Solar technologies for plant microbial pathogens inactivation on water

Science against microbial pathogens: communicating current research and technological advances ______________________________________________________________________________ A. Méndez-Vilas (Ed.)

Solar technologies for plant microbial pathogens inactivation on water M. I. Polo-López, I. García-Fernández and P. Fernández-Ibáñez* Plataforma Solar de Almería – CIEMAT, P.O. Box 22, 07200 Tabernas, Almería, Spain. According to the United Nations Food and Agriculture Organization (FAO), agriculture consumes 70% of fresh water used worldwide. In developing countries, this increases to over 95% of available fresh water. The water used for crops averages around 1000–3000 m3 per ton of cereal harvested, or in other words, 1-3 tonnes of water are used to grow 1 kg of cereal. Bearing in mind that the daily drinking-water requirement per person is only 2-4 litres, it is often forgotten that it still takes 2000 to 5000 litres of water to produce a person’s daily food requirement. The standard methods used for water disinfection are well known: chlorination, ozonation and UV-C lamps. Chlorine is a very effective disinfectant for most microorganisms like bacteria and viruses, but protozoa like Cryptosporidium, Giardia and Acanthamoeba, are highly resistant to chlorine, present a high risk of infection and are extremely persistent in water supply systems. These protozoa, as well as Campylobacter jejuni, Campylobacter coli, Yersinia enterocolitica, Pseudomonas aeruginosa, have been successfully inactivated by solar and solar photocatalytic disinfection. Furthermore, the main drawback of chlorine is the appearance of organohalides, especially trihalomethanes (THMs), which are toxic or potentially carcinogenic, as disinfection by-products in chlorinated drinking water. Such significant resistance and other findings have led to severe criticism of its use in drinking water and even in irrigation water. Research seeking alternatives to chlorine as a general disinfectant and especially for drinking water disinfection, is therefore necessary to solve the above mentioned limitations and issues. Any such solution will have to take into account many factors including: i) low cost, ii) low power consumption, iii) sustainability, and iv) absence of negative effects on health, and taste. It is well known that solar inactivation of microbial cells occurs through a variety of mechanisms. Sunlight in water induces a series of photooxidative processes as well as the generation of reactive oxygen species (ROS), which eventually damage most cells. During the last 20 years, research on heterogeneous photocatalysis for water disinfection has been increasing. This chapter is an overview of the use of sunlight to disinfect water contaminated by plant pathogens using photocatalytic processes promoted by solar photons like TiO2/UVzA, or photo-induced reactions like H2O2/sunlight. The experience of our group in testing solar reactors during recent years at the Plataforma Solar de Almería (Spain) is also reviewed. Keywords: Solar reactors, solar radiation, photocatalysis, water disinfection.

1. Introduction Water scarcity and lack of access to safe water, already a serious global problem, will become critical in the coming decades. Problems associated with water scarcity, and the gradual destruction and contamination of fresh water resources are gaining importance in many areas of the planet, causing concern even in countries which, so far, have not experienced such problems. The most important issue in water is, of course, disinfection of drinking water. According to the World Health Organization (WHO) and UNICEF, polluted drinking water and lack of sanitation are responsible for the deaths of approximately 4500-5000 children every day, and one billion people still lack access to safe drinking water [1]. The second most critical issue is the disinfection of water for agriculture. According to the United Nations Food and Agriculture Organization (FAO), agriculture consumes 70% of fresh water used worldwide. In developing countries, this increases to over 95% of the available fresh water. 80% of land cultivated worldwide is today still exclusively rainfed and supplies over 60% of the world’s food. Irrigation could triple or quadruple this production. However, the FAO does predict a sharp increase in irrigation replacing rainfed agriculture [2]. Stored rainwater or surface water used for irrigation accumulates phytopathogens like bacteria and fungi. Recovery, purification and reuse of wastewater and surface water may be a good approach for reducing pressure on fresh water resources. This is especially of interest in arid and semi-arid areas of the world; where irrigation of crops could be done with reused water that fulfils a number of quality and safety prerequisites. Water disinfection is mainly defined as the destruction of microorganisms causing diseases and epidemics, like cholera and typhoid fever. The mechanism involved is most commonly explained as the destruction of the organism protein structure and inhibition of enzymatic activities [3]. This definition leads to their general resistance to widelyused high-level disinfectants such as ozone and chlorine compounds. The most resistant infectious agents include prions, followed by coccidian (Cryptosporidium) and bacterial spores (Bacillus), mycobacteria (Mycobacterium tuberculosis), viruses (poliovirus), fungi (Aspergillus), and finally Gram-negative (Pseudomonas) and Gram-positive bacteria (Enterococcus). This resistance is decisively determined by cell wall permeability to the specific disinfectant, although size and complexity of the microorganism also influence its resistance.

