Environmental benefit from modern biotechnology and ICT applications
B A S I C L E V E L
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.
Contents
Renewable energy: biotechnology for biogas and bioethanol production
Recent updates on the legislation on renewable energy à 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. The 2030 climate and energy framework includes EU-wide targets and policy objectives for the period from 2021 to 2030. The key targets are:
– Reduce CO2 emissions (from 1990 levels) by 40%
– Increase renewable energy sources by 32%
– Improve energy efficiency by 32,5%
Biogas
Biogas is the mixture of gases produced by the breakdown of organic matter in the absence of oxygen (anaerobically), primarily consisting of methane and carbon dioxide. Biogas can be produced from raw materials such as agricultural waste, manure, municipal waste, plant material, sewage, green waste or food waste. Biogas is a renewable energy source (Fig. 1). Biogas is considered to be a renewable resource because its production-and-use cycle is continuous, and it generates no net carbon dioxide. As the organic material grows, it is converted and used. It then regrows in a continually repeating cycle. From a carbon perspective, as much carbon dioxide is absorbed from the atmosphere in the growth of the primary bio-resource as is released, when the material is ultimately converted to energy.
Biogas is produced by anaerobic digestion with methanogenic or anaerobic organisms, which digest organic materials inside a closed system, or fermentation of biodegradable materials. This closed system is called an anaerobic digester or a biodigester.
Biogas is primarily methane (CH4) and carbon dioxide (CO2) and may have small amounts of hydrogen sulfide (H2S), and moisture (Fig. 2). After purification from H2S and moisture, the biogas can be used to produce electricity and thermal energy. In this way, biogas can be used in a in a co-generation system (a kind of gas engine) to convert the energy in the gas into electricity and heat.
Methane from biogas (after elimination of CO2) can be used as a fuel and for any heating purpose. Biogas can therefore be cleaned and upgraded to natural gas standards, when it becomes bio-methane.
Biogas can be produced from a vast variety of raw materials (feedstocks). The biggest role in the biogas production process is played by microbes feeding on the biomass (details in the related power point). Materials suitable for biogas production include:
- biodegradable waste from enterprises and industrial facilities, such as surplus lactose from the production of lactose-free dairy products
- spoiled food from shops
- biowaste generated by consumers
- sludge from wastewater treatment plants
- manure and field biomass from agriculture
The material is typically delivered to the biogas plant’s reception pit by lorry or waste management vehicle. A delivery of solid matter such as biowaste will next undergo crushing to make its consistency as even as possible. At this point, water containing nutrients obtained from a further stage in the production process is also mixed with the feedstock to take the rate of solid matter down to only around one-tenth of the total volume.
This is also when any unwanted non-biodegradable waste, such as packaging plastic of out-of-date food waste from shops, is separated from the mixture. This waste is taken to a waste treatment facility where it is used to generate heat and electricity. Biomass that has passed through slurrification is combined with biomass delivered in the form of slurry to the biogas plant and pumped into the pre-digester tank where enzymes secreted by bacteria break down the biomass into an even finer consistency. A scheme of a biogas plant is presented in Fig. 3.
The residual solids and liquids created in biogas production are referred to as digestate. This digestate goes into a post-digester reactor and from there further into storage tanks. Digestates are well suited for uses such as fertilization of fields. Digestates can also be centrifuged to separate the solid and liquid parts.
Solid digestates is used as fertilizers in agriculture or in landscaping and can also be turned into gardening soil through a process of maturation involving composting.
Digestates are centrifuged to obtain process water for the slurrification of biowaste at the beginning of the process. This helps reduce the use of clean water. The centrifuged liquid is rich in nutrients, particularly nitrogen, that can be separated further using methods such as stripping technology and used as fertilizers or nutrient sources in industrial processes. Methane that can possibly be obtained from different by-products/waste is shown in Fig. 4.
Stages in biogas production. Anaerobic digestion is a multiple-stage process in which hydrolysis is one of the main steps. During hydrolysis the complex insoluble substrate macromolecules are hydrolysed into simpler and more soluble intermediates by bacteria.
A large number of microbial species, acting in concert, are capable of utilising organic substrates such as carbohydrates, proteins and lipids to produce volatile fatty acids (VFAs), which can be converted to methane and carbon dioxide by methanogenic microorganisms. Bacteria excrete enzymes that hydrolyse the particulate substrate to small transportable molecules, which can pass through the cell membrane. Once inside the cell, these simple molecules are used to provide energy and to synthesize cellular components. Polysaccharides are converted to simple sugars; hydrolysis of cellulose by cellulase enzymes yields glucose; hemicellulose degradation results in monosaccharides such as xylose, glucose, galactose, pentoses, arabinose and mannose, while starch is converted to glucose by amylase enzymes. A scheme of the process and examples of microorganisms involved is depicted below (Fig. 5).
Methanogens are Archea. They can live in different habitats and are an heterogeneous group of microorganisms. They lack cell nuclei and are therefore prokaryotes. Archaea were initially classified as bacteria, receiving the name archaebacteria (in the Archaebacteria kingdom), but this term has fallen out of use. Archaeal cells have unique properties separating them from the other two domains, Bacteria and Eukaryota. Archaea are further divided into multiple recognized phyla. Classification is difficult because most have not been isolated in a laboratory and have been detected only by their gene sequences in environmental samples. Archaea and bacteria are generally similar in size and shape, although a few archaea have very different shapes. Despite this morphological similarity to bacteria, archaea possess genes and several metabolic pathways that are more closely related to those of eukaryotes, notably for the enzymes involved in transcription and translation. Other aspects of archaeal biochemistry are unique, such as their reliance on ether lipids in their cell membranes. Archaea use more energy sources than eukaryotes: these range from organic compounds, such as sugars, to ammonia, metal ions or even hydrogen gas. Salt-tolerant archaea (the Haloarchaea) use sunlight as an energy source, and other species of archaea fix carbon, but unlike plants and cyanobacteria, no known species of archaea does both. Archaea reproduce asexually by binary fission, fragmentation, or budding; unlike bacteria, no known species of Archaea form endospores. The first observed archaea were extremophiles, living in extreme environments, such as hot springs and salt lakes with no other organisms. Improved molecular detection tools led to the discovery of archaea in almost every habitat, including soil, oceans, and marshlands. Archaea are particularly numerous in the oceans, and the archaea in plankton may be one of the most abundant groups of organisms on the planet. Archaea are a major part of Earth’s life. They are part of the microbiota of all organisms. In the human microbiome, they are important in the gut, mouth, and on the skin. Their morphological, metabolic, and geographical diversity permits them to play multiple ecological roles: carbon fixation; nitrogen cycling; organic compound turnover; and maintaining microbial symbiotic and syntrophic communities, methane formation.
