as gas chromatography and high-performance liquid chromatography combined with capillary electrophoresis or mass spectrometry) are conventional analytical methods used for environmental monitoring of pollutants, but they require costly reagents, time-consuming sample pre-treatment and costly equipment. Therefore, for monitoring pollutants responsible for adverse effects on habitats and human health, more sensitive, cost-effective, fast, easy to function, and compact biosensing devices are desperately needed to overcome the magnification of environmental issues. In the case of accidental release of pesticides or acute poisoning, for example, common methods are not appropriate for in situ measurements where fast, miniaturized, and portable equipment like environmental monitoring biosensors is required. In this regard, the role of nanotechnology in the creation of rapid and intelligent biosensing devices is crucial for the success of environmental pollutant detection; most recent biosensors include nanomaterials and novel nanocomposites in their systems, which are beneficial for improving analytical performance, such as sensitivity and detection limits.

For the detection and monitoring of different environmental pollutants, biosensors, including immunosensors, aptasensors, genosensors and enzymatic biosensors have been documented using antibodies, aptamers, nucleic acids, and enzymes as recognition elements.

Pesticides

Pesticides are among the most significant environmental pollutants because of their large presence in the environment. Organophosphorus insecticides, for example, are commonly used in agriculture and represent a group of pesticides which, due to their high toxicity, are of immense environmental concern. Easy, responsive, and miniaturized in situ methodologies like biosensors have therefore been established as analytical strategies for their detection and monitoring, without the need for comprehensive sample pre-treatment.

Disposable amperometric enzymatic (acetylcholinesterase) biosensors were proposed for the detection of organophosphorus insecticides using paraoxon as a model analyte applying a cysteamine self-assembled monolayer on gold screen-printed electrodes. The disposable biosensors showed a linear spectrum of up to 40 ppb with a 2 ppb detection limit and a 113 μA mM cm^-2 sensitivity. Using the self-assembled monolayer, good analytical output could be due to the highly oriented enzyme immobilization. Recoveries of 97 ± 5 percent (n = 3) were reported after being tested in river water samples spiked with 10 ppb of paraoxon, indicating the effectiveness of such enzymatic biosensors. Furthermore, the use of disposable screen-printed electrodes dispenses with time-consuming methods like the reactivation of immobilized enzymes utilizing, for example, obidoxime solution and pralidoxime iodide (PAM) or the use of the renewable enzyme membrane needed for the second application of biosensors.

Nanoparticles based on iridium oxide have been used in the disposable enzymatic biosensor with tyrosinase based on low-cost screen-printed carbon electrodes for the detection of chlorpyrifos in river water samples. Linear biosensor response (0.01–0.1 μM) and low detection limit (3 nM) were reported, which could be due to the high conductivity of nanoparticles of iridium oxide and tyrosinase efficiency. Recovery tests were carried out in river water samples with the addition of 0.1 μM of chlorpyrifos and recoveries of 90 ± 9.6 percent were obtained with a residual standard deviation (RSD) smaller than 10 percent (n = 3) to demonstrate the applicability of the biosensor.

Acetamiprid was detected by colorimetric aptasensors and water samples by impedimetric aptasensors in real environmental samples, like fresh surface soil samples. A linear range of 75 nM to 7.5 μM and a detection limit of 5 nM were observed with the colorimetric aptasensor, while a wider linear range (50 fM to 10 μM) and a lower detection limit (17 fM) were observed with the impedimetric aptasensor. Gold nanoparticles, multi-walled carbon nanotubes (MWCNT) and reduced graphene oxide nanoribbons were used in that biosensor as a composite to sustain the electrode surface acetamiprid aptamer, which could be responsible for higher electron transfer and improved analytical performance of the biosensor. A related detection limit (33 fM) was observed by an aptasensor based on silver nanoparticles anchored on nitrogen-doped nanocomposite graphene oxide constructed for acetamiprid detection in wastewater samples.

Pathogens

The existence of pathogens in environmental matrices, and especially in water compartments, could pose a serious risk to human health, and some biosensors have recently been suggested for monitoring the environment. For example, for the detection of metabolically active Legionella pneumophila in complex environmental water samples, rapid and precise optical biosensors based on surface plasmon resonance have been proposed. In one study, the detection principle was based on the identification of bacterial RNA by the immobilized RNA detector probe on the gold surface of the biochip. For signal amplification, streptavidin-conjugated quantum dots were used, and the detection period was approximately three hours, indicating the viability of the biosensing device for successful bacteria detection in the range of 10⁴-10⁸ CFU mL^-1.

Potentially toxic elements

The contamination by heavy metals and corresponding ions of the natural waters can pose significant risks to human health, and compact, low-cost, and rapid heavy metal analyses are a global priority concern. As a model target for testing an optical DNA biosensor for the detection of heavy metal ions that are extremely toxic and common pollutants in the environment, mercury ions (Hg²⁺) were used. The biosensor was compact, low-cost, and rapid with in situ screening of Hg²⁺ in natural waters in less than 10 min. The detection principle is focused on the capacity of certain metal ions to bind selectively to certain bases to form stable metal-mediated DNA duplexes; in the case of Hg²⁺, thymine bases can be selectively coordinated to form stable thymine-Hg²⁺-thymine complexes. In the detection range between 0 and 1000 nM, a detection limit of 1.2 nM was achieved, which is lower than the maximum value requested by the United States Environmental Protection Agency (10 nM)

For the detection of Pb²⁺ in water samples (pond and lake water samples) using DNAzymes/carboxylated magnetic beads and DNA aptamers, two fluorescence based optical biosensors have recently been suggested. The detection limits of 5 nM and 61 nM, respectively, were observed by biosensors based on DNAzymes and DNA aptamers, with a respective linear detection range of 0 to 50 nM and 100 to 1000 nM. The use of label-free unique dye (SYBER Green I), which was intercalated with double stranded DNA, showing strong fluorescence intensities, as seen in Figure 5. Moreover, the absence of biosensor fluorescence intensity is observed only with the dye (curve a). With the DNAzyme + Pb²⁺ (curve b) the fluorescence intensity increases with the addition of the dye + DNAzyme + Pb²⁺, illustrating the sensitivity of the biosensor towards Pb²⁺.

Figure 5. Fluorescence emission spectra for detection of Pb²⁺

Toxins

Harmful toxins like brevetoxins and microcystins are created by the eutrophication of aquatic systems by the algal blooms of cyanobacteria, and thus accurate and cost-effective systems are needed for the early detection of such toxins. For the sensitive detection of brevetoxin-2, a marine neurotoxin, an electrochemical aptasensor has been used composed by gold electrodes functionalized with cysteamine self-assembled monolayers. A detection limit of 106 pg mL^-1 was achieved and strong selectivity was observed for brevetoxin-2 against other toxins of various groups, like okadaic acid and microcystin. The feasibility of the aptasensor for detecting brevetoxin-2 in real samples was achieved by analysing shellfish and strong recoveries (102-110 percent) were reported, indicating no interaction with the aptasensor response from the shellfish matrix.

Endocrine disrupting chemicals

In water samples, bisphenol A was detected as an endocrine disrupting chemical by aptasensors based on the fluorescence principle with functionalized aptamers (fluorescein amidite) and gold nanoparticles and based on evanescent-wave optical fibre. The evanescent-wave optical fibre aptasensor was compact and found to be rapid, cost-effective, sensitive and selective for the detection of bisphenol A in water samples, with the benefit of no requirement of any pre-concentration or treatment steps.  Furthermore, the aptasensor can be reused for 90 s by regeneration with a 0.5% sodium dodecyl sulphate (SDS) solution and further washing with a phosphate buffered saline (PBS) solution (pH 7.2) for over a hundred assay cycles without any noticeable loss of efficiency. Similar detection limits (0.1 and 0.45 ng mL^-1) were observed in both optical biosensors where the DNA molecule probe, which is the complementary sequence of a small fraction of the bisphenol A aptamer, was adsorbed by electrostatic interaction in the surface of gold nanoparticles and covalently immobilized on the surface of the fibre. Lately, for the detection of bisphenol A in river water samples using molybdenum carbide nanotubes, another fluorescence-based aptasensor was proposed. With such a label-free, inexpensive, and easy to use aptasensor, a low detection limit of 0.23 ng mL⁻¹ has been obtained. The specificity of the aptasensor was evaluated by analysing other molecules with structures similar to that of bisphenol A (e.g., 4,4J-biphenol, bisphenol AF, and 4,4J-sulfonyldiphenol) and only background signals showing high specificity for bisphenol A were identified for these molecules.

A disposable and label-free electrochemical immunosensor based on a field effect transistor with SWCNT has recently been employed in seawater samples for assessing another endocrine disrupting chemical – 4-nonylphenol. The immunosensor has a high reproducibility (0.56 ± 0.08%), an average recovery of 97.8% to 104.6% and a low detection limit (5 μg L^-1), which is lower than the recommended maximum concentration of 7 μg L^-1 specified by the corresponding regulations. In seawater samples such as 4-nonylphenol, the biosensor could be used to detect hazardous priority substances, even at low concentrations and with an easy and low-cost methodology.

Other environmental compounds

New, fast, and accurate analytical methodologies have been needed for the early detection and monitoring of various other hazardous compounds liberated during algal blooms. Due to the excellent sensitivity and specificity of nucleic acid probes to their complementary binding partners, biosensors have been developed to detect algal RNA. For the enhanced selective and sensitive detection of RNA from 13 harmful algal organisms, an electrochemical genosensor based on screen-printed gold electrode was recently reported; the genosensor could distinguish RNA targets from environmental samples (spiked seawater samples) containing 10⁵ cells, considered to be the limit of detection.

Biotechnology is a modern advanced technology grounded on science disciplines like molecular biology, biochemistry, cell biology. It accepts a lot of new techniques such as cell and tissue cultivation, cell fusion, gene recombination, microbial fermentation, and also duplication and recombination of organisms at a cellular, chromosomal, and gene level.

Biotechnology is a modern advanced technology grounded on science disciplines like molecular biology, biochemistry, cell biology. It accepts a lot of new techniques such as cell and tissue cultivation, cell fusion, gene recombination, microbial fermentation, and also duplication and recombination of organisms at a cellular, chromosomal, and gene level. In this way, it can make organisms answering to human needs, produce new products and breed new plants having novel features of high output and stress resistance. Modern biotechnology is composed of four technological systems. They are genetic, cell, enzyme, and fermentation engineering, of which genetic engineering and cell engineering are the most advanced ones.

Besides the great social and economic benefits of modern biotechnology, there is a possibility it to harm human health and the environment. This can result in many socio-economic problems, such as destroying original social and economic patterns, posing threats to biodiversity and traditional crop varieties, injuring countries or communities’ socioeconomic welfare, violating traditional ethical, moral, and religious values.