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Science against microbial pathogens: communicating current research and technological advances _______________________________________________________________________________ A. Méndez-Vilas (Ed.)

Existing drinking water pretreatments, i.e., coagulation, flocculation, and sedimentation, remove a maximum of 90% of bacteria, 70% of viruses and 90% of protozoa. Filtration for drinking-water treatment (e.g. granular, slow sand, precoat and membrane filtration) with proper design and adequate operation can be a consistent and effective barrier for microbial pathogens leading to approx. 99% bacteria removal. For highly resistant microorganisms, filtration in combination with chlorine is recommended, although some pathogens, such as Cryptosporidium, are highly resistant to chlorine and UV-C [4]. Nonchemical disinfection methods are rare in agriculture. Chemical additives, also called pesticides, may be used to combat phytopathogens in water and soil, but they have a number of disadvantages, such as resistance of some pathogens to pesticides and phytotoxicity. Chlorine has been extensively used in drinking water and agriculture. Nevertheless, chlorine leads to the generation of disinfection by-products which are hazardous to both humans and plants. Chlorine also gives an unpleasant taste to drinking water and some pathogens are resistant to it. Hydrogen peroxide has commonly been used as a disinfectant in agriculture, but it rapidly becomes phytotoxic at doses over 50 mg/L, as is the case in hydroponic cultures. In some cases, hydrogen peroxide has been combined with germicidical UV-C radiation or ozone to enhance its disinfectant action [5]. Both techniques are in use, but energy costs are very high. It is well known that solar inactivation of microbial cells occurs through a variety of mechanisms. Sunlight in water induces a series of photooxidative processes as well as the generation of reactive oxygen species (ROS), which eventually damage most cells. During the last 20 years research on heterogeneous photocatalysis for water disinfection has been increasing. Recent research on solar photocatalytic disinfection attempts to combine sustainability with low cost, leading to an efficient disinfection method, not only for drinking water, but also for irrigation. To date, the photocatalytic oxidation processes most widely studied and developed for the solar purification (decontamination and disinfection) of water effluents have been heterogeneous photocatalysis and homogeneous photo-Fenton [6][7]. Both processes make use of the most energetic part of the solar spectrum near the ultraviolet/visible light bands in strong oxidation reactions which take place when radiation activates a photocatalyst in the presence of oxygen. The lowselectivity hydroxyl radicals (•OH) generated attack organic molecules and microorganisms in water. Hydroxyl radicals gradually degrade hazardous organic molecules to less harmful analogues, like inorganic ions and carbon dioxide. These oxidizing radicals also produce severe damage to cell membranes, causing cell death or lack of viability of a number of microorganisms exposed to the treatment. Another photochemical process promoted by solar photons is hydrogen peroxide (in amounts below its own toxicity) in the presence of natural sunlight. It has been demonstrated recently that there is a synergistic effect between H2O2 (at low concentrations, i.e., tens of milligrams per litre) and solar irradiation which makes microorganisms in water become unviable. Therefore, solar photocatalysis and photolysis represent an alternative approach for removing not only hazardous compounds, but also pathogens from water. This chapter shows some case studies of water disinfection using the above mentioned solar processes. Some of our group’s experience with solar reactors for photocatalytic water disinfection based mainly on non-concentrating collectors is also presented.