Substrates for methanogenesis are a large array and they are listed below.
Fig. 6. Substrates for methanogenesis
Bioethanol
The use of bioethanol in Europe will be described and the main microorganisms used for bioethanol production (yeasts and bacteria) will be presented and their performances compared.
Fig. 7. Ethanol vs bioethanol production
Ethanol is widely used as a solvent, reagent, in the food industry and automotive fuel (Fig. 7 and 8). The production of ethanol by fermentation is based on the use of raw materials, microorganisms and technologies different from those used for the production of alcoholic beverages (wine and beer) in order to have the maximum yield in ethanol in the shortest time and with the lowest costs. 95% of the world actual ethanol production is BIOETHANOL (sugar fermentation process), whereas only 5% is manufactured by the chemical process of reacting ethylene with steam.
Fig. 8. Bioethanol use on the EU market
Bioethanol is the principle fuel used as a petrol substitute for road transport vehicles. Ethanol or ethyl alcohol (C2H5OH) is a clear colourless liquid, it is biodegradable, low in toxicity and causes little environmental pollution if spilt. Ethanol burns to produce carbon dioxide and water. Ethanol is a high octane fuel and has replaced lead as an octane enhancer in petrol. By blending ethanol with gasoline we can also oxygenate the fuel mixture so it burns more completely and reduces polluting emissions. Ethanol fuel blends are widely sold in the United States. The most common blend is 10% ethanol and 90% petrol (E10). Vehicle engines require no modifications to run on E10 and vehicle warranties are unaffected also. Only flexible fuel vehicles can run on up to 85% ethanol and 15% petrol blends (E85).
The main sources of sugar required to produce ethanol come from fuel or energy crops. These crops are grown specifically for energy use and include corn, maize and wheat crops (1st generation bioethanol), waste straw, willow and popular trees, forest and agricultural waste, residues from pulp production, solid urban waste (2nd generation bioethanol), third-generation bioethanol has been derived from algal biomass including microalgae and macroalgae (Fig. 9).
Fig. 9. First, second and thirg generation
Benefits of Bioethanol. Bioethanol has a number of advantages over conventional fuels. It comes from a renewable resource i.e. crops and not from a finite resource and the crops it derives from can grow well in Europe (like cereals, sugar beet and maize). Another benefit over fossil fuels is the greenhouse gas emissions. The road transport network accounts for 22% of all greenhouse gas emissions and through the use of bioethanol, some of these emissions will be reduced as the fuel crops absorb the CO2 they emit through growing. Also, blending bioethanol with petrol will help extend the life of the diminishing oil supplies and ensure greater fuel security, avoiding heavy reliance on oil producing nations. By encouraging bioethanol’s use, the rural economy would also receive a boost from growing the necessary crops. Bioethanol is also biodegradable and far less toxic that fossil fuels. In addition, by using bioethanol in older engines can help reduce the amount of carbon monoxide produced by the vehicle thus improving air quality. Another advantage of bioethanol is the ease with which it can be easily integrated into the existing road transport fuel system. In quantities up to 5%, bioethanol can be blended with conventional fuel without the need of engine modifications.
Biothanol can be produced from biomass by the hydrolysis and sugar fermentation processes. Biomass wastes contain a complex mixture of carbohydrate polymers from the plant cell walls as cellulose, hemi cellulose and lignin. In order to produce sugars from the biomass, the biomass is pre-treated with acids or enzymes in order to reduce the size of the feedstock and to disrupt the plant structure. The cellulose and the hemi cellulose portions are broken down (hydrolysed) by enzymes or dilute acids into sucrose sugar that is then fermented into ethanol. The lignin which is also present in the biomass is normally used as a fuel for the ethanol production plants boilers.
There are three principle methods of extracting sugars from biomass: concentrated acid hydrolysis, dilute acid hydrolysis and enzymatic hydrolysis. The first one works by adding 70-77% sulphuric acid to the biomass that has been dried to a 10% moisture content. The acid is added in the ratio of 1.25 acid to 1 biomass and the temperature is controlled to 50 °C. Water is then added to dilute the acid to 20-30% and the mixture is again heated to 100 °C for 1 hour. The gel produced from this mixture is then pressed to release an acid sugar mixture and a chromatographic column is used to separate the acid and sugar mixture. The dilute acid hydrolysis process is one of the oldest, simplest and most efficient methods of producing ethanol from biomass. Dilute acid is used to hydrolyse the biomass to sucrose. The first stage uses 0.7% sulphuric acid at 190 °C to hydrolyse the hemi cellulose present in the biomass. The second stage is optimised to yield the more resistant cellulose fraction. This is achieved by using 0.4% sulphuric acid at 215 °C. The liquid hydrolates are then neutralised and recovered from the process. Instead of using acid to hydrolyse the biomass into sucrose, we can use enzymes to break down the biomass in a similar way.
Corn, one of the most agricultural source to obtain ethanol, can be processed into ethanol by either the dry milling or the wet milling process. In the wet milling process, the corn kernel is steeped in warm water, this helps to break down the proteins and release the starch present in the corn and helps to soften the kernel for the milling process. The corn is then milled to produce germ, fibre and starch products. The germ is extracted to produce corn oil and the starch fraction undergoes centrifugation and saccharifcation to produce gluten wet cake. The ethanol is then extracted by the distillation process. The wet milling process is normally used in factories producing several hundred million gallons of ethanol every Year. The dry milling process involves cleaning and breaking down the corn kernel into fine particles using a hammer mill process. This creates a powder with a course flour type consistency. The powder contains the corn germ, starch and fibre. In order to produce a sugar solution the mixture is then hydrolysed or broken down into sucrose sugars using enzymes or a dilute acid. The mixture is then cooled and yeast is added in order to ferment the mixture into ethanol.
Sugar Fermentation Process
The hydrolysis process breaks down the cellulostic part of the biomass or corn into sugar solutions that can then be fermented into ethanol. Yeast is added to the solution, which is then heated. The yeast contains an enzyme called invertase, which acts as a catalyst and helps to convert the sucrose sugars into glucose and fructose.
