Piggery Wastewater Treatment Using Photosynthetic Microorganisms

1. Introduction

The human population in the world reached 8 billion in 2022 and recent projections suggested a population up to 9.7 billion by 2050, which represents future challenges for the sustainable development for mankind [1]. This demographic expansion will entail global changes generated by an intensive anthropogenic activity on the Earth, which involves negatives impacts in the atmosphere due to the intense emission of greenhouse gases, in the cryosphere due to the melting of ice fields, in the land surface caused by soil pollution and desertification, in the biosphere due to a loss of biodiversity and species extinction, and in the hydrosphere due to eutrophication and pollution of surface water and groundwater [2,3]. The development of new technologies that reduce pollution and pave the way towards a sustainable development at a global scale represents a severe challenge nowadays. In this context, multiple technologies have been engineered in recent years to reduce the pollution generated by anthropogenic activity. These technologies include greenhouse gas capture [4,5], new renewable energy sources with special focus on solar energy and biofuels [6,7], sustainable farmland and livestock management [8] and an extensive innovation in wastewater treatment technology [9,10].

In this context, wastewater treatment in cities, industries and farms aims at reducing water pollution generated by anthropogenic activity. Wastewaters are mainly classified depending on their origin as domestic, industrial and agro-industrial [11]. Depending on their origin, wastewaters exhibit a different physical, chemical and microbiological composition [12]. Domestic wastewaters are generated from human activities in households (e.g. use of the toilet, bathing, kitchen and washing), and therefore its production directly increases with human population. This type of wastewater is characterized by low concentration of organic matter and nutrients due to the large water volume use in households (Table 1) [13]. On the other hand, industrial wastewaters are one of the most diverse sources of wastewaters and their compositions are industry specific. For instance, brewery wastewaters are mainly composed of a high concentration of carbon (sugars and alcohols) [14], while wastewater generated by the mining industry contains highly toxic and recalcitrant pollutants such as heavy metals. Finally, agro-industrial wastewaters are produced by intensive agriculture, animal husbandry and their processing. Of them, wastewaters from husbandry of cows, poultry, pigs, turkeys and fishes rank among the most pernicious as a result of their large volumes of production and the gradual increase in animal production. Livestock wastewaters are characterized by a high content of organic matter from animal manure and a high content of ammonium from animal urine (Table 1) [15,16].

2. Piggery Wastewater

The increased livestock farming for human food production represents a big environmental problem in regions such as Europe, Asia and North America, where the highest densities of animal farms are located. For instance, the production of pigs in the European Union (EU) reached 23,720 thousand metric tons of pig meat in 2021, with Spain as the second largest pork producer in the EU [17]. Other countries such as China and United States produced 47,500 and 12,559 thousand metric tons of pig meat in 2021, respectively (United States, Department of Agriculture). Intensive animal husbandry generates large volumes of wastewaters with high concentrations of organic matter, nitrogen and phosphorous, which requires the decentralized implementation of cost-effective wastewater treatment. Indeed, the uncontrolled discharge of pig manure entails a high impact in the environment due to the massive greenhouse gas emissions from anaerobic fermentation of pig manure and the released nitrogen and phosphorous, which negatively impacts biodiversity [10,15,18,19]. Piggery wastewater (PWW) is considered one the most polluting agro-industrial wastewaters [15], characterized by high concentrations of organic matter, carbon (in form of volatile fatty acids), nitrogen (mainly in the form of ammonium) and even presence of heavy metals (such as copper, lead and zinc) [15,20,21]. In addition, the inadequate management of this wastewater can cause malodour pollution in the surrounding areas.

Depending on the farming practices and type of pig farmed (litters, piglet or sows), PWW concentration can vary due to differences in animal nutrition, farming practices and water usage in the cage washing and maintenance (Table 2). Overall, these wastewaters are characterized by their high concentration of dissolved carbon, nitrogen and phosphorus, and suspended solids.