2. Occurrence of phytopathogens in irrigation water Stored rainwater or surface water used for irrigation accumulates countless phytopathogens that can produce significant damage in crops. The main phytopathogens affecting agriculture are fungi, the most frequent and important of which are phytopathogens like Fusarium, Pythium, Phytophthora, and Olpidium. Nevertheless, other less frequent pathogens can have a severe negative impact on agriculture, such as viruses (watermelon mosaic virus, tomato mosaic virus and cucumber green mottle mosaic virus), nematodes, (Meloidogyne incognita), and bacteria like Erwinia spp., Pseudomonas syringae and Clavibacter michiganensis. All these pathogens are present in irrigation water. Water acts as the growing medium and vehicle for pathogens to spread through crops. Thus, elimination of these pathogens from irrigation water is crucial to avoid significant damage to cultures and consequent economic losses. Fusarium is a large genus of filamentous soil fungi found worldwide. Its distribution in water systems, including reservoirs, rivers, coastal seawater, wastewater effluents, and even hospital water distribution systems is an important issue [8]. Some species of Fusarium may affect humans and plants. Some of them produce mycotoxins which can be harmful to human and animal health. The main toxins produced are fumonisins and trichothecenes [9], which cause diseases like skin affections and eye infections due to fungal contamination of contact lenses [10], and is especially aggressive in people vulnerable to disease. However, agriculture is the most affected field by Fusarium. It produces vascular withering in roots and stem rot in a variety of crops. Examples of the impact of Fusarium sp on agriculture are the “Panama disease, or Banana wilt” produced by F. oxysporum in commercial bananas [11], “coffee wilt disease” produced by F. xylarioides [12], and other plants like wheat, barley, basil, tomato, peas, aubergine, melon, etc., are affected by Fusarium [13]. Its resistance to stress is conferred by its strong spore survival structure. Pythium and Phytophthora are two closely related genera belonging to the chromista kingdom, although commonly known as fungi, and indeed are called fungus-like because of their controversial classification. Both are characterized by the fast production of sporangia and swimming biflagellated zoospores. This makes initial infection spread rapidly through the plants and strongly attack the crop. Furthermore, other survival forms called oospores, which are more

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resistant to chemical disinfection, are also generated by these fungi. These genera are especially sensitive to the presence of free water, which is why they are very common and infective in hydroponic cultures and nurseries, where infections can destroy plants in a short time (2-3 days). Plant, foliage, crown, roots and fruit are commonly affected by them [14]. The most important diseases caused by Pythium affect cucumbers and beans. Phytophthora is a parasite of various hosts, causing important crop diseases like potato blight (P. infestans), black pod in cacao (P. palmivora), blight of peppers (P. capsici), and other diseases in avocado, tobacco, tomato, etc. One of the severest diseases in tomato is caused by Clavibacter michiganensis subsp. michiganensis. Virulence of Clavibacter michiganensis is due to two plasmids pCM1 (27.5 kb) and pCM2, which increase its ability to invade the plant and produce exopolysaccharides, which cause the disease’s symptoms [15]. Phytopathogenic bacteria spread through contaminated soils and surfaces, infected seed, infected fruit waste, etc. They are also found in contaminated water resources, which make plant infection easier, since water is indispensable to generate infection. Viruses are made resistant by the capsid, which is a protein coating that protects the genetic material housed inside. Crops affected by viruses are mainly cucumber, tomato and watermelon. Nematodes can occur accidentally in natural water resources like rivers, lakes, ponds, reservoirs, etc., although their normal habitat is soil [16]. They produce diseases in tobacco, potatos, soybean, and cotton.