Fig. 10. Yeast alcoholic fermentation
The main features of microorganism to be used in the industrial production of ethanol are:
– high yields of molar conversion of sugar (moles of ethanol produced/moles of sugar consumed)
– high production rate (moles of ethanol produced/time × volume of culture) mol/Lh or g/Lh
– high ethanol yields in weight (grams of produced ethanol/volumes of culture, ≥ 120 g/L) g/L
– high tolerance to ethanol
– as low as possible production of side products derived from side fermentations (e.g. glycerol)
Yeasts for bioethanol fermentation: Microorganisms such as yeasts play an essential role in bioethanol production by fermenting a wide range of sugars to ethanol. They are used in industrial plants due to valuable properties in ethanol yield (>90.0% theoretical yield), ethanol tolerance (>40.0 g/L), ethanol productivity (>1.0 g/L/h), growth in simple, inexpensive media and undiluted fermentation broth with resistance to inhibitors and retard contaminants from growth condition. As the main component in fermentation, yeasts affect the amount of ethanol yield. Saccharomyces cerevisiae is the most widely used yeast. Since thousands of years ago, S. cerevisiae have been used in alcohol production especially in the brewery and wine industries. It keeps the distillation cost low as it gives a high ethanol yield, a high productivity and can withstand high ethanol concentration. Nowadays, yeasts are used to generate fuel ethanol from renewable energy sources. Some yeast strains belonging to the species Pichia stipitis, S. cerevisiae and Kluyveromyces fagilis were reported as good ethanol producers from different types of sugars. S. cerevisiae tolerates a wide range of pH thus making the process less susceptible to infection. Baker’s yeast was traditionally used as a starter culture in ethanol production due to its low cost and easy availability. However, baker’s yeast and other S. cerevisiae strains were unable to compete with wild-type yeast which caused contamination during industrial processes. Stressful conditions like an increase in ethanol concentration, temperature, osmotic stress and bacterial contamination are the reasons why the yeast cannot survive during the fermentation. Flocculent yeasts were also used during biological fermentation for ethanol production as it facilitates downstream processing, allows operation at high cell density and gives higher overall productivity. It reduces the cost of cells recovery as it separates easily from the fermentation medium without centrifugation. There are common challenges to yeasts during sugar fermentation which are rise in temperature (35–45 °C) and ethanol concentration (over 20%). Yeasts growth rate and metabolism increase as the temperature increases until it reaches the optimum value. Increase in ethanol concentration during fermentation can cause inhibition to microorganism growth and viability. Inability of S. cerevisiae to grow in media containing high level of alcohol leads to the inhibition of ethanol production. The other problems in bioethanol fermentation by yeast are the ability to ferment pentose sugars. S. cerevisiae is the most commonly used in bioethanol production. However, it can only ferment hexoses but not pentoses. Only some yeasts from genera Pichia, Candida, Schizosaccharomyces and Pachysolen are capable of fermenting pentoses to ethanol. The efficiency of ethanol production on an industrial scale will be increased by using yeasts that are tolerant to inhibitors. The common challenges of yeasts can be overcome by using ethanol-tolerant and thermotolerant yeast. Ethanol-tolerant and thermotolerant strains which can resist stresses can be isolated from natural resources such as soil, water, plants and animals. Ethanol fermentation at high temperature is a beneficial process as it selects thermo-tolerant microorganisms and does not require cooling costs and cellulase. For example, K. marxianus is thermotolerant yeast which is capable of co-fermenting both hexose and pentose sugars and can survive the temperature of 42–45 °C.
Zymomonas mobilis for bioethanol fermentation: Zymomonas mobilis is a Gram negative, facultative anaerobic, non-sporulating, polarly-fagellated, rod-shaped bacterium. It is the only species of the genus Zymomonas. It has notable bioethanol-producing capabilities, which surpass yeast in some aspects. It was originally isolated from alcoholic beverages like the African palm wine, the Mexican pulque, and also as a contaminant of cider and beer (cider sickness and beer spoilage) in European countries. Zymomonas mobilis degrades sugars to pyruvate using the Entner-Doudoroff pathway. The pyruvate is then fermented to produce ethanol and carbon dioxide as the only products (analogous to yeast). The advantages of Z. mobilis over S. cerevisiae with respect to producing bioethanol are:
- higher sugar uptake and ethanol yield (up to 2.5 times higher),
- lower biomass production,
- higher ethanol tolerance up to 16% (v/v),
- does not require controlled addition of oxygen during the fermentation.
However, in spite of these attractive advantages, several factors prevent the commercial usage of Z. mobilis in cellulosic ethanol production. The foremost hurdle is that its substrate range is limited to glucose, fructose and sucrose. Wild-type Z. mobilis cannot ferment C5 sugars like xylose and arabinose which are important components of lignocellulosic hydrolysates. Unlike E. coli and yeast, Z. mobilis cannot tolerate toxic inhibitors present in lignocellulosic hydrolysates such as acetic acid and various phenolic compounds. Concentration of acetic acid in lignocellulosic hydrolysates can be as high as 1.5% (w/v), which is well above the tolerance threshold of Z. mobilis. Engineered Z. mobilis could overcome its inherent deficiencies and therefore expanded its substrate range to include C5 sugars like xylose and arabinose. Acetic acid resistant strains of Z. mobilis have been developed by rational metabolic engineering efforts, mutagenesis techniques or adaptive mutation. Mpreover, an extensive adaptation process was used to improve xylose fermentation in Z. mobilis. By adapting a strain in a high concentration of xylose, significant alterations of metabolism occurred. One noticeable change was reduced levels of xylitol, a byproduct of xylose fermentation which can inhibit the strain’s xylose metabolism. An interesting characteristic of Z. mobilis is that its plasma membrane contains hopanoids, pentacyclic compounds similar to Eukaryotic sterols. This allows it to have an extraordinary tolerance to ethanol in its environment, around 13%.
A comparison between production of bioethanol by S. cerevisiae and Z. mobilis is shown in Fig. 11.
Fig. 11. Comparison between different microbial agents operating the bioethanol
Biotechnology for the remediation of contaminated sites
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
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).
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).
STRENGTHS and WEAKNESSES of bioremediation technologies
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).