The modern biotechnological practice in the production of fiber, pharmaceuticals, and food has a powerful development of the late 20th and beginning of the 21st century. This emerging technology is often accepted as the next technological revolution possessing the potential to change fundamentally the way of societal arrangement regarding production and distribution of goods. Considerable investments have been already made in biotechnological research for new products’ development. Currently, science and technology are expected to bring consumers a big variety of genetically modified (GM) products. Besides, many GM products have already entered the food distribution chains. Despite its promise to bring significant benefits to society, public acceptance of modern biotechnology is quite diverse at the world dimension. To enable biotechnology to contribute major benefits for human beings, the international society has paid serious attention to the biosafety of the overall biotechnological production to promote its better acceptance.

Nowadays, biotechnology has been classified into few categories based on its applications. They are depicted in Fig. 1

In general, biotechnology intends to emphasize the potential benefits to society through reduction of hunger and malnutrition, suspension and healing of diseases, and increase of health and general well-being. On the other hand, there is an opinion that GM products are used as a needless interference with nature and can cause unknown and potentially disastrous consequences. At the same time in the U.S., GM crops entered the grain supply chains without raising major public concern. Besides, agricultural biotechnology is facing significant opposition in Europe and many other developed countries. Regarding the obvious public troubles about the perceived risks for humans and the environment, European Union (EU) accepted rather limited regulations on all transgenic crops in any part of the EU food system. At a global level, the situation is even more diverse: in the U.K., objection movements throw down GM crops on different occasions. Until recently, Brazil and India refused to approve any GM crop. Due to similar consumer concerns, some fast-food supply chains decided not to use GM potato in their products.

The reason against the use of gene technologies in agricultural production is grounded on the fact that some people and institutions see risks to humans and the environment, while others oppose it minding moral, ethical, and social concerns. Biotechnology frequently is running down because of its tools’ application in plants and animals, especially gene transfer across species, based on understanding “realms of God” and against “Law of Nature”. Also, there is an opinion that since genes are naturally occurring parts, which are an object of discovery (not of invention), patent ownership of genetic findings and processes is morally and ethically insolvent.

Public discussions on agricultural biotechnology also pose some social and political debates. There is a talk concerning the application of modern genetic technologies in production of commodities in developed countries that are currently imported to developing countries. It is claimed that such developments will have significant negative effects on the poverty situation in the Third world and will lead to global instability. At the same time, other researchers have the opposite opinion. Another source of care is the possibility that farmers will become steadily dependent on multinational corporations for their “means of production” bringing harmful economic, social and political effects.

The importance of this subject defines the need for a full understanding of public interests and concerns regarding an agreement between private and public decisions concerning food biotechnology. Consumer acceptance of biotechnology is strongly related not only to expected risks and benefits associated with GM products but also to their moral and ethical dimension. Further, public views about multinational corporations, trust in government, science, and technology development also reveal their attitudes towards biotechnology. It is found that consumers’ cognitive factors (e.g., levels of risk aversion, opinions about GM foods) impacted the acceptance of GM food products, while the socio-economic factors did not have significant effects. Thus, the public perceptions of biotechnology possess a lot of dimensions and are possibly affected by multiple forces, preferences, and events. For instance, positive benefits (e.g., nutritional benefits from improved/new products, environmental benefits via reduced use of pesticides, etc.) can encourage consumer acceptance of food biotechnology. On the other hand, sensibility about risks to humans and the environment is expected to have adverse effects on public acceptance of GM products. Thus, the activities to analyze and determine the factors influencing consumer attitudes towards biotechnology have to follow the procedure, denoted in Fig. 2.

Societal Impact

To evaluate the impact of modern biotechnology on society, it is not enough only to measure it by specific indicators. When the focus is on such dimensions like increase the number of patents on genetically modified organisms (GMOs), this will not indicate the dynamic forces shaping the impact of biotechnology on industrial and societal structures. The impact analysis is combining complex problems and has a variety of diverse inputs, which possess both short- and long-term effects and influence the results of the problem or use of technology, in an unrespecting way. To realize the sense about the nature of links between societies, individuals, and the balance of forces between social groups facing new technologies is of basic importance.

Establishment and spreading of knowledgeSocietal Impact

A starting point is the result of the technological impact, which depends upon the establishment and spreading of knowledge. It is regarded as preferred values, social practices, and norms of behavior of involved actors in the development and use of biotechnology. The knowledge extends beyond the collection of information on biotechnology, such as techniques, risks, etc. It also embraces the way they are shown and commented on by individuals. Risk and safety for the public cannot be assayed in the same manner as scientific risk is done. To account for these differences, it is necessary to explain how trust is created between consumers and manufacturers. In brief, such a definition of knowledge gives a piece of analysis on the impact of biotechnology and more broadly, environmental policies and practices.

From a conceptual point of view, this approach could be treated as a knowledge-based discourse. It is useful considering the expression of the biotechnological impact in the sense of the educational, social, and political background. The environmental discourses of biotechnology knowledge, and more specifically, how it is constructed, accepted, and perhaps most importantly, spread and sustained within society are taken into account as well.

Science and Technology (S&T) impact on society

In the EU there is a discussion on biotechnology and especially concerning genetic engineering and impacts arising from this technology. This issue has to be addressed strongly linked to the result of social interactions. Therefore, the way of realization of these dynamic forces should account for the interrelations and the form of resulting complexity between actors.

Another point to estimate, are two major topics when examining potential technology impacts. These are i) the immediate impact of the use and development of the technology and ii) the technology transcending risks. That means dependence on the long-term application of the technology and how to achieve and sustain a dominant position in its impact upon society from education, political and social perspectives. This could be sustained generally through more concern about the long-term consequences of S&T activities noted below:

– Publication of educational efforts in society: Performance of intensive efforts to include scientists, key public opinion leaders, and media in an existing debate on biotechnology and especially about genetic engineering. The core objective is to avoid an informational vacuum, which can facilitate anti-biotechnology activity and generate negativism in knowledge structures.

– Role of regulatory policy in society: Focusing on effective risk communication and understanding the fact that the public perception of risk can be very different from that of the scientific community.

Perception of modern biotechnology applications

Three major topics are identified and based around: i) the science, ethics, and gender of biotechnology ii) the International political economy, trade, and the environment; iii) the state and system security and warfare through biotechnology. These general themes are coming from consideration for the international relations of biotechnology. They imply the concept of power in the International Political Economy (IPE) to frame the differing discourses and complex issues of technology impacts. Based on these topics the several indicators are regarded (Fig. 3):

Modern biotechnology is regarded as a complex emerging field that exhibits high science combined with limited knowledge of part of the society. It is related to attitudes towards the natural environment, technological progress, religious and moral beliefs, and several other sets. A matrix of variables including interest of the public domains, such as science and politics, optimism about technologies, social and cultural values, engagement with the issue of biotechnology and confidence in the industry, regulation, and other civil society groups, all these factors contribute to the public’s representation of and opinions about biotechnologies (Fig. 4). Background characteristics such as gender, education, age, and religion have been found to affect people’s attitudes as well.

The serious debate on the acceptance of genetic engineering is accompanied by many pieces of evidence that object to the focus on specific applications of the technology than genetic engineering per se. Studies are showing that consumer attitudes regarding gene transfers are influenced by the type of transfers. The range of acceptance encompasses the following: i) plant to plant gene transfers; ii) animal to animal transfer; iii) animal-plant or human-animal, the last being least acceptable.

A survey on perception on modern biotechnology applications, performed among people from different countries/continents indicated that medical applications (leading to the development of medicines and vaccines, and genetic testing) are very much acceptable in comparison to food or crop biotechnology applications. However, medical applications linked to xenotransplantation or animal cloning for milk production meet serious problems. The Asian people, for instance, are less troubled for medical genetically engineered products than for genetically modified food. The investigation concerning cloning of human cells and bioremediation among the Europeans is put at the intermediate level, less assumed than genetic testing but better confessed acceptable than genetically modified crop and food.

Another survey reported that genetic testing still scored the highest support within the European population, followed by human cell cloning, the production of GM enzymes for environmentally friendly soaps, and xenotransplantation, At the bottom of this range are placed GM crops and food. Also, higher support is given to technics for the manipulation of bacteria to clean oil spills followed by disease-resistant crops; afterward are applications for fat meat and better taste tomatoes and the less support receive technologies for enhancing milk production in cows.

The Malaysian consumers are more open for use of modern biotechnology applications, which do not involve inter-species gene transfers, like food production and GM crop that only involve the transfer of plant genes. For them, the gene modifications are more acceptable in the following order: i) transfer oil palm genes to reduce its fat (saturated) content, ii) transfer human genes into bacteria to produce insulin, iii) transfer bacterial genes into soybean to make it resistant to herbicides.

An individual’s attitude towards a new technology depends on several related factors such as his/her perception of its risks and benefits, and socially communicated values and trusts in institutions representing these technologies. Concerning public perception of biotechnology, it is speculated that the attitude to genetic engineering is determined by the worth of potential benefits offered, knowledge on genetic engineering, and scientific world-view, from which the perceived risk (rational worries) and anxieties or fears (irrational worries) must be excluded. Additionally, various minor factors such as background factors must be added. It is considered that the main effect on acceptance seemingly is based on the knowledge level, awareness of benefits, confidence, and trust.
The studies of public attitude towards biotechnology indicate a lot of similarities with risk perception studies. Some authors have used the psychometric approach based on cognitive psychology and it is accepted as the most mature and dominant paradigm in risk perception studies. These psychometric methods are settled on the ground of the following views:

• To perform “risk” as a subjective idea, and not an objective one;
• To involve technical/physical and social/psychological outlooks in risk criteria;
• To take opinions of the “public” (i.e., laypeople, not experts) as the subject of interest.

Thus, the public acceptance of modern biotechnology is based on analyzing the cognitive structure of risk judgments, commonly using multivariate statistical procedures like factor analysis, multi-dimensional scaling, and multiple regression. This psychometric approach assumes that the public does not perceive technological risk due to a single dimension linked to expected injuries or fatalities close to a risk assessor’s viewpoint but interprets risk as a multidimensional concept, concerned with broader qualitative dimensions.

The key varieties of a risk perception study are the perceived magnitude of risk or dread, risk acceptance, familiarity with the hazard, and at least – the factor benefit. The importance of another dimension, ‘interference with nature’ in risk perception studies on genetic engineering is also very important.
Biotechnology is at the confluence of science and ethics. The development of technology is linked with an ethical vision, which in turn is shaped by specific issues. Loads of biotechnology can be evaluated for their benefit to human society. But it has to be considered that biotechnology has also a dangerous part. It can provoke unanticipated consequences, which can cause harmful effects or dehumanize people. So, the ethical issues of proposed effects must be carefully investigated.

The ethical evaluation of new technologies, including biotechnology, requires a different approach to ethics. There is a necessity for changes because a new technology can have a more profound impact on the world due to i) restrictions to a rights-based ethics approach; ii) the importance and difficulty of predicting consequences; iii) opportunity now to manipulate humans themselves.