3. Solar energy for water disinfection 3.1

Solar radiation and cellular response

The solar irradiance incident on the Earth’s outer atmosphere is approximately 1360 Wm-2. A diversity of molecules like H2O, CO2, O3, O2, aerosols and other pollutants in the atmosphere, scatter and absorb different ranges of extraterrestrial solar irradiance. In the standard case of a typical clear-sky atmosphere in summer at the equator, irradiance received on a horizontal surface at ground level is reduced to 1120 Wm-2. The UV-C (200-280 nm) range of the solar spectrum, which is the most energetic band of the UV, is absorbed by the atmosphere, and is therefore not a component of the sunlight responsible for carcinogenic damage in organisms. Highly efficient UV-C lamps are used for water disinfection. These photons are absorbed by DNA molecules and directly induce pyrimidine, purine dimers and pyrimidine adducts. Resistance to UV-C treatment may vary depending on the type of microorganism. Legionella pneumonphila is one of the most sensitive to UV-C, while Giardia muris and Cryptosporidium parvum oocysts are the most resistant. UV-C disinfection, like all technologies based on irradiation, is strongly limited by water turbidity. The solar ultraviolet radiation that reaches the surface of the earth contains UV-B (280-320 nm) and UV-A (320400 nm) light. The UV-B spectrum overlaps with the tail of DNA absorption. Therefore, UV-B is considered to be the solar component mainly responsible for the majority of human skin cancers. Nevertheless, the UV-A region of sunlight is potentially carcinogenic and is certainly involved in photo-aging, so much research has been undertaken to understand the cell-damaging action of both components (UV-A and UV-B). Today it is accepted that the damage caused by UV-A and UV-B light is mainly due to its absorption by intracellular chromophores [17]. Damage produced by light absorption through chromophores leads to generation of ROS which result in oxidative injury to cells and cell components. ROS can lead to lipid peroxidation, pyrimidine dimer formation and even DNA lesions. When ROS react with DNA, single strand breaks (SSBs) are generated as well as nucleic base modification which may be lethal and mutagenic. Furthermore oxidation of proteins and membrane damage is also induced. ROS can be inhibited by scavenging enzymes, such as catalase, peroxidase and superoxide dismutase, which are found in all aerobic organisms. Scavenging agents not only act during UV-A-irradiation-induced oxidative stress, but also during normal cell metabolism. They correct and repair a number of oxidative lesions caused during cell respiration by peroxyradicals (HO2•), hydrogen peroxide (H2O2), and hydroxyl radicals (•OH). Intracellular •OH radical formation can be attributed to the Fenton and Haber-Weiss reactions, due to the presence of intracellular iron and hydrogen peroxide. 3.2

Solar water disinfection

Solar disinfection is not a new discovery. It was known and used nearly 2000 years ago by Indian communities to purify drinking water. The bactericidal effect of sunlight was rigorously studied for the first time by Downes & Blunt in 1877 [18]. But the first successful application was demonstrated by Acra et al. in 1980 [19]. They used sunlight for disinfection of oral rehydration solutions taken to developing countries as part of the WHO disease control program. The full potential of SODIS for inactivation of a wide range of waterborne pathogens has been under study since then. Bacterial disinfection by solar radiation is proportional to the intensity of radiation and temperature and inversely proportional to the depth of the water due to the dispersion of light. The amount of this radiation attenuation depends on the wavelength range, for example, from 200 to 400 nm, the reduction is less than 5% m-1 depth, and at longer