Test: LO7 Basic level
References
- Meegoda JN, Li B, Patel K, Wang LB. 2018. A review of the processes, parameters, and optimization of anaerobic digestion. International journal of environmental research and public health, 15(10): 2224
- Wang P, Wang H, Qiu Y, Ren L, Jiang B. 2018. Microbial characteristics in anaerobic digestion process of food waste for methane production–A review. Bioresource technology, 248: 29-36
- Azhar S HM, Abdulla R, Jambo SA, Marbawi H, Gansau JA, Faik AAM, Rodrigues KF. 2017. Yeasts in sustainable bioethanol production: A review. Biochemistry and Biophysics Reports, 10: 52-61
- Prasad RK, Chatterjee S, Mazumder PB, Gupta SK, Sharma S, Vairale MG, .Gupta DK. 2019. Bioethanol production from waste lignocelluloses: A review on microbial degradation potential. Chemosphere, 231: 588-606
- Sivagami K, Sakthivel KP, Nambi IM. 2018. Advanced oxidation processes for the treatment of tannery wastewater. Journal of environmental chemical engineering, 6(3): 3656-3663
- Krzeminski P, Tomei MC, Karaolia P, Langenhoff A, Almeida CMR, Felis E, Fatta-Kassinos D. 2019. Performance of secondary wastewater treatment methods for the removal of contaminants of emerging concern implicated in crop uptake and antibiotic resistance spread: A review. Science of the Total Environment, 648: 1052-1081
- Abatenh E, Gizaw B, Tsegaye Z, Wassie M. 2017. The role of microorganisms in bioremediation-A review. Open Journal of Environmental Biology, 2(1): 038-046
- Wan X, Lei M, Chen T. 2020. Review on remediation technologies for arsenic-contaminated soil. Frontiers of Environmental Science & Engineering, 14(2): 1-14.
- Meegoda JN, Li B, Patel K, Wang LB. 2018. A review of the processes, parameters, and optimization of anaerobic digestion. International journal of environmental research and public health, 15(10): 2224
- Wang P, Wang H, Qiu Y, Ren L, Jiang B. 2018. Microbial characteristics in anaerobic digestion process of food waste for methane production–A review. Bioresource technology, 248: 29-36
- Azhar S HM, Abdulla R, Jambo SA, Marbawi H, Gansau JA, Faik AAM, Rodrigues KF. 2017. Yeasts in sustainable bioethanol production: A review. Biochemistry and Biophysics Reports, 10: 52-61
- Prasad RK, Chatterjee S, Mazumder PB, Gupta SK, Sharma S, Vairale MG, .Gupta DK. 2019. Bioethanol production from waste lignocelluloses: A review on microbial degradation potential. Chemosphere, 231: 588-606
- Sivagami K, Sakthivel KP, Nambi IM. 2018. Advanced oxidation processes for the treatment of tannery wastewater. Journal of environmental chemical engineering, 6(3): 3656-3663
- Krzeminski P, Tomei MC, Karaolia P, Langenhoff A, Almeida CMR, Felis E, Fatta-Kassinos D. 2019. Performance of secondary wastewater treatment methods for the removal of contaminants of emerging concern implicated in crop uptake and antibiotic resistance spread: A review. Science of the Total Environment, 648: 1052-1081
- Abatenh E, Gizaw B, Tsegaye Z, Wassie M. 2017. The role of microorganisms in bioremediation-A review. Open Journal of Environmental Biology, 2(1): 038-046
- Wan X, Lei M, Chen T. 2020. Review on remediation technologies for arsenic-contaminated soil. Frontiers of Environmental Science & Engineering, 14(2): 1-14.
Environmental benefit from modern biotechnology and ICT applications
A D V A N C E D L E V E L
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.
Contents
Renewable energy: biotechnology for biogas and bioethanol production (level B)
Biogas
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. Anaerobic digestion has received a new attention in recent years since the energy crisis of the early 1970s, and especially following the Gulf war. The process involves the treatment of agricultural and industrial waste of varying types in the production of biogas. Interest in the anaerobic treatment of agro-industry waste is increasing because it is economical, has lower energy requirements and is ecologically sound, among several other advantages, compared with aerobic treatment processes. The process produces digested sludge, which is mainly used as fertilizer for crop production since the nutrients in the raw material remain in the mineralized sludge as accessible compounds. Treating waste to yield fuel while recycling nutrients constitutes a sustainable cycle.
Anaerobic digestion is a complex, natural, two-stage process of degradation of organic compounds through a variety of intermediates into methane and carbon dioxide, by the action of a consortium of microorganisms. The interdependence of the bacteria is a key factor in the anaerobic digestion process. In the first stage, the volatile solids in manure are converted into fatty acids by anaerobic bacteria known as “acid formers.” In the second stage, these acids are further converted into biogas by more specialized bacteria known as “methane formers.” The anaerobic digestion process, which has been at work in nature for millions of years, can be managed to convert a farmer’s often problematic waste-stream into an asset. Instability during both the start-up and operation of the anaerobic degradation process can be problematic due to the low specific growth rate of the methanogenic microorganisms involved.
Here we give some extra details about the reactors used for biogas production. Several parameters are known to be important for the development and management of a biogas producing plant. In particular:
the working temperature. The process can be performed in:
– psychrophilic conditions (20º C) (not much used in conventional plants)
– mesophilic conditions (35 – 42° C)
– thermophilic conditions (> 50° C);
Mesophilic digestion. The digester is heated to 30–35 °C and the feedstock remains in the digester typically for 15–30 days. Mesophilic digestion tends to be more robust and tolerant than the thermophilic process, but gas production is less, larger digestion tanks are required and sanitisation, if required, is a separate process stage.
Thermophilic digestion. The digester is heated to 55 °C and the residence time is typically 12–14 days. Thermophilic digestion systems offer higher methane production, faster throughput, better pathogen and virus ‘kill’, but require more expensive technology, greater energy input and a higher degree of operation and monitoring. During this process 30–60% of the digestible solids are converted into biogas.
Therefore, the process in thermophilic condition is faster, but mesophilic conditions are used when the characteristics of the feeding substrate(s) change with time, season, etc.
à the solid content in the reactor. We may distinguish:
– wet/humid processes (5 – 8% dry matter in the reactor)
– semi dry processes (dry matter = 8 – 20%)
– dry processes (dry matter >20%)
à the metabolic phases in the reactor.
– ONE PHASE: the entire microbial chain is kept in a single reactor;
– TWO PHASES: the hydrolytic fermentative phase is separated from the methanogenic one.
How does a biogas plant work? Please check the website: https://www.youtube.com/watch?v=3UafRz3QeO8
The following picture (Fig. 1) shows the different bioreactor configurations that can be developed to perform biogas production. They can differ for two parameters: the hydraulic scheme and the way microorganisms are working in the reactor (free or immobilized cells).