The ethical questions concerning biotechnology are very different. Due to the potential for deep change in the human future, such questions must be carefully considered. In the first place, it is necessary to articulate and predict the responsibilities towards nature and others, including future generations, and then to focus on rights and freedom. The real power and potential of biotechnology require strong caution to ensure ethical progress, as biotechnology is accepted as a significant force to improve the quality of people’s lives in the 21st century.

Biotechnology is intrinsically linked to science and scientific knowledge. But sometimes there is doubt that biotechnology is closely tied to ethics. Lastly, different biotechnological trends are promoting a certain vision of life, some of which are good for life and deserved to be encouraged or pursued but others are bad and should be eliminated. This vision impacts people’s choices and influences their sense of ethically appropriate biotechnology.

At times, the link between biotechnology and ethics is described as a conflicting point. Once, there is an impression that ethics is needed only in case one wants to tell others that they are doing wrong things. To a certain degree, this is lucid, since dispute, debate, and argument are common parts of ethics consultation. But ethics is just as important in the case of consensus that the chosen pathway is good and right. Of course, there isn’t any necessity for an ethical debate when the search is a cure for cancer. Thus, the solution to perform such a search is predicted by a common understanding that to cure cancer there is an ethical reason. The efforts, resources, and creativity devoted to working out better treatments are ethically eligible and the majority of these achievements due to biotechnology.

The societal acceptability of benefits and risks imposed by modern biotech applications concern:

• Benefits vs. risks societal dispute
• Risk acceptance as a key element in risk perception
• Tools for measuring public attitudes towards risks and benefits: risk management

Benefits vs. risks societal dispute

To determine the acceptability of biotechnological applications it is very important to evaluate the perceived benefits and risks. The diagram on Fig. 5 summarizes them.

Risk acceptance

Another key variable is risk acceptance which is important to measure risk perception. But it is rarely used in attitude towards biotechnology studies. Highlighted the harsh situation with modern technologies, they are always linked with some kind of risks that pose serious problems for societies. Policy-makers have considered cost-benefit analysis as the basis of decision-making methodology for societal risk acceptance. The core question to be solved in the risk-benefit analysis is: whether this product (activity, technology) is eligible safe or how safe is safe enough? There are two main approaches for performance of risk-benefit analysis (Fig. 6).

Risk Management

The management of risk is the process that aims to diminish the effect and to balance the way people respond to it. If one wants to push away all risks, this is not a viable goal. People readily take risks performing a wide range of activities. Thus, the question about the acceptable risk is not entirely technical; its answer depends on values. In a simplified way, it is possible to set a simple threshold for acceptable risk, for instance – one in a million-lifetime risk. All the activities riskier than this threshold are “unacceptable “as well as all those less risky are “acceptable”.

However, there are two reasons why this approach is not working. The first is linked to the above-mentioned risk perception. The people are not reacting to risk simply; the acceptability of risk in the public’s mind is not simply a matter of the presumable fatal things but involves other elements of the activity. For instance, if the activities are taken voluntarily if the process leading to the risk is new/old or familiar, if the process provokes an intuitive, emotional dread reaction or not. That is why, to define an acceptable risk, the processes of risk communication and engagement described above must be included. In the second issue, some activities are essential and must be encountered even if they cause relatively high risks.

Besides, there could be other cases where inexpensive alternatives exist to perform less risky or modified activities. In these cases, the risk might be well predicted as unacceptable, due to ready alternatives. This is an economic issue that deals with several resources dedicated to risk reduction. Besides the economic problems, it is possible to consider risks, not in terms of absolute values but to their internal differences between alternatives.

However, very often it is not possible fully to escape risk; rather one must choose among different risks. Therefore, no definite acceptable threshold of allowable risk exists, as well as there is no single convenient monetized value for risk diminution. Societal desire to invest in risk reduction depends on the wide range of factors that manage risk perception, like the fear associated with the risk, its catastrophic potential, etc.

Social benefits of modern biotechnology

Biotechnologies are being used widely in such fields as medicine and hygiene, agriculture, forestry, breeding, fisheries, energy and chemical industries, metallurgical and mining industries, food, and light industries, environmental protection. Modern biotechnology has gradually demonstrated its huge potential contribution to productivity, though it has a short history of only tens of years so far. Modern biotechnology will become powerful means to reduce the world’s constraints in the fields of food, health, energy, resources, and the environment. The development and application of modern biotechnology will, over a long time, affect human beings and society deeply.

Biotechnological industries are likely to become the leading industries in the 21st century. For example, crop varieties, produced by biotechnology, with novel traits of high productivity and stress resistance (aridity, coldness, high salt, etc.) are expected not only to increase grain production but also decrease the use of agricultural chemicals (such as pesticides, herbicides, and fertilizers), which will be beneficial to the environment. In the respect of medicine and hygiene, many drugs such as DNA vaccines, protein engineering medicine, monoclonal antibodies, anti-sense RNA drugs have been developed by biotechnology. Transgenic animals and plants producing some kinds of medicines (such as vaccines and hormones) may help to reduce illness in the process of everyday diet. Human Genome Project (HGP) and the research into some disease genes will discover the genetic reasons for some illnesses. Environmental pollution is getting worse at present, but biological transformation reactors created by biotechnology promise to absorb pollutants or wastes and decompose them into materials of low or no toxicity.

Therefore, in the present society characterized by knowledge-intensive industries, countries all over the world, especially the industrialized ones, take biotechnology seriously. Large quantities of manpower and financial resources are allocated to R & D in biotechnology. Relevant development strategies and policies are drawn up and support mechanisms created to stimulate the development of biotechnology.

Although there have been many studies on public attitude or perception towards biotechnology and some researchers tried to identify factors predicting attitude using either regression or correlation, there are limited studies that try to construct a structural model predicting attitude towards biotechnology.

The first documented model was developed by Kelley (1995) who proposed a structured way for the approval of genetic engineering by the Australians (Fig. 7). Later, Pardo et al. (2002) proposed another model for explanation of European attitude towards biotechnology applications. Kelley found out that approval of genetic engineering was mainly predicted by agricultural and health goals (beneficial aspects). On the other hand, scientific and genetic engineering knowledge did not predict approval. Demographic variables also did not directly affect the approval of genetic engineering (their effects were not statistically significant). Gender was found to affect knowledge and scientific world-view while age was found to affect knowledge, goal, and scientific world-view, thereby exerting a very weak (indirect) effect on approval. Education had a large effect on knowledge but not concerning attitudes. Occupation and religion did not have any effect on any of the intermediate variables. Green supporters were less favorable to genetic engineering (negative direct effect on the approval of genetic engineering) as compared to Labor Coalition supporters (politic).

It is often speculated that biotechnology is the technology to deeply transform the economy and society. Following such opinions, the development and application of biotechnology have considerable potential for far-reaching economic, social, and environmental impacts. That is why biotechnology is of strategic importance to knowledge-based economies and their governments. Nations that failed to develop biotechnology capabilities fail in realizing economic impacts.

Biotechnology-related applications are diffused over several very different industrial sectors, e.g., food production, textile finishing, pulp and paper, agriculture, power generation, chemicals and petrochemicals, and pharmaceuticals. The products manufactured by these sectors do not usually distinguish between alternative production processes. Thus, for many products, a distinction between biotech and conventional (e.g., chemical) production processes and also the introduction of some new ones would be necessary. Moreover, biotechnology is a dynamic field and certain biotechnological production methods will probably soon be re-developed, and completely new, products will appear. Current official statistics and existing companies’ surveys can provide specialized information about the diffusion of biotechnological applications, and their economic impact. The data have been constantly renovated and different techno-economic studies are performed to provide future production foresight and scenarios. Applying a blend of methods and sources, they frequently focus on expert judgment, case-studies, and various statistics.

An alternative to measuring the diffusion of biotechnology in use for major manufacturing sectors is the biotechnology-related-sales (BRS) concept. Existing studies have used various approaches for measurement (definition of the biotechnology aggregation level, examined sectors, scenario assumptions, etc.). Moreover, to assess the impact of biotechnology diffusion, an input-output model and relevant indicators have been introduced to study its effect on employment. The model studies in a comparative manner how biotechnology is affecting major application sectors. It compares the market penetration in 2004 and 2020 through the diffusion rates, measured with upper and lower limits. This approach helps not to overestimate the impact of biotechnology while capturing certain innovative biotechnological applications. The data of this survey indicate a nearly similar diffusion pattern in pharmaceuticals, food-processing, and agriculture. Diffusion is expected to increase in all sectors greatly by 2025, as indicated by the penetration of certain product groups. However, it was shown that there is a serious level of suspicion about the diffusion of biotechnology; the expectations for 2025 are highly uncertain. The analysis of this tendency, indicate that there are diffusion barriers specified in Fig. 7.

Figure 7. Main barriers interfering Biotechnology diffusion

Socioeconomical Impacts

Motivated to perceive great economic profit, a lot of famous international business corporations made considerable investments of capital in the R & D of biotechnology, and in this way are changing the previous world’s social and economic trends. Such an example is the company Monsanto (formerly US-based, and now a part of the global consortium BASF). Traditionally working in the chemical industry, after 1985 the company made serious business rearrangements, invested in 3 biotechnological sectors, and became a major consortium dealing with R & D in bio-science. After 1998, about 20 million hectares of fields all over the world were planted with Monsanto’s GM seeds. Soon after that, Monsanto took an advance on the crop seeds international market through a completely integrated system for trading GM seeds and agricultural chemicals. In this way, Monsanto received control on a big part of the human population food chain gaining tremendous profit feeding them with GM crop. This is an example of how in the current globalized world, a possibility for a few multinational corporations to decide on human food consumption emerges.

In the case of use in dairy, bovine growth hormone (BGH) produced by genetic engineering, the milk yield of a dairy cow can increase by 30 percent., which means 10 percent less fodder usage. So, the result is very impressive for the dairy industry.

A lot of medicines, like vaccines for treating malaria and cholera are provided to patients via new antigens synthesized through modern biotech methods. They make the rates of illness and death in many countries (especially in the developing ones) falling rapidly, concurring the population growth rates. In this context, a question arises about the impacts on the already insufficient economics and infrastructures of the said countries.

Impacts on biodiversity and sustainable agriculture

Many GM crops contain foreign genes from other plants, animals, and microbes. All these foreign genes can be transferred to other plants in nature through the pollens of genetically transformed plants and will cause pollution to the natural pool of genetic resources. Hence, as the majority of transgenic plants possessed economically useful features, like high productivity, disease-pest resistance, stress resistance, and farmers are attracted to economic interests to grow these plants rather than traditional crop varieties, the predicted danger for the natural resources is serious. Also, the technologies for breading of a monoculture of crop varieties in some fields may add to the loss of biodiversity. So, in a long term, this degradation of the genetic base may reduce the natural resistance of plants to diseases and insect pests and this may cause diminish in the crop yields. In this way, the global sustainable development could be compromised.