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wavelengths it can be up to 40% m-1 [20]. The most harmful wavelengths are in the near UV-A spectrum (320 to 400 nm), whereas the spectral band from 400 to 490 nm is the least harmful. It is widely accepted that solar inactivation of microbial cells occurs through a variety of mechanisms depending on the UV range. Sunlight used during solar disinfection consists mainly of UV-A (only 4-5% of solar UV is UV-B) and hence the main inactivation mechanism is a photooxidative process with generation of ROS, as explained above. Differences in bacterial inactivation rate at temperatures varying from 12 to 40ºC have been found to be negligible. However, when temperature rises to 50ºC the bactericidal action is doubled, due to the strong synergy between UV radiation and the thermal effect [21]. In developing countries where it can be difficult to find safe drinking water, the need for an effective but practical water disinfection method is still of vital importance. Solar water disinfection has been shown to be an effective household treatment method that is both practical and low-cost. Through a synergistic effect of mild heat and UV light, microbial pathogens in drinking water contained in poly(ethylene) terephthalate (PET) bottles are inactivated within six hours after exposure to sunlight [6]. Although SODIS in PET bottles is effective, there are a number of limitations like: i) The volume of water disinfected at a given time is restricted to < 3 L, which creates a requirement to have sufficient bottles and time to provide adequate volume of treated water for an average household. ii) Periods of cloudy weather will require SODIS users to expose bottles for 2 consecutive days in order to inactivate pathogens. iii) During rainy seasons, an alternative disinfection method has to be used. The use of filtration before solar exposure is also recommended for water that has a turbidity ≥ 30 NTU [22]. 3.3 Heterogeneous photocatalysis (TiO2/UV) Irradiation of a semiconductor particle by light with energy greater than or equal to the band gap energy results in the promotion of an electron from the valence band to the conduction band, leaving behind a hole in the valence band. Both electron and hole are responsible for charge conduction in the semiconductor. They may recombine in the bulk dissipating the energy as heat or light, or they may move to the solid-liquid interface. For titanium dioxide, TiO2 (the semiconductor most widely used in photocatalysis) the band gap energy is 3.2 eV, which corresponds to light wavelengths of ≤ 387 nm, and therefore, TiO2 can only be excited by UV light. The positive hole is able to oxidise water or hydroxyl groups yielding hydroxyl radicals (OH). The hydroxyl radical is a non-selective oxidant which attacks organic molecules at or near the particle surface-water interface. The most commonly employed oxidant (electron acceptor) is molecular oxygen, available from the air, and dissolved in water. The reduction of molecular oxygen yields a superoxide radical anion (O2-, or at low pH, the hydroperoxyl radical, HO2), hydrogen peroxide (H2O2) and hydroxyl radicals (OH). These ROS are very active, indiscriminate oxidants, especially the hydroxyl radical [23]. These ROS cannot only destroy a large variety of chemical contaminants in water, but also cause fatal damage to microorganisms [24]. The first reports on the potential of TiO2 for disinfection were by Matsunaga et al. in 1985 [25]. Since then, interest in research on TiO2 disinfection has grown, and a number of articles have focused on TiO2-assisted inactivation of a wide range of microorganisms in water. These studies have shown good disinfection results for bacteria, like total coliforms, Escherichia coli, Serratia marcescens, Streptococcus aureus, Streptococcus faecalis, Enterobacter cloacae, Pseudomonas aeruginosa and Salmonella typhymurium; fungi such as Aspergillus niger, Candida Albicans, Fusarium solani [6]. Even prions [26], yeasts, tumour cells, viruses, and cellular molecules [24] have been inactivated using heterogeneous photocatalysis. It is widely accepted that the first target of oxidative radicals is the surface of the external membrane of the cell wall. Damage occurs on the lipopolysaccharides layer of the external cell wall and on the peptidoglycan layer first. Then the lipid membrane is peroxidised (the radicals oxidize to fatty acids), and the protein membrane (amino acids) and polysaccharides are oxidised [6]. Regarding solar photocatalytic applications, let us recall that the solar spectrum contains only a small proportion of UV (4-5%, depending on location) and this obviously limits the applications of TiO2 photocatalysis in solar driven water treatment. Current research in the field is focused on the development of stable visible-light-active photocatalytic materials which can utilise the solar spectrum more effectively. The configuration of the catalyst in the reactor can significantly alter disinfection results. There are two approaches for water purification treatment purposes, i) aqueous suspensions of TiO2 particles in what is called a slurry, and ii) as immobilised TiO2 on an inert matrix. The choice of catalyst preparation depends on the final application. If the system is designed as part of routine drinking water purification for house-hold (point-of-use) water treatment, TiO2 in suspension is unfeasible. The photocatalyst particles would have to be removed after solar exposure and before consumption. The choice of light source and mode of illumination also affects disinfection results. The spectral distribution of the photon source, whether lamp or natural solar, and intermittence of illumination strongly affect disinfection [27][28]. The photocatalytic inactivation rate as a function of the initial bacterial concentration obeys first order kinetics. This has already been proven with total coliforms, spores, etc. The range of concentration of microorganisms for which the first order kinetics is valid strongly depends on the microorganism itself [24].

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3.4

Hydrogen peroxide/near-UV light (H2O2/UV)