Fig. 1. Reactors for biogas production
The continuous stirred-tank reactor (CSTR) is a common model for a chemical reactor in environmental engineering. It is a batch reactor equipped with an impeller or other mixing device to provide efficient mixing. An ideal CSTR assumes perfect mixing. In a perfectly mixed reactor, the feeding is instantaneously and uniformly mixed throughout the reactor upon entry. Consequently, the performance is a function of residence time and reaction rate. The contact with the solid phase of the bioreactor can be improved by a sedimentation tank that separates the liquid medium from the solid part, which is then sent back to the bioreactor (referred to anaerobic contact process in the figure).
CSTRs consist of: a tank reactor (usually of constant volume), a stirring system to mix reactants (impeller or fast flowing introduction of reactants), feed and exit pipes to introduce reactants and remove products CSTR are commonly used in industrial processing. Biodigestors for biogas production are continuous agitated-tank reactors made of concrete or steel.
The anaerobic packed-bed reactor is filled with an inert support that provides a very large surface area for microbial growth. The influent passes through the media and anaerobic microbes attach themselves to the support creating a thin layer of anaerobic bacteria called biofilm—this film gives the digester its name, fixed film reactor or packed bed reactor. These microbes then continue to grow by removing material from the wastewater as it flows by. In most digesters the microbes are floating in the liquid and a portion of these active growing microorganisms are continuously discharged with the effluent. In a packed-bed digester the bacteria remain attached to the plastic support when effluent is discharged. Microorganisms are already “at work” when the new influent is added. Packed-bed digesters have smaller reactor vessels, shorter retention times and must be loaded with a feedstock that will readily flow through the media without clogging. Three to five day retention times are typical and digesters can be run at ambient temperatures in hot climates but are usually heated to mesophilic or thermophilic temperatures.
Which are the advantages of anaerobic packed-bed reactor? Increased stability and performance in anaerobic reactors can be achieved if the microbial consortium is retained in the reactor. Two means of achieving this are to use dense bacterial granula as in UASB reactors or a microbial biofilm attached to inert carriers in the above described packed-bed reactors. Upflow anaerobic sludge blanket (UASB) technology, normally referred to as UASB reactor, is indeed a form of anaerobic digester that is used for wastewater treatment and as a methanogenic (methane-producing) digester. A similar but variant technology to UASB is the expanded granular sludge bed (EGSB) digester (Fig. 2). An expanded granular sludge bed (EGSB) reactor is a variant of the UASB concept. The distinguishing feature is that a faster rate of upward-flow velocity is designed for the wastewater passing through the sludge bed. The increased flux permits partial expansion (and from this the name of the reactor is derived) of the granular sludge bed, improving wastewater-sludge contact as well as enhancing segregation of small inactive suspended particle from the sludge bed. The increased flow velocity is either accomplished by utilizing tall reactors, or by incorporating an effluent recycle (or both).
Fig. 2. Fixed bed/Expanded bed reactors (left) and UASB reactor
UASB is an anaerobic process that forms a blanket of granular sludge which suspends in the tank. Wastewater flows upwards through the blanket and is processed by the anaerobic microorganisms. The upward flow combined with the settling action of gravity suspends the blanket with the aid of flocculants. The blanket begins to reach maturity at around three months. Small sludge granules begin to form and they contain organic matter and bacteria without any support matrix, the flow conditions create a selective environment in which only those microorganisms capable of attaching to each other survive and proliferate. Eventually the aggregates form dense compact structures referred to as “granules”. Biogas with a high concentration of methane is produced, and this may be captured and used as an energy source, to generate electricity for export and to cover its own running power. The technology needs constant monitoring when put into use to ensure that the sludge blanket is maintained, and not washed out (thereby losing the effect). The heat produced as a by-product of electricity generation can be reused to heat the digestion tanks. The packing medium in the packed-bed reactor and the granular sludge in the UASB reactor serve as a filter preventing bacterial washout and also providing a larger surface area for faster biofilm development and improved methanogenesis. Specific surface area, porosity, surface roughness, pore size, and orientation of the packing material were found to play an important role in anaerobic reactor performance. Biofilm or fixed-film reactors depend on the natural tendency of mixed microbial populations to adsorb onto surfaces and to form a biofilm. Many carrier materials have been investigated regarding their suitability as supports for biofilm, including cheap, readily available materials like sand, clay, glass, quartz and a number of plastics. In nature, microorganisms inhabit the outer and inner surfaces of stone, gravel or sand. This biofilm formation becomes an important factor for water self-cleaning ability. The growth of microorganism in a biofilm is the basis for biological water treatment such as denitrification and for intensification of aerobic and anaerobic wastewater treatment. The use of packed-bed reactors to treat different kinds of wastewater has also been reported, for example, dairy and brewery wastewater. The biofilm formation on carrier materials improves the conversion rates by reducing its sensitivity toward concentration variations and inhibiting substance. The efficiency of removing organic matter in fixed-bed reactors is directly related to the characteristics of the support material used for immobilization of anaerobes. Reticular polyurethane foam has a high specific surface area. It is an excellent colonization matrix for an anaerobic filter reactor. Pore size was one of the most important parameter for microbiological and engineering requirements in high-efficiency beds. Many kinds of bedding model have been considered for degrading a variety of organic wastes in anaerobic digestion reactors.
The development of fixed biomass reactors has ensured that significant advances in the knowledge and application of anaerobic processes for waste treatment have taken place. Compared to conventional units, fixed film bioreactors perform efficiently at higher organic loading rates, due to more effective biomass retention in the reaction zone resulting in higher cellular retention times. Immobilized biomass anaerobic reactors also show better responses to organic shock loads and toxic inputs. In many cases, immobilized biomass reactors completely recover their performance after such troubles
How does a UASB reactor work? Please have a look at this video https://www.youtube.com/watch?v=0QsEdlJgllI
A useful digestion output of the anaerobic digestion process is digestate. Digestate is the remaining part of the degraded biomass after biogas production: it is stable organic matter rich in various nutrients (N, P, K). Depending on the feedstock used for biogas production, digestate can be directly usable as organic fertiliser in the same way raw animal slurries are spread on fields in agriculture. It can also be further upgraded to recover high quality mineral nutrients. Digestate use as organic fertiliser displays multiple advantages: it allows reuse of nutrients and substitutes mineral fertiliser of fossil origin. Compared to raw manure, digestate is also sanitised thanks to the biogas production process neutralising most of the pathogens of the original feedstock such as bacteria and crop diseases. Digestate homogeneity and density also allow for faster penetration in the soil compared to raw manure, making nutrients more easily accessible to plants in the soil. If unfit for agricultural purposes, digestate can be further processed and used as a raw material for industrial processes.