Bacillus thuringiensis (Bt) is a soil bacterium, which is used to synthesize a toxin against insects within plants. It has been used as a microbial pesticide for many years and the genes coding these insecticidal toxins could be introduced in the cell of many crops such as cotton, soybean, and rape, to create pest-resistance crops. In this case, the experts in environmental protection and the Green Peace Corporation are troubled by the fact that the toxin production is realized in the late life cycle of the transgenic plants and there is an opportunity to increase commonly the resistance of pests to the Bt toxins. So, this situation would lead to bigger usage of Bt-based pesticides, due to loss of their efficacy and causing heavy economic losses.

Impacts on public welfare

The exploitation of genetic engineering technology to humans could facilitate the diagnose and cure of genetically related diseases such as some cancers, hemophilia, etc. But such gene diagnosis may also have an opposing effect on the employment and marriage prospects of human beings. Through genetic engineering animals, plants and microbes can be transformed into bio-factories for the production of different kinds of chemicals. Thus, they can be treated as constant biological producers and at the same time, the rising potential for products outcome can cause socioeconomic problems. Assume that specific microbial strains are engineered to produce a valuable substance on large scale. In such a case, it is important to forecast the impact of this production on the world’s major producers of the same substance. If this substance counts for a significant percentage of the income of a given country, what would be the influence and consequences of this biotech production on the country’s agriculture and financial infrastructure.

Inconvenient application of biotechnology can cause many risks, like other advanced technologies. For instance, the newly introduced genes in crops made by genetic engineering can lead to allergic reactions. The economically important features of pest-resistance, herbicide-resistance, or stress-resistance, introduced in plants may get these transgenic hosts away from agricultural cultivation systems. The genes for resistance in transgenic plants can be transferred to their wild weedy relatives, and the latter can be transformed into “super” weeds, which control will be very difficult. Thus, the large-scale releases of transgenic pest-resistance crops into the environment will cause severe selection pressures to enforce the resistance of target pests.

The transgenic virus-resistance plants that are already transformed with foreign virus genes possess virus-coded proteins that may recombine with the genetic material of other viruses to yield new types of viruses with higher toxicity. If the selective killing of the target pests and pathogens is not possible, the transgenic pest-and-disease resistance crops can at once poison other organisms and also may cause harmful action via food chains on beneficial microorganisms, insects, birds, and mammals.

Moreover, many biotechnological products are alive organisms and can move and reproduce by themselves. Once they are released into the environment and are proven to be toxic, then it is nearly impossible to discard them, and the harm they create can rise with time becoming more and more serious. In this way, environmental releases of transgenic plants on a large-scale may damage the natural ecological balance in a longtime perspective.

Biosafety concerns have hindered the R & D applications of biotechnology. The public in lots of countries has signified its negative opinion, repulsion, indeed dread regarding biotechnological products. This is voiced even through demonstrations, crushing fields crops, banding the import, or baying the biotechnological products. This makes the biosafety issues at the very top of the agenda of many countries and international societies. Laws and regulations on biosafety are formulated in many countries, and biosafety issues are regarded in many international documents and treaties, like Agenda 21 and The Convention on Biological Diversity.

Biotechnology is on the way to become seriously relevant to the economy and can gain considerable importance in several application sectors in the next 10 to 15 years. However, the overall effects of modern biotechnology on the economy are quite diverse. For instance, employment estimations show that increasing the part of biotechnology in application sectors and its link to suppliers indicate the trend for reduction of employment due to productivity effects. The economic importance of biotechnology rises because of its diffusion in mature manufacturing sectors. However, diffusion in these main application sectors can influence only limited parts of overall employment. Only if biotechnology increases in other sectors might a major economy-wide impact emerge.

The deepness of biotechnological transformation of the economy depends strongly on its potential to propose the creation of new products and to improve the efficiency in the production of existing ones. Hence, it is expected that biotechnology would enable the creation of many products but the potential to raise labor productivity is estimated to be restricted. One important view is that biotechnology may transform the economy and society differently, cooperating with other technologies like ICT. Major boosts for such economic growth may emerge in combination with other technologies or efficiency gains, which raise productivity and purchasing power to realize the fruits of biotechnology.

Recent updates on the legislation on renewable energy Directive (EU) 2018/2001 of the European Parliament and of the Council of 11 December 2018 on the promotion of the use of energy from renewable sources.

Wastewater

Fig. 12. Water location and use

Water is a resource to be protected. Please find below the main location where freshwater is located on earth and some of the reasons why water is slowly running out. Almost 70 % of the water today is consumed for agriculture, about one-quarter for commercial uses, and roughly 10% is utilized for domestic purposes. Therefore, the main sector that uses water is agriculture/farming. Agricultural water is mainly used for irrigation as well as pesticide and fertilizer applications and for animal farming. There are three sources for agriculture water: i) Groundwater from underground wells; ii) surface water that is derived from open canals, streams, irrigation ditches, and diverted from reservoirs; iii) rainwater which is usually collected in barrels, tubs, and large cisterns. Water is often poisoned. The main causes of poisoning are: domestic use of water (organic matter, surfactants….), agriculture and industrial use of water (fertilizers and pesticides, water deriving from industrial processes), atmosphere (contamination of rainwater by toxic substances present in the atmosphere, deriving from industries, airplanes, motor vehicle engines).

Directive 2000/60/EC of the European Parliament established a framework for Community actions in the field of water policy. This legislation establishes a framework for the protection of inland surface waters, coastal waters and groundwater. The objectives of the directive are:

– the safeguard against further deterioration;

– the improvement of the state of the ecosystems;

– the promotion of sustainable water use;

– the reduction of groundwater pollution;

– the reduction of discharges;

– mitigation of the effects of floods and droughts.

The depuration of wastewater is obtained through different treatments that are often applied in sequences: primary, secondary (biological), tertiary treatments as indicated in the picture) (Fig. 13 and 14).

Fig. 13 (above ) and 14 (below). Primary, secondary and tertiary treatment for the biological depuration of wastewater

Fig. 15. Aerobic oxidation tank

The activated sludge process is one of the most commonly used for secondary wastewater treatment of civil and industrial origin. It is a suspended-growth biological treatment process, using a dense microbial culture in suspension to biodegrade organic material under aerobic conditions and forming spontaneously a biological floc (referred to as activated sludge). Diffused or mechanical aeration maintains the aerobic environment in the reactor. Typical retention times are 5-14 hours in conventional units rising to 24-72 in low rate systems. The activated sludge process depends on aerobic biological action (Fig. 15). The microorganisms break down the complex organic substances into simple molecules including water and CO₂. This process results in the removal of soluble and suspended organic matter from wastewater. The growth of microorganisms in the presence of dissolved oxygen removes the majority of pollutant matter; in turn, protozoa grow and feed on these organisms. The resulting balance is of a living culture in suspended form in the activated sludge flocs. The main elements of the system include an aeration tank (secondary treatment) in which the wastewater is thoroughly mixed with continuously activated sludge and oxygen. From this part of the process, it passes into a clarifier tank (secondary sedimentation), where the settled sludge is removed from the purified water to be recycled by the return activated sludge pumps. For this system to work, two requirements must be met: the aeration device must be capable of both transferring oxygen from the atmosphere to the liquid, and distributing this oxygen throughout the wastewater to the suspended living microorganism. This type of system is suited for low-strength waste, typically on the order of 50-200 mg L⁻¹ BOD. Pre- or post-treatment of wastewater may also be applied. After the aeration basin, the mixture of microorganisms and wastewater (mixed liquor) flows into a settling basin or clarifier where the sludge is allowed to settle. Some of the sludge volume is continuously recirculated from the clarifier, as Returned Activated Sludge, back to the aeration basin to ensure adequate amounts of microorganisms are maintained in the aeration tank. The microorganisms are again mixed with incoming wastewater where they are reactivated to consume organic nutrients. Then the process starts again.

The activated sludge process, under proper conditions, is very efficient. It removes 85 to 95 percent of the solids and reduces the biochemical oxygen demand (BOD) about the same amount. The efficiency of this system depends on many factors, including wastewater climate and characteristics. Toxic wastes that enter the treatment system can disrupt the biological activity. Wastes heavy in soaps or detergents can cause excessive frothing and thereby create aesthetic or nuisance problems. In areas where industrial and sanitary wastes are combined, industrial wastewater must often be pretreated to remove the toxic chemical components before it is discharged into the activated sludge treatment process. Nevertheless, microbiological treatment of wastewater is by far the most natural and effective process for removing wastes from water.

Microbial populations in activated sludges: The microbial community partecipating to the biological depuration process forms floc agglomerates which are called activated sludges. The activated sludge of a of a secondary treatment plant is a microbial culture that develops around organic and inorganic particles and that metabolizes the organic matter present in wastewater. Activated sludge flocs tend to settle down in the “secondary” sedimentation phase because of gravity. Several groups of microorganisms are responsible for the depuration process:

Bacteria are primarily responsible for removing organic nutrients from the wastewater. They also develop a sticky layer of slime around the cell wall that enables them to clump together to form bio-solids or sludge that is then separated from the liquid phase. The successful removal of wastes from the water depends on how efficiently the bacteria consume the  organic  material  and  on  the ability of the bacteria to stick together, form floc, and settle out of the bulk  fluid. The flocculation (clumping) characteristics of the microorganisms inactivated sludge enable them to amass to form solid masses large enough to settle to the bottom of the settling basin.

FUNGI are also heterotrophic organisms helping in the degradation of organic matter.

Protozoa play a critical role in the treatment process by removing and digesting free swimming dispersed bacteria and other suspended particles. This improves the clarity of the wastewater effluent. Like bacteria, some protozoa need oxygen, some require very little oxygen, and a few can survive without oxygen. The types of protozoa present are classified as follows:

Amoebae à Little effect on treatment & die off as amount of food decreases

Flagellate à Feed primarily on soluble organic nutrients

Ciliates à Clarify water by removing suspended bacteria.

Rotifers and Nematods are multi-cellular organisms which are larger than most protozoa and do not basically remove organic material from the wastewater. Although they can eat bacteria, they also feed on algae and protozoa. A dominance of metazoa is usually found in longer age systems; namely, lagoon treatment systems. Although their contribution in the activated sludge treatment system is small, their presence does indicate treatment system conditions.

In addition to activated sludge plants, there are other types of secondary wastewater treatment processes. Some of them can be IMMOBILIZED CELLS PROCESSES, as indicated in Fig. 16.