Photolysis of hydrogen peroxide occurs when it is irradiated by photons of wavelengths lower than 300 nm yielding two  OH radicals for each H2O2 molecule [29]. Solar radiation on the Earth's surface does not contain photons with that wavelength. Therefore, solar energy is inefficient for hydroxyl radical generation by this pathway, and although the combined effect of H2O2 with UV-C lamps or O3 has been used as an advanced oxidation method, the combined effect of solar energy and H2O2 has not been investigated as a disinfection method to date. The lethal synergy of H2O2 and near-UV light was first reported for phage T7 in 1977 [30]. Since then, only a few contributions have reported the damaging effect of H2O2/UV-Vis on microorganisms in water and shown experimental evidence of disinfection capacity of near UV or visible light and hydrogen peroxide using different microorganisms such as E. coli, Streptococcus mutans, Fusarium solani, and Fusarium equiseti [31][32]. The H2O2/UV-Vis process requires the addition of small amounts of hydrogen peroxide and may use solar light. The synergistic effect of hydrogen peroxide and solar photons is attributed to the generation of •OH radicals from H2O2 after the Fenton reaction inside microbial cells. This disinfection process is potentially low cost, since at concentrations below 50 mg L-1 which have been shown to be non-toxic for crops, hydrogen peroxide is cheap and safe to use [33]. Moreover, the decomposition of hydrogen peroxide into water and oxygen avoids concerns about secondary pollution due to the disinfectant itself. Other advanced oxidation processes, like titanium dioxide or photo-Fenton need posttreatment to remove the catalyst or change the pH. Solar irradiation contributes to redox cycling of Fe3+–Fe2+ in the presence of H2O2 at low pH (photo-Fenton catalytic reactions). However, iron concentrations are increased in cells irradiated with near UV photons. Therefore, the influence of photo-Fenton on this disinfection process is not excluded [33]. Intracellular photo-Fenton reactions may be promoted by the addition of H2O2 due to the presence of “free” iron inside the spore. This is mainly associated, but not exclusively, with the existence of biosynthesised siderophores or Fe-transported siderophores as the source of iron. The trace concentrations of “free” iron catalyse the production of hydroxyl radicals via the Fenton/Habber-Weiss reaction cycle. The critical factor seems to be the availability of the cellular labile iron pool (LIP), which may also be favoured by irradiation of cells by UV light [34]. This alternative approach to purifying water polluted with phytopathogens resistant to standard disinfection methods like chlorine, plaguicides, etc., may be used to treat irrigation water to avoid the presence of phytopathogens or to disinfect effluents of a wastewater treatment plant for water reuse. The main advantages of this system are its low cost, and no post-treatment removal of reagents.

4. Compound parabolic Collector (CPC) reactors. The compound parabolic collectors (CPCs) first used in the sixties to collect solar radiation using a fixed device were a combination of parabolic solar concentrators and flat stationary systems. CPCs are stationary collectors with a reflective surface using non-imaging optics which can be designed for any given reactor shape. They collect all incident solar radiation on a given surface area and reflect it towards the receiver placed in the focus. In tubular photoreactors for water treatment applications, the CPC mirror geometry is an ordinary involute of two parabolas truncated so that all the Sun’s rays below the acceptance angle reach the reactor tube (Fig. 1(a)). A CPC with a concentration factor of 1 has an acceptance angle of 90º. CPC mirrors have a number of advantages: 1) They make highly efficient use of direct and diffuse solar radiation without sun tracking, and therefore, are a lower-cost system than solar collectors used for thermal applications. 2) They have high optical efficiency. 3) Water temperatures are quite low, so photochemical reactions are quite efficient. 4) And turbulent water flow can easily be maintained under low pressure. Tubular CPC photoreactors have been demonstrated to be a good option for solar photochemical applications like wastewater treatment for removing hazardous chemical compounds and disinfect contaminated water [6]. The development of CPC pilot plants for water disinfection led to the construction and evaluation of the solar CPC photoreactor pilot plant shown in Fig. 1(b). This photoreactor consists of two CPC mirror modules placed on an anodized-aluminium platform titled 37º. The total collector surface of the photoreactor is 4.5 m2. Each module is made up of ten 1.5-m-long and 50-mm-outer-diameter borosilicate glass tubes. This glass transmits 90% of UV-A radiation. The CPC mirror is made of highly reflective anodised aluminium with a concentration factor of one. The ratio of irradiated water to total water is 75%, and the total treatment volume is 60 L. Water is circulated through the tubes to a tank by a centrifugal pump (150 W). pH, dissolved oxygen (DO) and temperature probes are installed in the pipes to continuously monitor these parameters which are recorded with data acquisition software [35]. Prior to pilot-plant solar tests, small-scale proof-of-principle experiments are done in magnetically stirred 250-mL glass bottle reactors exposed to natural sunlight. Total water volume treated in this case is 0.2 L and the illuminated surface is 0.0095 m2 [35].