An overall scheme of a biogas producing plant is presented below (Fig. 3).
Fig. 3. A biogas producing plant.
Bioethanol
Countries worldwide have considered and directed policies toward the increased and economic utilization of biomass for meeting their future energy demands in order to meet carbon dioxide reduction targets as specified in the Kyoto Protocol as well as to decrease reliance and dependence on the supply of fossil fuels. Although biomass can be a huge source of transport fuels such as bioethanol, biomass is commonly used to generate both power and heat, generally through combustion. Ethanol is at present the most widely used liquid biofuel for motor vehicles. The importance of ethanol is increasing due to a number of reasons such as global warming and climate change.
The global market for bioethanol has entered a phase of rapid, transitional growth. Many countries around the world are shifting their focus toward renewable sources for power production because of depleting crude oil reserves. The trend is extending to transport fuel as well. Ethanol has potential as a valuable replacement of gasoline in the transport fuel market. However, the cost of bioethanol production is more compared to fossil fuels. Brazil and the USA are the two major ethanol producers accounting for 62% of the world production. Large scale production of fuel ethanol is mainly based on sucrose from sugarcane in Brazil or starch, mainly from corn, in the USA. Please see a schematic figure below (Fig. 4)
Fig. 4. Schematic production of bioethanol from sugar crops
Current ethanol production based on corn, starch and sugar substances may not be desirable due to their food and feed value. Cost is an important factor for large scale expansion of bioethanol production. The green gold fuel from lignocellulosic wastes avoids the existing competition of food versus fuel caused by grain-based bioethanol production. Hence bioethanol production could be the route to the effective utilization of agricultural wastes. Rice straw, wheat straw, corn straw, and sugarcane bagasse are the major agricultural wastes in terms of quantity of biomass available.
Lignocellulosic materials are renewable, low cost and are abundantly available. It includes crop residues, grasses, sawdust, wood chips, etc. Extensive research has been carried out on ethanol production from lignocellulosics. Lignocellulosics are processed for bioethanol production through three major operations:
- pretreatment for delignification is necessary to liberate cellulose and hemicellulose before hydrolysis;
- hydrolysis of cellulose and hemicellulose to produce fermentable sugars including glucose, xylose, arabinose, galactose, mannose and fermentation of reducing sugars.
- The non-carbohydrate components of lignin also have value added applications
The most important processing challenge in the production of biofuel is pretreatment of the biomass. Lignocellulosic biomass is composed of three main constituents namely hemicellulose, lignin and cellulose. Pre-treatment methods refer to the solubilization and separation of one or more of these components of biomass. It makes the remaining solid biomass more accessible to further chemical or biological treatment. The lignocellulosic complex is made up of a matrix of cellulose and lignin bound by hemicellulose chains. The pretreatment is done to break the matrix in order to reduce the degree of crystallinity of the cellulose and increase the fraction of amorphous cellulose, the most suitable form for enzymatic attack. Pretreatment is undertaken to bring about a change in the macroscopic and microscopic size and structure of biomass as well as submicroscopic structure and chemical composition. It makes the lignocellulosic biomass susceptible to quick hydrolysis with increased yields of monomeric sugars.
The goals of an effective pretreatment process are:
- formation of sugars directly or subsequently by hydrolysis to avoid loss and/or degradation of sugars formed
- to limit formation of inhibitory products
- to reduce energy demands and minimize costs.
Physical, chemical, physicochemical and biological treatments are the four fundamental types of pretreatment techniques employed. In general, a combination of these processes is used in the pretreatment step.
Among physical pretreatment, the first step for ethanol production from agricultural solid wastes is the mechanical size reduction through milling, grinding, or chipping. This reduces cellulose crystallinity and improves the efficiency of downstream processing. Pyrolysis is a physical treatment: the materials are treated at a temperature higher than 300 °C, whereby cellulose rapidly decomposes to produce gaseous products and residual char. The residual char is further treated by leaching with water or with mild acid. The water leachate contains enough carbon source to support microbial growth for bioethanol production. Glucose is the main component of water leachate. Pretreatment of lignocellulosic biomass in a microwave oven is also a feasible method which uses the high heating efficiency of a microwave oven. Microwave treatment utilizes thermal and non-thermal effects generated by microwaves in aqueous environments. Heat is generated in the biomass by microwave radiation, resulting from the vibrations of the polar bonds in the biomass and the surrounding aqueous medium. This unique heating feature results in an explosion effect among the particles and improves the disruption of recalcitrant structures of lignocellulose. In the non-thermal method, i.e., the electron beam irradiation method, polar bonds vibrate, as they are aligned with a continuously changing magnetic field and the disruption and shock to the polar bonds accelerates chemical, biological and physical processes.
Among physicochemical treatments, steam explosion is a promising one making biomass more accessible to cellulase attack. This method of pretreatment does not use any catalyst and the biomass fractionates to yield levulinic acid, xylitol and alcohols. In this method the biomass is heated using high-pressure steam (20–50 bar, 160–290 °C) for a few minutes; the reaction is then stopped by sudden decompression to atmospheric pressure. When steam is allowed to expand within the lignocellulosic matrix it separates the individual fibers. The high recovery of xylose (45–65%) makes steam-explosion pretreatment economically attractive.