Fig. 16. Immobilized cell tecnologies for the depuration of wastewater

Percolating filters are a biological purification technology through microorganisms that develop, in an aerobic environment, on appropriate support materials, through which sewage percolates.The percolating filter tank is filled with inert, natural or synthetic materials (e.g. stones) through which wastewater is fed from above. The DEPURATION PHASES include: 1) Primary treatments to prevent obstruction of the bed; 2) Formation of the biofilm  (3-4 mm thick); 3) Cell detachment from biofilm and setting-up of the biofilm again; 4) Final sedimentation. The main advantages of this technology are the low cost of set-up and maintenance and the fact that they can tolerate variation of the organic load of the influent. The main drawbacks include the large areas for the set-up and the problems of bad odours. Biocircles are a modified version of the percolating filters, in which the surfaces bearing the biofilm rotates around an axis, half immersed in the liquid to be treated; the rotation allows the oxygenation of the biomass adhered to the disc. Anaerobic systems work well when the entering flow rate is low and the organic load entering the system is sufficiently high. The required depuration efficiency is not high (anaerobic microorganisms are characterized by a lower growth rate and a slower metabolism with respect to aerobic ones, so the organic matter is not completely degraded). These conditions are typical of some industrial wastewater, anaerobic reactors do not work well for the treatment of large scale civil effluents.

Another important system for the depuration of wastewater is phytodepuration (Fig. 17), which is a: purification technology characterised by biological treatments, in which plants growing in water-saturated soil develop a key role helped by the direct action of the bacteria that colonize the root system and rootstock.

Fig. 17. General features of phytodepuration.

These treatments are seen both as alternatives as well as support to traditional systems based on biological processes and chemical and physical reactions. The term “Wetlands” indicates “Phytodepuration” systems of wastewaters designed to artificially create the same ecological conditions that are naturally established in watery areas. “Phytodepuration” systems engineered, designed and built to reproduce natural self-depurative processes in a controllable environment. In comparison to natural wetlands, phytodepuration systems allow for the choice of the site, the flexibility in the dimension, control of hydraulic flows and retention times. Phytodepurifyng functions can be preferred and additionally exploited with opportune strategies, like the choice of plant species and substratum and control of the flow of water. With Phytodepuration systems, pollutants are removed through a combination of chemical, physical and biological processes. The most effective processes are sedimentation, precipitation, adsorption, assimilation from plants and microbial activity. Phytodepuration technology adds the medium’s adsorbing ability to the traditional biological oxidation depurative treatment (filtering action by plant roots that also provide a large surface suitable for developing microbial masses involved in the treatment) and removal of nutriments due to their growth. Different strategies are described In Fig. 18:

Fig. 18. Different phytodepuration strategies

Floating macrophytes, including water hyacinth (Eichhornia crassipes), are dominant invasive organisms in tropical aquatic systems and they may play an important role in modifying the gas exchange between water and the atmosphere. Hydrophytes are plants which live in water and adjust with their surroundings. They either remain fully submerged in the water or most of their body parts remain under the water. In the picture you can distinguish different examples of emergent hydrophyte system.

Contaminated soils

What are xenobiotic compounds (Fig. 19)?

Fig. 19. Definition of xenobiotics

Which fate can they have in the environment (Fig. 20)?

Fig. 20. Fate of xenobiotics in the environment

Xenobiotics are not necessarily molecules extrinsic to the biosphere, but they can also be natural molecules present in non-natural concentrations in the environment. They are not necessarily toxic molecules, but, generally, they are recalcitrant to the biodegradation.

Antropogenic molecules can derive from different sources:

1) Petrochemical industry: – fuels(aliphatic and aromatic hydrocarbon mixtures), – pure chemicals for the chemical and pharmaceutical industry (alcohols, ethers, esters, aldehydes,  frequently substituted with Cl^– ,amino or nitro groups

2) Pulp and paper industries: use wood as raw material and produce pulp, paper and other cellulose-based products. – the bleaching of paper with chlorine-based products produces halogenated molecules including chloro-lignin

3) Synthetic plastic industry: styrene, vinyl chloride, solvents, cross-linking agents to produce polymers.

4) Pesticide industry: benzene and heterocyclic derivatives, urea, organophosphorus compounds

5) Pharmaceutical and cosmetic industry

6) Textile industry: reagents to produce synthetic fibers, detergents to soften fibers and pesticides for insect/moth control

7) Paint industry

8) domestic use of chemicals (personal hygiene products, cleaning products, ….)

A typical example of natural molecules present in non-natural concentrations is olive mill wastewater: they derive from olive milling to produce olive oil, but they have an high COD (see below) and need to be treated before being discarded in the environment.

Several questions have to answered before deciding whether a contaminated soil can be subjected to a bioremediation treatment. The first aspect to be considered is if we are referring to a chronic contaminated soil or a recent sudden contamination has occurred. In the first case, we can take some time to decide what to do, study in depth the situation, do some preliminary laboratory test and decide the best strategy. In the seconf case, we have to act promptly following the experiences that have been collected for the same contaminants and the same soils.

Please read the questions that need to be answered before DECIDING THE STRATEGY TO APPLY (Fig. 21).

Fig. 21. How to decide which bioremediation strategy is the best one for your

A large array of bioremediation technologies can be used for the decontamination of contaminated sites (Fig. 22).

Fig. 22. In situ and ex situ bioremediation technologies

 IN SITU means that the soil is not removed from its original location and is treated in the same location where the contamination occurred.

 NATURAL ATTENUATION: within this term “a variety of physical, chemical, or biological processes” are included. These processes, under favorable conditions, act without human intervention to reduce the concentration, the toxicity and the mobility of contaminants in soil or groundwater. These in situ processes include biodegradation. A precise monitoring of the process is done, in order to make sure that the soil is remediated. The use of natural attenuation is often proposed as a remedial solution for benzene, toluene, ethylbenzene, and xylene (BTEX). More recently, natural attenuation has been proposed for chlorinated solvents, nitroaromatics, heavy metals, and other contaminants for which the scientific understanding and field experience are robust enough. Natural attenuation is applied when there is solid evidence that natural attenuation processes are transforming the contaminants to harmless products.

BIOAUGMENTATION: it is the addition of microbial cultures in order to speed up the rate of degradation of a contaminant. Indigenous microorganisms present in the contaminated areas may already be able to break down the contaminants, but their action may be inefficient and slow. Bioaugmentation requires studying the indigenous varieties present in the location to determine if biostimulation is possible. The same indigenous bacterial cultures can be isolated, cultivated and implemented into the location to boost the degradation of the contaminants. If the indigenous variety do not have the metabolic capability to perform the remediation process, exogenous varieties with different degradation pathways can be introduced.

biostimulation: is the addition of nutritional supplements for the indigenous microbiota to promote its metabolism. Usually, it refers to the addition of rate limiting nutrients like phosphorus, nitrogen, oxygen, electron donors to severely polluted sites to stimulate the existing bacteria to degrade the hazardous and toxic contaminants. The addition of these nutrients improves the degradation potential of the indigenous microorganisms. Among all the bioremediation techniques, biostimulation is considered to be the most efficient method for remediation of hydrocarbons, especially petroleum products and its derivatives. This is mostly due to the easy availability of carbon source which is one of the rate-limiting nutrients required by the indigenous microorganisms for their metabolic activities from the petroleum contaminants. In addition to the mentioned rate limiting nutrients, implementation of other nutrients rich organic matter can also trigger the remediation process extensively. In this context, it has been shown that the addition of bio solids (nutrient rich organic matter) obtained from the treatment of domestic sewage and inorganic fertilisers, rich in nitrogen and phosphorus, can improve and speed up the degradation rate of petroleum hydrocarbons.

BIOVENTING: also referred to as “Bioenhanced Soil Venting”, it is an in situ technology based on the natural stimulation of the indigenous biological activity with the introduction of oxygen through an air fluid; it is successfully applied to any aerobically biodegradable organic substance, in particular for the remediation of sites polluted by petroleum derivatives. The intake of air is at low flow rate as it is only designed to provide the oxygen needed to support microbial activity. Inside the polluted area, toxic compounds are removed from the air flow while organic compounds are biodegraded aerobically. The air is injected directly through one or more wells connected with vacuum pumps which provides the forced circulation of the air in the unsaturated contaminated soil. Passive bioventilation systems are also applied which exploit the natural exchange of air to transport oxygen, a one-way valve is installed on top of the external vent that allows air to enter only when the pressure inside the contaminated soil is higher than atmospheric.

Fig. 23. Phytoremediation technology 

PHYTOREMEDIATION: It is the treatment of contaminated soil with the use of plants to clean up soil contaminated with hazardous contaminants (Fig. 23). It is more properly defined as “the use of green plants and the associated microorganisms, along with proper soil amendments and agronomic techniques to either contain, remove or render toxic environmental contaminants harmless”, because plants act in synergy with rhizospheric microorganisms that, very often, strongly collaborate with plants in the realization of the process. Phytoremediation is proposed as a cost-effective plant-based approach of environmental remediation that takes advantage of the ability of plants to concentrate elements and compounds from the environment and to detoxify various compounds. The concentrating effect results from the ability of certain plants called hyperaccumulators to bioaccumulate chemicals. The remediation effect is quite different. Toxic heavy metals cannot be degraded, but organic pollutants can be and are generally the major targets for phytoremediation. Several field trials confirmed the feasibility of using plants for environmental cleanup.

EX SITU means that the soil is removed from its original location and treated very close to the site where the contamination has occurred (on site) or far away (off site).

LANDFARMING: it is a full-scale bioremediation technology, which requires excavation and placement of the contaminated soils, sediments, or sludges in a site where they can be treated; this is typically an on site remediation technology used to enhance the microbial degradation of hazardous compounds. Usually, liners and plastic covers are employed to control leaching of contaminants underground in order to avoid contamination of aquifers. Soil conditions are often controlled to optimize the rate of contaminant degradation, in particular:

• Moisture content (usually by irrigation or spraying).
• Aeration (by tilling the soil with a predetermined frequency, the soil is mixed and aerated).
• pH (buffered near neutral pH by adding crushed limestone or agricultural lime).
• Other amendments (e.g., soil bulking agents, nutrients, etc.).

Contaminated soil is usually treated in lifts that are up to 1 meter thick. When the desired level of treatment is achieved, the lift is removed and a new one is placed. Very often only the top of the remediated lift is removed, then the new lift is constructed by adding more contaminated soil to the remaining material and mixed. This serves to inoculate the freshly added material with an actively degrading microbial culture, and can reduce treatment times. This technique has been successfully used for years in the management and disposal of oily sludge and other petroleum refinery wastes. Generally, the higher the molecular weight of the target molecule (i.e., the more rings within a polycyclic aromatic hydrocarbon), the slower the degradation rate. Also, the more chlorinated or nitrated the compound, the more difficult it is to be degraded. Factors that may limit the applicability and effectiveness of the process include: (a) large space requirements; (b) the conditions advantageous for biological degradation of contaminants cannot be reached, which increases the length of time to complete remediation, particularly for recalcitrant compounds; (c) inorganic contaminants are not biodegraded; (d) the potential of large amounts of particulate matter released by operations; and (e) the presence of metal ions may be toxic to microbes and may leach from the contaminated soil into the ground. Land farming, combined with other biological treatments, is widely used and has been successfully applied to many waste types, especially for disposal of oily sludge and other petroleum refinery wastes.