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a)

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Fig. 1 Diagram of the CPC collector showing acceptance angle (c) and aperture width (a) Photo of 60-L CPC solar reactor at the PSA facilities (Almería, Spain) (b).

UV radiation is measured with a global UV-A radiometer (295-385 nm) located close to the reactor and at the same inclination angle (37º). The radiometer provides incident UV-A irradiance data in W/m2. Inactivation kinetics during disinfection depends not only on experimental time but also on total amount of UV-A photons received by the system during tests. It is also important to compare results under different UV-A radiation conditions, e.g., on different days. Therefore, cumulative UV-A energy per unit of volume (QUV) received in the photoreactor is calculated and used to evaluate disinfection results as defined by Eq. 1. QUV ,n  QUV ,n  1  Δt nUVG ,n Ar / Vt

Δtn  tn  tn 1

(1)

where QUV,n, QUV,n-1 is the UV-A energy received per litre (kJL-1) at times n and n-1, UVG,n is the average incident radiation on the irradiated area, ∆tn = tn- tn-1, where tn (s) is the sample experimental time n, Ar is the illuminated area of the solar collector (m2), and Vt is the total volume (L) [6].

5. Case studies. 5.1

Fungal spore inactivation using TiO2/UV-Vis light.

Solar photocatalysis with suspended TiO2 was used to disinfect water contaminated with Fusarium spores. To discriminate responses between different spores, we individually investigated photocatalytic inactivation of chlamydospores and macroconidia of F. equiseti and microconidia of F. solani (Fig. 2). In both cases, spore concentrations were decreased from 102-103 CFU mL-1 to detection limit in distilled and well water (DW and WW). Microconidia required the least energy to achieve complete inactivation followed by macroconidia and chlamydospores. In TiO2 photocatalysis, the first point of ▪OH attack is on the cell wall [36], so inactivation efficacy is better if spores and catalyst particles are close together or in contact with each other (adsorption). It has been shown experimentally that spore and catalyst suspensions have a tendency to attach to each other and form aggregates during photocatalysis [37]. The cell wall is thus continuously attacked by ▪OH generated on the catalyst surface under irradiation. They gradually damage the spore, eventually causing cell death. Resistance of cells to photocatalysis depends on the chemical composition and structure of the cell wall. The chemical composition of microconidia, macroconidia and chlamydospore walls is similar, mainly glucosamine, glucose, mannose, galactose, amino acids and glucuronic acids, although in different proportions [38]. Fusarium contains an electron-dense material that has a melanin-like nature resistant to lysis [39], which is deposited on the spore wall during chlamydospore formation. This component increases its resistance to oxidation and light. F. equiseti has previously been reported to be more resistant to solar photocatalysis than other Fusarium species like F. verticillioides or F. anthophilum, which generate primarily microconidia and very few macroconidia [40]. Spores were inactivated in DW and WW down to the detection limit, but samples in WW required higher solar UV-A energy (QUV) to achieve complete disinfection than with DW in all cases evaluated (Fig. 2). The DW matrix has no ions, so the catalyst is unaffected by other chemical reactions, while WW is characterized by high ion concentrations of, e.g., carbonates/bicarbonates, sulphates, nitrates, etc., which decrease the efficacy of photocatalytic processes. Carbonates/bicarbonates present in water are a limiting factor for photocatalysis because HCO3─ reacts with the hydroxyl radicals producing the less reactive anion radical CO3─▪ and eliminating ▪OH from the water, i.e., they are ▪OH

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scavengers [37]. The high carbonate concentration in the WW used in these experiments (approximately 500 mg/L) could therefore be inhibiting the photocatalytic activity and be responsible for the lower spore inactivation rate in WW. The most important finding of this experimental work was the very efficient Fusarium spore inactivation rate observed using a 60-L CPC solar reactor. This solar system showed the excellent potential of photocatalytic treatments for disinfection of contaminated irrigation water and water reuse. 3

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Fig. 2 Solar photocatalytic water disinfection of F. equiseti chlamydospores (-■-), F. solani macroconidia (-●-), and F. equiseti microconidia (-▲-) with suspended TiO2 (100 mg/L) in a CPC solar reactor with distilled water (a); and with well water (b).