Chemical pretreatment methods involve the usage of dilute acid, alkali, ammonia, organic solvent, CO2 or other chemicals. These methods are easy in operation and have good conversion yields in short span of time. Acid pretreatment is considered as one of the most important techniques and aims for high yields of sugars from lignocellulosics. It is usually carried out by concentrated or diluted acids (usually between 0.2% and 2.5% w/w) at temperatures between 130 °C and 210 °C. The acid medium attacks the polysaccharides, especially hemicelluloses which are easier to hydrolyze than cellulose. However, acid pretreatment results in the production of various inhibitors like acetic acid, furfural and 5- hydroxymethylfurfural. These products are growth inhibitors of microorganisms. Hydrolysates to be used for fermentation therefore need to be detoxified. Alkaline pretreatment of lignocellulosics digests the lignin matrix and makes cellulose and hemicellulose available for enzymatic degradation. Alkali treatment of lignocellulose disrupts the cell wall by dissolving hemicelluloses, lignin, and silica, by hydrolyzing uronic and acetic esters, and by swelling cellulose. Crystallinity of cellulose is decreased due to swelling. By this process, the substrates can be fractionated into alkali-soluble lignin, hemicelluloses, and residue, which makes it easy to utilize them for more valuable products. The end residue (mainly cellulose) can be used to produce either paper or cellulose derivatives. Organic solvent are alternative methods for the delignification of lignocellulosic materials. The utilization of organic solvent/water mixtures eliminates the need to burn the liquor and allows the isolation of the lignins (by distillation of the organic solvent). Examples of such pretreatments include the use of 90% formic acid and that of pressurized carbon dioxide in combination (50% alcohol/water mixture and 50% carbon dioxide). Other various organic solvents which can be used for delignification are methanol, ethanol, acetic acid, performic acid and peracetic acid, acetone, etc.
Biological treatments. Enzymatic hydrolysis is the preferred saccharification method because of its higher yields, higher selectivity, lower energy cost and milder operating condition than chemical processes.
Different mode of fermentation. Fermentation of bioethanol can be carried out in batch, fed-batch, repeated batch, or continuous mode. In batch process, substrate is provided at the beginning of the process without addition or removal of the medium. It is known as the simplest system of bioreactor with flexible and easy control process. The fermentation process is carried out in a closed-loop system with high sugars concentration at the beginning and ends with high product concentration. There are several benefits of batch system including complete sterilization, does not require labour skills, it is easy to manage the feedstocks, and can be controlled easily. However, the productivity is low and needs intensive and high labour costs. The presence of high sugar concentration in the fermentation medium may lead to substrate inhibition of cell growth and ethanol production. Cells recycle batch fermentation is a strategic method for effective ethanol production as it reduces time and cost for inoculum preparation. The other advantages of repeated-batch process are easy cell collection, stable operation, and long-term productivity. Sugar materials and immobilized yeast cells are used to facilitate cell separation for cell recycling. However, its application in the process of lignocellulosic materials is extremely difficult because lignocelluosic residue remain in the fermentation medium together with yeast cells. The use of free cells in this system reduces yeast cell concentration and results in lower ethanol production in the subsequent batches. Repeated-batch fermentation can be performed by replacing free cells with the immobilized cells. Fed-batch fermentation is a combination of batch and continuous mode which involves the addition of substrate into the fermenter without removing the medium. It has been used to overcome the problem of substrate inhibition in batch operation. Volume of culture in fed-batch processes can vary widely but it must be fed properly at certain rate with the right component composition. Productivity of fed-batch fermentation can be increased by maintaining substrate at low concentration which allows the conversion of sufficient amount of fermentable sugars to ethanol. This process has higher productivity, higher dissolved oxygen in medium, shorter fermentation time and lower toxic effect of the medium components compared to other types of fermentation. However, ethanol productivity in fed-batch is limited by feed rate and cell mass concentration.
Continuous operation is carried out by constantly adding substrates, culture medium and nutrients into a bioreactor containing active microorganisms. Culture volume in continuous operation must be constant and the fermentation products are taken continuously from the media. Various type of products can be obtained from the top of the bioreactor such as ethanol, cells and residual sugar. The advantages of continuous system over batch and fed-batch system are higher productivity, smaller bioreactor volumes and less investment and operational costs. At high dilution rate, ethanol productivity is increased while ethanol yield is decreased due to incompletely substrate consumption by yeasts. However, the possibility for contamination to occur is higher than other types of fermentation. Moreover, the ability of yeasts to produce ethanol in continuous process are reduced due to long cultivation time.
Factors affecting bioethanol production
Several factors influence the production of bioethanol: temperature, sugar concentration, pH, fermentation time, agitation rate, and inoculum amount. The growth rate of the microorganisms is directly affected by the temperature. High temperature which is unfavorable for cells growth becomes a stress factor for microorganisms. The ideal temperature range for fermentation is between 20 and 35 °C for Saccharomyces cerevisiae. Free cells of S. cerevisiae have an optimum temperature near 30 °C whereas immobilized cells have slightly higher optimum temperature due to its ability to transfer heat from particle surface to inside the cells. Moreover, enzymes which regulate microbial activity and fermentation process are sensitive to high temperature which can denature its tertiary structure and inactivates the enzymes. Thus, temperature is carefully regulated throughout the fermentation process.
The increase in sugar concentration up to a certain level caused fermentation rate to increase. However, the use of excessive sugar concentration will cause steady fermentation rate. This is because the concentration of sugar use is beyond the uptake capacity of the microbial cells. Generally, the maximum rate of ethanol production is achieved when using sugars at the concentration of 150 g/L. The initial sugar concentration also has been considered as an important factor in ethanol production. High ethanol productivity and yield in batch fermentation can be obtained by using higher initial sugar concentration. However, it needs longer fermentation time and higher recovery cost.
Ethanol production is influenced by pH of the broth as it affects bacterial contamination, yeast growth, fermentation rate and by-product formation. The permeability of some essential nutrients into the cells is influenced by the concentration of H+ in the fermentation broth. Moreover, the survival and growth of yeasts is influenced by the pH in the range of 2.75–4.25. In fermentation for ethanol production, the optimum pH range of S. cerevisiae is 4.0–5.0 [34]. When pH is lower than 4.0, a longer incubation period is required but the ethanol concentration is not reduced significantly. However, when then pH was above 5.0, the concentration of ethanol reduces substantially.
Fermentation time affects the growth of microorganisms. Shorter fermentation time causes inefficient fermentation due to inadequate growth of microorganisms. On the other hand, longer fermentation time gives toxic effect on microbial growth especially in batch mode due to the high concentration of ethanol in the fermented broth. Complete fermentation can be achieved at lower temperature by using longer fermentation time which results in lowest ethanol yield.
Agitation rate controls the permeability of nutrients from the fermentation broth to inside the cells and removal of ethanol from the cell to the fermentation broth. The greater the agitation rate, the higher the amount of ethanol produced. Besides, it increases the amount of sugar consumption and reduces the inhibition of ethanol on cells. The common agitation rate for fermentation by yeast cells is 150–200 rpm. Excess agitation rate is not suitable for smooth ethanol production as it causes limitation to the metabolic activities of the cells.
Inoculum concentration does not give significant effects on the final ethanol concentration, but it affects the consumption rate of sugar and ethanol productivity.