COMPOSTING: Composting is a process that works to speed up the natural decay of organic material by providing the ideal conditions for microorganisms to thrive (Fig. 24). The end-product of this concentrated decomposition process is nutrient-rich product (compost) that can help crops, garden plants and trees to grow.

Fig. 24. Composting

Compost bioremediation refers to the use of a biological system of micro-organisms in a mature, cured compost to sequester or break down contaminants in water or soil. Micro-organisms consume contaminants in soils, ground and surface waters, and air. The contaminants are digested, metabolized, and transformed into humus and inert by-products, such as carbon dioxide, water, and salts. Compost bioremediation has proven effective in degrading or altering many types of contaminants, such as chlorinated and non-chlorinated hydrocarbons, wood-preserving chemicals, solvents, heavy metals, pesticides, petroleum products, and explosives. Compost used in bioremediation is referred to as “tailored” or “designed” compost in that it is specially made to treat specific contaminants at specific sites. The ultimate goal in any remediation project is to return the site to its pre-contamination condition, which often includes revegetation to stabilize the treated soil. In addition to reducing contaminant levels, compost advances this goal by facilitating plant growth. In this role, compost provides soil conditioning and also provides nutrients to a wide variety of vegetation.

BIOREACTORS: the treatment of a contaminated soil in a bioreactor is the finest remediation technology although it is the most expensive (Fig. 25).

Fig. 25. Example of soil treatment in a biorector

It is an ex situ off site technology: the soil, after its removal from the original site, can be treated far away from its original location. The soil is treated in slurry phase inside a bioreactor made of different materials (glass, steel, concrete or other materials) and all the parameters of the remediation process are monitored and controlled to make the process as effective as possible (pH, redox potential, temperature, concentration of the pollutant(s), presence of degradation metabolites). Microorganisms can therefore work in their optimal conditions. The treatment of a contaminated soil in a bioreactor is usually applied for soils contaminated by particularly recalcitrant molecules that are difficultly removed by other remediation technologies (e.g. highly chlorinated molecule).

STRENGHTS

– Reduced costs with respect to chemical and physical strategies (lower energy costs);

– Reduced environmental impact: the soil can be re-used in situ;

– The problem (i.e. the contamination) is solved (pollutants disappear, they are not simply moved from one site to another);

– Acceptability by the public opinion.

WEAKNESSES

– Problems of the bioavailability  of pollutants;

– Problems in case pollutants are more than one;

– Problems of adequate environmental conditions (pH, temperature, oxygen availability).

The biogas producing process has already been described in the basic level section. To sum up what already described, in developing countries there has been an increased interest in the development of technologies to produce renewable energy sources.

Main steps towards modern BIOPLASTICS

• Bioplastics are not a real innovation: natural resins were used since ancient times (for example amber, shellac, etc.)
• Starting from 1860, the first plastics deriving from cellulose were released (eg. celluloid, cellophane)
• In the 1940s, Henry Ford made car parts with plastics obtained from soy
• In the ’50s plastics derived from oil spread
• Oil crisis in the 70s: the interest in bioplastics was rediscovered. Currently there is an increase in the demand for bioplastics, mainly due to the pressing environmental problems (depletion of resources, greenhouse effect, waste disposal, etc.). Fig. 5, 6 and 7 give some basic info about bioplastics.

Fig. 5. Bioplastics and European

Plastic and rubber are polymeric materials consisting of monomers. These are mainly produced from petroleum and the originated material is therefore non-renewable. Around 4% of the world’s oil consumption is used as raw material in plastic production, and a similar amount is used as energy in the production process. In addition to petroleum, plastic production requires the use of chemical additives such as plasticizers, flame retardants, heat and UV stabilizers, biocides, pigments, and extenders. Several additives are classified as hazardous according to the EU regulations (carcinogenic, mutagenic, harmful for reproductive health or for aquatic life, or having persistent negative impacts on the environment).

In the ‘60s plastic was considered for the first-time as a concern in sea and ocean pollution and negative health impacts on humans and the environment were starting to be described. Indeed, plastics release toxic chemicals throughout the life cycle of the product.

Plastic recycling emerged as a possible solution. Recycling, however, in not the only solution needed to solve the plastic waste crises that is polluting the environment. Plastic can range from being unrecyclable, recyclable only once or twice, or at a defined number of times but not forever. After this limit, the plastic will end up in a landfill. Furthermore, a lot of plastic consumers do not even allow their plastic to have this long of a life. Renewable plastics, meaning plastics derived from renewable sources and easily biodegradable in the environment, may offer a solution to the problem of bioplastic poisoning.

Fig. 6. Biobased and biodegradable

Fig. 7. Main bioplastics produced

Bio-based is defined in European standard EN 16575 as “derived from biomass”. Biodegradable materials are materials that can be broken down by microorganisms like bacteria or fungi into water, carbon dioxide or methane and biomass. However, biodegradability depends on the environmental conditions: presence of microorganisms, temperature, and availability of oxygen and water. Compostable materials are materials that break down at composting conditions. Industrial composting conditions require elevated temperature (55˚C – 60˚C) combined with a high relative humidity and the presence of oxygen, and they are in fact optimal when compared against other degradation conditions like in soil, surface water and marine water. Compliance with EN 13432 is considered a good measure for compostability of packaging materials. According to this standard, plastic packaging can be called compostable. Some details on the 3 main categories of bioplastics are given below.

Starch-based plastics

75% of all organic material on earth is present in the form of polysaccharides. An important polysaccharide is starch. Plants synthesize and store starch in their structure as an energy reserve. Starch is found in seeds, tubers, or roots of the plants. Sources of starch are corn, wheat, rice, potato, tapioca, pea, and many other plant resources. Most of the starch produced worldwide is derived from corn. Starch is generally extracted from plant resource by wet milling processes. Starch consists of two types of glucose polymers: amylose and amylopectin. Amylose is essentially a linear polymer in which glucose units are predominantly connected through α-D-(l, 4) glucosidic bonds. Amylopectin is a branched polymer, containing periodic branches linked with the backbones through α-D-(l, 6) glucosidic bonds. The content of amylose and amylopectine in starch varies and depends on the starch source.

Fig. 8. Novamont’s starch‐based technology

An important class of plastics is represented by starch‐based plastics. Beginning in the early 1990s, research and technology developments have permitted to complex natural polymers like starch (from maize, potato etc.) with biodegradable macromolecules (polymeric complexing agents) in order to obtain thermoplastic and biodegradable innovative materials on an industrial scale. In particular, Novamont’s starch‐based technology (Fig. 8) employs processing conditions able to almost completely destroy the crystallinity of amylose and amylopectin, in the presence of macromolecules, which are able to form a complex with amylose. They can be of natural or synthetic origin and are biodegradable. The complex formed by amylose with the complexing agent is generally crystalline and it is characterised by a single helix of amylose formed around the complexing agent. Unlike amylose, amylopectin does not interact with the complexing agent and remains in its amorphous state. The source of the starch, i.e. its ratio between amylose and amylopectin, the processing conditions and the nature of the complexing agents allow engineering of various supramolecular structures with very different properties. Over the last few years many successful efforts have been made to increase the amount of renewable raw materials for producing biodegradable polyesters. Novamont is therefore one of the most important players in starch‐based bioplastics. The company is currently working in the development of a biorefinery project consisting of an innovative development model capable of synthesising various chemical intermediates using renewable raw materials cultivated with low input and in marginal areas instead of fossil raw materials.

Polylactic acid plastics

Synthetic biodegradable poly-lactones such as poly-lactic acid (PLA), poly-glycolic acid (PGA), and poly-caprolactone (PCL) are polymers that are degraded by simple hydrolysis of the ester bonds. The hydrolytic products from such degradation process are then transformed into non-toxic subproducts (Fig. 9).

Fig. 9. PLA life cycle

PLA plastics are derived from the fermentation of agricultural by-products such as starch-rich substances like maize, wheat or sugar and corn starch. The process involves conversion of corn, or other carbohydrate sources into glucose followed by fermentation into lactic acid (Fig. 9 and 10).

Fig. 10. PLA production from starch

PLA derived from lactic acid is thermoplastic, biodegradable aliphatic polyester having ample potential for packaging applications. The lactic acid monomers are either directly polycondensed or undergo ring opening polymerization of lactide resulting in formation of PLA pellets. The properties of PLA as packaging material depend on the ratio between the two optical isomers of the lactic acid monomer. When 100% L-PLA monomers are used it results in very high crystallinity and melting point, whereas 90/10% D/L copolymers fulfils the requirements of bulk packaging. PLA is the first biobased polymer commercialized on a large scale and can be shaped into injection moulded objects, films and coatings. PLA has replaced high-density polyethylene, low-density polyethylene (LDPE), polyethylene terephthalate and PS as packaging material.

The main properties of PLA are: i) the mechanical resistance and heat sensitivity are similar to traditional plastics; ii) hardness, stiffness and degree of elasticity are similar to PET, iii) it can contain fats, oils, alcohol and aliphatic molecules, iv) scarce resistance to acids and bases is but good resistance to UV radiation, v) it can be printed and dyed, vi) it can be transformed into goods through standard machines used for traditional plastics, vii) the post-use phase may involve composting in industrial plants.

Polyhydroxyalcanoates

Polyhydroxyalkanotes (PHAs) are bio-degradable polymers that are accumulated by some bacteria as storage compound in form of intracellular granules. PHA is one of the biopolymers that can effectively replace the conventional petrochemical plastics with their material properties that parallel them. Even then, their production at large scale is still limited by its high production cost compared with conventional fossil-fuel based plastics as the PHA price, depending on polymer composition, ranges from 2.2 to 5.0 €/kg that is at least three times higher than the major petrochemical based polymers which cost less than 1.0 €/kg (calculations made in 2016).

In the majority of companies producing PHAs, mostly pure cultures are used. The problem with the use of pure cultures is the requisites for sterility, refined substrates if plant-based feedstocks are not used, thus limiting the process of commercialisation. All these issues shall be overcome using Mixed Microbial Cultures (MMCs): this combines the transformation of waste into value added product production. Biological treatment of wastewater and sludge management for recovering carbon from wastewater as PHAs is a route to transform end-of-pipe environmental protection infrastructure into bio-refineries. Integration strategies for MMC PHA production within wastewater treatment processes have been proposed for industrial process wastewater and municipal wastewater treatment.

One of the best characterized members of the PHA family is polyhydroxybutirate (PHB), produced by microorganisms that store it inside the cell cytoplasm. In 1926, a microbial production of linear polyester of D (-)-3-hydroxybutyric acid as intracellular granules, which occurred in both gram-positive and gram-negative bacteria under a starvation conditions, was first discovered (Fig. 11).