5.2

Solar photo-assisted water disinfection using H2O2/UV-Vis light

Fig. 3(a) shows the inactivation rate of fungal spores (F. equiseti chlamydospores and F. solani microconidia), funguslike oomycete spores (Phytophtora capsici zoospores), and bacteria (Clavibacter michiganensis) with 10 mg/L of H2O2 in distilled water under natural sunlight in a solar bottle reactor. Treatment resistance was observed to be in the following order: C. michiganensis < P. capsici zoospores < microconidia of F. solani < chlamydospores of F. equiseti. Inactivation can be explained by: (i) production of ROS and DNA mutations by the direct action of sunlight on the microorganisms; and (ii) internal Haber-Weiss reactions (internal photo-Fenton). This mechanism is favoured by H2O2 diffusion into the cell, where it can react with free iron to generate •OH by the Haber-Weiss reaction. •OH reacts with any organic molecule inside the cell, like DNA. The powerful generation of •OH by intracellular Fenton reaction could accumulate damage leading to cell death due to the overloaded cell defence mechanisms. Fenton and photo-Fenton reactions occur quickly in metabolically active cells like bacteria. In spores, the metabolic activity and oxidative reactions occur once spore germination has begun. During germination the spore swells from uptake of water to hydrate the core, followed by the initiation of enzimatic and other metabolic activity, and finally, growth of the germ tube [42]. H2O2 enters the spore with water uptake [41] leading to internal Fenton reactions, as explained elsewhere for F. equiseti chlamydospores [35]. This biological difference accounts for the lower resistance of C. michiganensis, a vegetative bacterium, than fungal spores (Fig. 3a). Germination (time and energy requirement) varies for each microorganism, and for the fungal spores evaluated in this study. Fig. 3(a) shows that P. capsici swimming spores respond to the treatment like C. michiganensis, while the inactivation rate of Fusarium spores is the lowest. P. capsici zoospores are motile biflagellate spores leading to dynamic dissemination of disease in crops [43]. The whole P. capsici germination process occurs in a short time in water (~2-3 h), while Fusarium takes around 1 day. Therefore, spore swelling and H2O2 uptake in this zoospore are faster than in Fusarium. This may explain why P. capsici required less treatment time than Fusarium spores. Fig. 3(a) also shows that microconidia were less resistant than chlamydospores, as shown with TiO2 photocatalysis (in the section above). The influence of the chemical composition of the water on the photocatalytic process was also evaluated. Fig. 3(b) shows inactivation of F. equiseti chlamydospores in DW, WW and simulated wastewater effluent (SWE) using H2O2 (10 mg/L)/sunlight in a 60-L-CPC solar reactor. As expected, the DW experiment showed the fastest inactivation efficacy. The presence of organic matter and inorganic compounds in WW and SWE reduced the treatment efficiency, due to the presence of scavengers like carbonates/bicarbonates. These results showed experimental evidence of the killing effect of solar radiation with additional low amounts of H2O2 (10 mg/L). We found these results with a model resistant phytopathogen (Fusarium) and a bacterium (C. michiganensis) at lab and pilot plant scale (0.2 and 60 L), in DW, WW and SWE. This method could be used to treat irrigation water to avoid the presence of phytopathogens or to disinfect effluents of a wastewater treatment plant

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for reuse of water. The main advantages of this system is its low cost, i.e., low consumption of H2O2, use of solar energy, and it does not require post-treatment removal of reagents like TiO2 or iron in photo-Fenton. 7

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Fig. 3 Inactivation of F. equiseti chlamydospores (--), F. solani microconidia (--), P. capsici zoospores (-▲-) and C. michiganensis (-■-) in distilled water with 10 mg/L of H2O2 in solar bottle reactor under natural sunlight (a). Inactivation of F. equiseti chlamydospores in different water sources: distilled water (-●-), well water (-▲-), and simulated wastewater effluent (-■-). Acknowledgements Financial support by the Access to Research Infrastructures activity in the 7th Framework Programme of the EU (SFERA Grant Agreement n. 228296) is gratefully acknowledged.

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