Biotechnology for bioplastic production (level B)
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.
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.
Biotechnology for the remediation of contaminated sites
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”.
Microbial technologies for honeybee’s health
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.
Test: LO7 Advanced Level
References
- EFSA (2009). Bee mortality and bee surveillance in Europe.CFP/EFSA/AMU/2008/02
- Motta EVS, Raymann K, Moran NA. 2018. Glyphosate perturbs the gut microbiota of honeybees. Proc Natl Acad Sci USA, pp. 1-6
- Zheng H, Powell JE, Steele MI, Dietrich C, Moran NA. 2017. Honeybee gut microbiota promotes host weight gain via bacterial metabolism and hormonal signaling. Proc Natl Acad Sci USA, 114: 4775-4780
- Saraiva MA, Zemolin APP, Franco JL, Boldo JT, et al. 2015. Relationship between honeybee nutrition and their microbial communities. Antoine Van Leeuwenhoek, 107: 921-933
- FAO/WHO (2001). Health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria. Food and Agriculture Organization of the United States, World Health Organization
- Kainthola J, Kalamdhad AS, Goud VV. 2019. A review on enhanced biogas production from anaerobic digestion of lignocellulosic biomass by different enhancement techniques. Process Biochemistry, 84: 81-90
- Shamurad B, Sallis P, Petropoulos E, Tabraiz S, Ospina C, Leary P, et al. 2020. Stable biogas production from single-stage anaerobic digestion of food waste. Applied Energy, 263: 114609
- Kumar M, Dutta S, You S, Luo G, Zhang S, Show PL, et al. 2021. A critical review on biochar for enhancing biogas production from anaerobic digestion of food waste and sludge. Journal of Cleaner Production, 127143
- Azhar, S. H. M., Abdulla, R., Jambo, S. A., Marbawi, H., Gansau, J. A., Faik, A. A. M., & Rodrigues, K. F. (2017). Yeasts in sustainable bioethanol production: A review. Biochemistry and Biophysics Reports, 10, 52-61.
- Galbe M, Zacchi G. 2007. Pretreatment of lignocellulosic materials for efficient bioethanol production. Biofuels, 41-65.
- Kim S, Dale BE. 2004. Global potential bioethanol production from wasted crops and crop residues. Biomass and bioenergy, 26(4): 361-375.
- Vilpoux O, Averous L. 2004. Starch-based plastics. Technology, use and potentialities of Latin American starchy tubers, 521-553.
- Sin LT. 2012. Polylactic acid: PLA biopolymer technology and applications. William Andrew.
- Koh JJ, Zhang X, He C. 2018. Fully biodegradable Poly (lactic acid)/Starch blends: A review of toughening strategies. International journal of biological macromolecules, 109: 99-113.
- Poltronieri P, Kumar P. 2017. Polyhydroxyalkanoates (PHAs) in industrial applications. Handbook of Ecomaterials. Cham: Springer International Publishing, 1-30.
- Yu CJ, Wan YJ, Yowanto H, et al. 2001. Electronic detection of single-base mismatches in DNA with ferrocene-modified probes. J Am Chem Soc, 123:11155–61.
- Yu X, Kim SN, Papadimitrakopoulos F, et al. 2005. Protein immunosen- sor using single-wall carbon nanotube forests with electrochemical detection of enzyme labels. Mol Biosyst, 1:70–8.
- EFSA (2009). Bee mortality and bee surveillance in Europe.CFP/EFSA/AMU/2008/02
- Motta EVS, Raymann K, Moran NA. 2018. Glyphosate perturbs the gut microbiota of honeybees. Proc Natl Acad Sci USA, pp. 1-6
- Zheng H, Powell JE, Steele MI, Dietrich C, Moran NA. 2017. Honeybee gut microbiota promotes host weight gain via bacterial metabolism and hormonal signaling. Proc Natl Acad Sci USA, 114: 4775-4780
- Saraiva MA, Zemolin APP, Franco JL, Boldo JT, et al. 2015. Relationship between honeybee nutrition and their microbial communities. Antoine Van Leeuwenhoek, 107: 921-933
- FAO/WHO (2001). Health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria. Food and Agriculture Organization of the United States, World Health Organization
- Kainthola J, Kalamdhad AS, Goud VV. 2019. A review on enhanced biogas production from anaerobic digestion of lignocellulosic biomass by different enhancement techniques. Process Biochemistry, 84: 81-90
- Shamurad B, Sallis P, Petropoulos E, Tabraiz S, Ospina C, Leary P, et al. 2020. Stable biogas production from single-stage anaerobic digestion of food waste. Applied Energy, 263: 114609
- Kumar M, Dutta S, You S, Luo G, Zhang S, Show PL, et al. 2021. A critical review on biochar for enhancing biogas production from anaerobic digestion of food waste and sludge. Journal of Cleaner Production, 127143
- Azhar, S. H. M., Abdulla, R., Jambo, S. A., Marbawi, H., Gansau, J. A., Faik, A. A. M., & Rodrigues, K. F. (2017). Yeasts in sustainable bioethanol production: A review. Biochemistry and Biophysics Reports, 10, 52-61.
- Galbe M, Zacchi G. 2007. Pretreatment of lignocellulosic materials for efficient bioethanol production. Biofuels, 41-65.
- Kim S, Dale BE. 2004. Global potential bioethanol production from wasted crops and crop residues. Biomass and bioenergy, 26(4): 361-375.
- Vilpoux O, Averous L. 2004. Starch-based plastics. Technology, use and potentialities of Latin American starchy tubers, 521-553.
- Sin LT. 2012. Polylactic acid: PLA biopolymer technology and applications. William Andrew.
- Koh JJ, Zhang X, He C. 2018. Fully biodegradable Poly (lactic acid)/Starch blends: A review of toughening strategies. International journal of biological macromolecules, 109: 99-113.
- Poltronieri P, Kumar P. 2017. Polyhydroxyalkanoates (PHAs) in industrial applications. Handbook of Ecomaterials. Cham: Springer International Publishing, 1-30.
- Yu CJ, Wan YJ, Yowanto H, et al. 2001. Electronic detection of single-base mismatches in DNA with ferrocene-modified probes. J Am Chem Soc, 123:11155–61.
- Yu X, Kim SN, Papadimitrakopoulos F, et al. 2005. Protein immunosen- sor using single-wall carbon nanotube forests with electrochemical detection of enzyme labels. Mol Biosyst, 1:70–8.