Fig. 11. Polyhydroxybutirate

Cost is the major drawback of PHB production during industrialization. Industrial production of PHB is costly than that of petroplastics. Large quantities PHB production is estimated about 4.4 USD/kg, i.e. far more expensive than polypropylene production cost, which is close to 1 USD/kg. The financial difficulties are undoubtedly related to production costs, both upstream and downstream processes. Approximately, 40% and 50% of overall production cost of PHB have been assigned to crude material and separation/purifcation systems, respectively. In bioextraction techniques, genetic engineering is the most commonly used to introduce microorganisms, and they are capable of effectively extracting PHB from PHB accumulating cells. There are several approaches that have been investigated including bacteriophage-mediated lysis system and predatory bacteria, which are better than the conventional extraction approaches that produce environmentally harmful solvents, with higher cost of degradation. Thus, due to the non-environmental and non-economical friendly properties of conventional extraction methods, more attention is given to bioextraction systems.

This unit is basically focused on the technologies that allow the study of the microbiota in soil or complex matrixes. The main technologies are depicted below (Fig. 12).

Fig. 12: Techniques to study the microbiome complexity

The culturomic approach is well described in the video that has been produced within Digit-Biotech. This “classical method”, provide for the identification of microorganisms through the isolation of pure cultures, followed by tests that analyze some morpho-physiological and biochemical characteristics. These analyzes are often not sufficient for the identification of most species of microorganisms and moreover are limited to cultivable species which represent a very small percentage of all species found in nature. These tests also have the serious limitation of requiring considerable time consuming. However, they have the great advantage of the obtainment through the isolation approach of target microorganisms that can be used in the bioremediation approach.

Over the past few decades, research in the field of microbiology environmental have shown that microbial communities play a functional role of control of ecosystems that is not attributable to individual species but to the communities themselves as “functional units”. This functional activity of microbial communities is, in many cases, responsible for important processes for humans, including the biodegradation of original waste in wastewater treatment plants and landfills, composting and, in general, all the processes in which chemical transformations of the substances produced by the activities take place. Denaturing Gradient Gel Electrophoresis (DGGE), real time PCR (or quantitative PCR), and whole genome approach (high-throughput sequencing and shotgun sequencing) are the main technologies used for the study of microbial populations in the environment.

DGGE: it is a electrophoretic separation technique used for the separation and analysis of DNA fragments that differ in the nucleotide sequence also of a single base pair. In classical electrophoresis conducted on agarose or acrylamide gel, DNA fragments are separated on the basis of molecular weight; the running speed decreases parallel to the increase in length of the fragment. On the contrary, in the DGGE, fragments of DNA of equal molecular weight are separated according to the denaturation pattern. The presence of heat or chemical denaturants allows the denaturation of the two filaments constituents of a double-stranded DNA (dsDNA) molecule. Temperature and concentration of denaturant to which the separation of the two filaments occurs strongly depend on the sequence of the fragment itself. In particular, the determining factors are: quantity of bonds hydrogen that are established between complementary bases and type of interactions that are established between bases adjacent on the same strand (stacking interaction). A DNA molecule therefore has domains with characteristic melting temperatures or Tm, determined by nucleotide sequence. DNA fragments almost identical in molecular weight, but that they also differ in a single nucleotide, they can be characterized by Tm and melting domains different from each other. The DGGE analysis is conducted on polyacrylamide gel containing a gradient denaturing in such a way that the dsDNA is subject, during the run, to an increase in denaturation conditions with consequent separation at the melting domains. In the upper part of the gel, where there are mild denaturation conditions, the melting domains at lower Tm begin to partially denature, creating branched molecules with less mobility. The increase in denaturation conditions along the polyacrylamide gel can determine the total dissociation of partially denatured fragments in single-stranded DNA (ssDNA). Experimentally, the complete dissociation of the two dsDNA strands is hindered by introducing, at the end of each filament, domains characterized by high contained in G + C and high Tm. G + C-rich regions are artificially created at one end of the dsDNA by means of incorporation of a GC-clamp during amplification reactions. The incorporation of the GC-clamp is made possible by the use of primers characterized by a sequence of about 30-40 GC at the 5 ‘end. The presence of the GC-clamp of the same sequence at the extremity of each molecule causes the differences between the stroke profiles of the analyzed fragments to be mainly determined by variations in the sequence of low melting domains. Since Tm is determined by the nucleotide sequence, the presence of a single mutation is capable of generating a different denaturation profile and, consequently, a different electrophoretic run. Hence the recurrence of polymorphisms in highly conserved genes can be analyzed by DGGE and can provide useful information to characterize the structure of microbial communities. In fact, with denaturing gradient gel electrophoresis an electrophoretic profile formed by a series of bands is obtained in which, as a first approximation, the number of bands is proportional to the number of species present and the position of each band is different for each species. The DGGE technique therefore provides a simple approach to obtaining microbial community profiles that can be used to identify spatial and temporal differences in the community structure or to monitor changes in structure that occur in response to environmental disturbances.

Real time PCR: It is a technique that allows to amplify and at the same time quantify a target DNA sequence. It involves the use of fluorescent dyes, such as Sybr Green that intercalate in the minor sulcus of the DNA double strand, or probes with specific sequences, consisting of oligonucleotides labeled with fluorescent agents. The emitted fluorescence is constantly measured and provides “real time” information on the amount of amplicon produced. From an amplification reaction a graph with a sigmoidal curve is obtained; this will start as soon as possible the greater the quantity of starting DNA and will continue to grow with an exponential trend until it reaches a maximum value (plateau), in which the reaction will slow down due to the exhaustion of the substrates.

In studying a Real Time PCR graph, three parameters are established:

– the fluorescence baseline or baseline region;

– the threshold line, parallel to the base line;

– the threshold cycle or CT, specific for each sample, identifies the value of the PCR cycle in which the exponential phase curve intersects the threshold line.

Most Real-Time PCR instruments are programmed to read the wavelengths of the SYBR Green emission and excitation spectrum (respectively 495nm and 537nm). This dye is very sensitive to light, it binds only to double-stranded DNA and therefore only to the newly synthesized amplicon. The samples are quantified on the basis of calibration curves obtained through the use of known quantities of 16 S rDNA gene copies. The comparison between the signal emitted by the unknown sample with the fluorescence values used for the construction of the calibration curve allows the quantification of a specific microbial species. In addition to the quantitative measurement of target bacteria, intercalators such as SYBR Green allow to distinguish amplicons of different lengths and to detect non-specific amplifications that may be present.

Next Generation Sequencing: The peculiarity of this technology introduced in 2006 consists not only in the ability to sequence a single DNA fragment at a time, extending this process to millions of fragments at the same time, but also in the ability to sequence DNA fragments in both directions.

The first step involves single-stranded DNA fragments, at the ends of which univocal sequences, called “index”, are loaded onto a flow of cells where they are captured on a surface containing “oligonucleotides still” complementary to the indexes, on which they are immobilized for the preparation of the libraries. The hybridization between the latter and the DNA fragments occurs through heating and cooling processes, followed by incubation with specific reagents and an isothermal polymerase. Through a “bridged” amplification each fragment is amplified distinctly from the others, creating a cluster of clones. When the cluster generation is complete, the generated models, after appropriate denaturation, are ready for actual sequencing.

Illumina uses a technology based on chain terminating fluorescent nucleotides with an OH at 3 ‘; this ensures that a single base per cycle is incorporated. An imaging step follows to identify the nucleotide incorporated in each cluster and a chemical step to remove the fluorescent group and terminal OH to allow the incorporation of another base in the next cycle.

At the end of the sequencing, which takes about 4 days, the sequence of each cluster is subjected to selection processes (trimming) to eliminate the low-quality products. During the data analysis the fragments of various lengths are aligned and superimposed, in this way it is possible to identify the sequence of the starting filament. In a standard procedure, at least 40-50 million sequences are analysed.

The shotgun sequencing is the sequence of all the genomes present in a complex matrix, such as a soil sample. Shotgun sequencing is therefore the most efficient way to sequence a large piece of DNA. For this, the starting DNA is broken up randomly into many smaller pieces, sort of in a shotgun fashion, with each of those pieces then sequenced individually. The resulting sequence reads generated from the different pieces are then analyzed by a computer program, looking for stretches of sequence from different reads that are identical with one another. When identical regions are identified, they are overlapped with one another, allowing the two sequence reads to be stitched together. This computer process is repeated over and over and over again, eventually yielding the complete sequence of the starting piece of DNA. The initial random fragmenting and reading of the DNA gave this approach the name “shotgun sequencing”.

Numerous biotic and abiotic stresses, such as the massive use of pesticides in agriculture and climate change, are compromising the survival of pollinating insects, with potentially harmful consequences on both agroecosystems and natural systems. In fact, bees are responsible for the pollination of 84% of cultivated plant species, 35% of which are of global importance and 78% of wild ones. Suffice it to say that 70% of seed crops alone (such as carrots, onions, garlic, etc.) are strictly dependent on insect pollination, as well as 80% of the 264 crop species of interest in Europe. From this, it follows that the activity of pollinating insects, including bees, plays an essential role at an economic level whose monetary estimate is about € 15 billion / year in Europe alone, while at worldwide the estimate grows to 153 billion €/year.

In addition to an incalculable value for the maintenance of biodiversity and balances present in the various ecosystems, then, bees supply honey, beeswax, propolis, pollen and royal jelly: in Europe, the data collected in 2010 showed a production of about 220 000 tons of honey with prices ranging from 1.50 to 40 €/kg depending on the area of ​​origin. Or, in Australia the production of honey and beeswax annually hovers around a commercial value of $ 90 million, underlining again the importance that the beekeeping sector plays in the panorama. world economy. Honeybee colonies have declined rapidly from 6 million in the 1940s to about 2.6 million today. High annual honeybee colony loss is still observed and has become the norm for beekeepers. Gut health plays a significant role in innate host immune response and adaptability to the multitude of stressors honeybees face today.

Broods affected by “Colony Collapse Disorder” (CCD), or “Hive Depopulation Syndrome” also showed significant signs of imbalance. The causes of this syndrome are not yet clear, but it is thought that they may be attributable to changes in environmental factors, malnutrition, the presence of pathogens and the massive use of insecticides. The symptomatology sees the presence of broods that abandon their larvae despite the presence of the queen, and lack of appetite for pollen and nectar stocks that are not consumed immediately (Fig. 13).

Fig. 13. Insights on Colony Collapse Disorder

One of the possible causes of this die-off may be related to gut microbiota dysbiosis, as microbial alteration in terms of quantity and composition. With this term we indicate the phenomenon that negatively affects the beneficial functions of the microbiota and that are associated with specific metabolic imbalances. In fact, these deficiencies could create serious problems on the development of young adults by affecting their ability to develop resistance genes, including those for the synthesis of vitellogenin, and by inhibiting the functions of the immune system, given the evidence that the same microbiota promotes its effectiveness (Fig. 14).

Fig. 14. Consequences of altered gut microbial compositions in bees

Few years ago, we tried to understand which factors are able to destabilize the microbiota, arriving at the conclusion that dysbiosis is caused by both biotic and abiotic factors. Considering biotic stresses, it has been seen that diet, the presence of specific pathogens and disorders (e.g., CCD) and adverse environmental conditions play a fundamental role. The lack of nutrients has a destructive impact on the normal development of the intestinal microbial flora, the consequence of which is to increase honeybee’s mortality, as well as increase the susceptibility to diseases and pathogens. Furthermore, the anomalous temperatures induce a state of stress in the hosts such as to have dramatic repercussions on the symbionts.

Considering abiotic stressors, the damage is almost entirely attributable to the use of insecticides, fungicides, acaricides and antibiotics. The bees, in fact, during foraging operations risk to ingest indirectly and to encounter the active ingredients, both on the main crops treated and on the neighboring ones subjected to drift. This could cause serious problems and imbalances in metabolism and immune defenses: in fact, there is the possibility that exposure to certain substances interferes with the ability of bees to regulate their microbial gut population.

For those reasons, one of the most innovative future prospect aim to understand the in-depth relationship between microorganisms and honeybees, in order to improve their dramatic lifespan and conditions.

Gut disbiosis: an example

Given the growing interest that public opinion is showing towards this product, the first objective analysis falls on the effect of Glyphosate (N-phosphonomethyl-glycine). It is a non-selective post-emergence systemic herbicide, therefore a total herbicide. Its mechanism of action interrupts the metabolic pathway responsible for the synthesis of phenylalanine, tyrosine and tryptophan, inhibiting the synthesis of 3-phosphoshikimate-1-carboxyvinyltransferase (EPSP synthase). This herbicide has always been seen as one of the least toxic products for animals, as they lack this metabolic pathway. Despite this, it has been shown that it can affect non-target organisms showing highly toxic effects towards earthworms, microalgae, aquatic bacteria, rhizosphere, and endophytes. Affecting the bacteria, then, it should be emphasized that the effects were also detected in intestinal microorganisms and symbionts of the fauna adjacent to agricultural areas, including bees.

Specifically, Motta et al. (2018) conducted a study aimed at characterizing the microbiota of bees exposed to Glyphosate, concluding that the absolute abundance of S. alvi, G. apicola, Lactobacillus sp. and Bifidobacterium sp. (Fig. 15)

Fig. 15. Analyses on the bee gut microbiota

has undergone a significant decrease. The product compromised the bacterial flora by stopping their growth without, however, directly killing them; it was therefore hypothesized that the effect fell on cell division during the early days of colonization. The bees that encountered the herbicide in the field, in fact, would have carried the active ingredient inside the hive which, being very stable and insoluble in water, would have been able to remain on the surfaces for a long time. Similarly, even in the field, persistence means that contamination can last for a long time. Inside the hive, therefore, the diffusion by trophallaxis and contact with other bees means that the product reaches the young larvae fed by adults, irreparably altering the development of beneficial symbiont species.

How is the microbiota acquired?

Fig. 16. The growth cycle of a bee

Natural bacteria picked up by honeybees from flowers while collecting nectar and pollen reside predominantly in honeybee midgut and hindgut. Gut bacteria naturally found in honeybees are dynamic. During development of the larvae (Fig. 16), bacterial population fluctuates. Larvae receive some bacteria from the nurse bees feeding them. During pupation, the gut lining is shed, and the gut of a newly emerging adult honeybee is sterile. The gut is quickly repopulated with characteristic microbiota. How does this happen? Main pathways are oral trophallaxis, interaction with hive material, and fecal-oral transmission. In particular, the characteristic microbiota of adult bees begins to develop about four days after the flicker.

Although the factors that allowed the evolution of the microbiota for every living being are still unknown, it is proven that social bees possess a distinctive microflora depending on the family they belong to. For example, it has been seen that analyzing the microbiota of different genera of eusocial corbiculate Apoidea such as Bombus spp., Megachile spp. and Apis spp., the main bacterial genera were recurrent (Snodgrassella spp., Gilliamella spp., Bifidobacterium spp. and Lactobacillus spp.) but the species varied in relation to the insect species. Hence the hypothesis that sees the microbiota as the result of a dynamic co-evolution between microorganisms and hosts, dependent on the environment and on the genotypic variations to which the species have been subjected over the centuries, whose richness is also correlated with the size of individual bees and entire colonies. In fact, the establishment of a species-specific microbial flora is the result of a long selection in which the optimal beneficial ratios have been established both for the microorganisms and for the hosts.

Who and where are they?

It has been estimated that within the intestines of adult worker bees there are about 1 billion bacterial cells, 95% of which are located, specifically, in the hindgut (Fig. 17). Here, a specific differentiation between ileus and rectum was noted; in the first three species of Proteobacteria such as G. apicola, F. perrara and S. alvi which form a dense biofilm in correspondence with the Malpighian tubes and which continues along the length of the ileum wall. In the rectum, however, a dense bacterial community prevails formed by three classes of Gram positive, such as Firmicutes (Firm-4, Firm- 5) and Bifidobacteria. As for the midgut, it has been seen that there are mainly Lactobacillus spp. and Acetobacteraceae, that is, those taxa that are also found in pollen, nectar and more generally within the hive. It can therefore be said that a pre-adapted microbiota does not exist in the midgut and that it varies in relation to the environment and the individual’s eating habits. In quantitative terms, then, here there is a much less abundant flora than in the rectum. The midgut also contains few bacteria, and those presents are more concentrated in the pro-ventricular area adjacent to the hindgut.

Fig. 17. The growth cycle of a bee

What are the gut microbiota functions?

In the last few years, the scientific community has begun to take an increasing interest in the role that the microbiota plays in the wellbeing of honeybees (Fig. 18). Numerous studies and research have shown that interactions with the host have supportive effects both at a metabolic and nutritional level and in terms of immune response to pathogens. As for food support, a balanced bacterial flora is necessary for a correct assimilation of nutrients as, thanks to its enzymatic activity, it participates in the degradation of complex sugars. In addition to being responsible for the presence of cellulases, hemicellulases and ligninolytic enzymes in the intestine useful for the digestion process of pollen grains, the richness of species is such as to allow the coexistence of different sugar catalysis pathways (especially for Gammaproteobacteria, Firmicutes and Bifidobacteriaceae). Indeed, it has been estimated that 91% of the protein transcripts linked to the digestion of plant macromolecules and to the fermentation phenomena of monomeric subunits are produced by bacteria. Another concrete example concerns pectin-lyases capable of degrading the pectins present in the cells of the wall of pollen grains. The latter is also an excellent indicator of the high genetic variability and adaptability within the same species of microorganisms. In fact, it has been seen that only some strains of G. apicola possess them while others are completely devoid of them. The importance of a greater digestive capacity and, consequently, the ability to metabolize nutrients that could not be demolished, has been demonstrated in various studies. Zheng et al. (2017), for example, by comparing bees with a normal microbiota and others without any type of intestinal flora, they highlighted appreciable physiological differences. In the former, the symbionts positively influenced the size of the intestine, the weight of individuals, the values ​​of vitellogenin and insulin and the sensitivity to sugars. These findings then suggested that the microbiota could influence the appetite and growth of the bees’ body through the increase of signals related to the presence of insulin.

In addition to the hydrolysis of complex carbohydrates, intestinal microorganisms produce useful metabolic substrates, such as vitamin B and other and short chains of fatty acids. For example, it has been seen that the genera Lactobacillus sp. and Bifidobacterium sp. are involved in the processes of fermentation of pollen and nectar so that they can be considered responsible for the vitaminic value of honey. The phenomenon of symbiosis with the host goes beyond nutritional and metabolic support: the microbiota plays an important role in supporting the immune system. In fact, in the first place, the bacteria could directly stimulate the production of the bee’s own defense molecules. Following the contact between the epithelial surface and the peptidoglycan (major component of the cell wall of Gram-positive bacteria), the immune system could activate the genes to produce 6 antimicrobial peptides such as: abaecin, hymenoptaecin, apidicin, defensin-1 and defensin-2. The production of these compounds, then, is accentuated by the alterations of the microbial membranes themselves and can also be induced by exposure to some pathogenic and non-pathogenic microorganisms. For example, Frischella perrara, a symbiont that colonizes the ileum region in the hindgut, above all stimulates the production of apidicin. Secondly, the microbiota may be directly responsible to produce antimicrobial compounds, which, among other things, is confirmed by numerous studies. Saraiva et al. (2015), for example, documented the presence of numerous genes involved in the biosynthesis of streptomycin and of secondary metabolites expressed by symbionts and which may play a role in maintaining the microbiota.

Fig. 18. Functions of the bee gut

What can we do?

From our and general experience in humans and animals, biotic and abiotic stresses could negatively affect the composition of the gut microbiota and therefore induce specific changes in the microorganism activities at gut level.

We must ask ourselves if any kind of microbiota modulation, by the administration of selected strains, could restore this perturbation, reduce bee mortality and/or improve honeybee health.

Probiotics are “live microorganisms that, when administered in adequate quantities, bring a benefit to the health of the host, excluding references to biotherapeutic agents and beneficial microorganisms not used in food” (FAO / WHO, 2001). Their administration, then, must not be associated with negative effects on organisms and the environment. The mode of action of these microorganisms can be summarized in the following functions:

• PROTECTIVE FUNCTION: dislocation of pathogens, competition for nutrients, competition with receptors and production of antimicrobial molecules (eg bacteriocins, organic acids …);
• STRUCTURAL FUNCTION: barrier effect, biofilm on the intestinal hair wall, development of the immune system;
• METABOLIC FUNCTION: differentiation and proliferation of intestinal epithelial cells, catalysis of carcinogenic substances present in the diet, synthesis of vitamins, fermentation of non-digestible sugars, ionic absorption, and energy saving.

However, it seems that the main mechanism of action of probiotics is the stimulation of the immune system: following cohesion with the intestinal wall, they are able to stimulate a series of cascade signals that activate the synthesis of antimicrobial peptides. In practice, they are able to carry out those tasks described previously that the bee’s microbiota naturally performs, proving to be a hypothetical optimal aid in safeguarding and optimizing it, as well as improving the bee’s life prospects.

As with human and animal nutrition, the main bacteria considered capable of these benefits are Lactobacillus spp., Bifidobacterium spp., Bacillus spp.

In conclusion, the use of probiotics has recently begun to be evaluated also within the hive itself. In fact, although bees prefer to consume fresh pollen, under certain conditions, such as seasonality, they need to supply themselves with the stored reserves. The humid environment (50-60% RH) that is created following the collection of pollen increases the risk of uncontrolled bacterial and, above all, fungal growth. There is a clear need to preserve the stocks inside the hive to avoid infections that could lead to fatal outcomes, such as calcified larvae due to Ascosphaera apis, or intestinal infections due to Nosema spp.