4. Photosynthetic Piggery Wastewater Treatment

The high environmental impacts, and high operating and energy costs of conventional anaerobic and aerobic treatments used for PWW treatment, have promoted research on photosynthetic PWW treatment in recent years [10,15,19,34,35,36,37]. Photosynthetic microorganisms are mainly classified in two groups according to their unique metabolism. The first group is composed of purple phototrophic bacteria (PPB), microorganisms that perform anoxygenic photosynthesis (without oxygen production) (Figure 1). This group is subdivided into two bacterial group: purple sulphur bacteria (PSB) and purple non-sulphur bacteria (PNSB) [38]. PPB were originally classified based on their sulfide tolerance and characteristic purple-red colour, although PPB were recently classified according to its class within the phylum Proteobacteria, where PSB belong to the class Gammaproteobacteria and PNSB belong to the class Alphaproteobacteria or Betaproteobacteria [39]. Another photosynthetic bacterial group capable of performing anoxygenic photosynthesis are green sulphur bacteria (GSB) and green non-sulphur bacteria (GNSB), classified in these group according to their sulphur tolerance and their characteristic green colour [31].

On the other hand, microalgae are a diverse group of photosynthetic microorganisms with eukaryotic and prokaryotic species living in marine, freshwater and terrestrial habitats capable of performing oxygenic photosynthesis (oxygen production). The major classification of eukaryotic microalgae is subdivided in Chlorophyta, Rhodophyta and Phaeophyta, which were originally described according to their characteristic colour (green, red and brown, respectively) [40]. Another group of eukaryotic microalgae are diatoms, which are characterized by cell walls composed of two siliceous pieces and classified into three classes: Coscinodiscophyceae, Mediophyceae and Bacillariophyceae [41]. In addition, prokaryotic microalgae belonging to the phylum Cyanobacteria have evolved in a wide variety of freshwater and saltwater environments.

PPB and microalgae possess extraordinary metabolic capabilities, and are able to grow at low temperatures or extremely high temperatures, in different pH ranges (alkaline or acidic), at high salt concentrations and even under the presence or absence of oxygen [16,39,42,43].

4.1. Purple Phototrophic Bacteria-Based Treatment

Purple phototrophic bacteria are gram-negative bacteria, prokaryotes and have the most versatile metabolism among microorganisms (Figure 2). Rhodopseudomonas palustris, Rhodobacter sphaeroides, Rhodospirillum rubrum from the PNSB group, and Allochromatium vinosum and Thiocapsa roseopersicina corresponding from the PSB group ranked among the most investigated PPB [39,44]. PPB are capable of growing under anaerobic and aerobic conditions via phototrophic and chemotrophic metabolisms, respectively [21,45,46]. In addition, PPB are capable of assimilating and growing using organic or inorganic compounds as a carbon source under heterotrophic and autotrophic metabolism, respectively. Some PPB species can obtain energy for growth from the oxidation of inorganic compounds chemolithotrophically using H₂ or S₂O₃⁻² as electron donors [39]. PPB can grow in environments with extreme conditions: high concentrations of salt [47], extremely high temperatures or low temperatures in the range of 10–13 °C [16,48,49] and very acid or alkaline conditions [42].

PPB can obtain energy though of anoxygenic photosynthesis process, absorbing energy from the sunlight radiation reaching the earth surface by photon absorption using photosynthetic complexes. In this process, PPB produce ATP via photophosphorylation under phototrophic growth [38,39]. Indeed, PPB are more efficient photosynthetic microorganisms in the photoconversion compared to microalgae [13]. The first descriptions of the basic mechanisms underlying photosynthesis were based on investigation using PPB as model microorganisms [50]. Electron flow during the photosynthetic process in PPB begins with photons absorption by multiple light-harvesting (LH) complexes composed of pigments (mainly of bacteriochlorophylls and carotenoids) bonded to proteins. The absorbed energy is conducted to the reaction centre (P870), where photosynthetic electron transport reactions begin. When P870 is excited (representation P870*, Figure 3), it becomes a very strong electron donor, capable of transferring energize electrons to a quinone (Q) located within the cytoplasmic membrane. These electrons, in turn, are donated to the cytochrome bc₁, which translocates four protons for every two electrons from the reaction centre. This process entails an increase in the proton concentration in the periplasm of PPB, which triggers the proton motive force to the cytoplasm, where ATPase enzyme synthetizes adenosine triphosphate (ATP) from adenosine diphosphate (ADP) and inorganic phosphate. In contrast to oxygenic photosynthesis (a process performed by microalgae), the electrons from cytochrome in PPB are not transferred to an electron acceptor (such as molecular oxygen) but to cytochrome C₂, and return to the reaction centre P870, closing the cyclic photophosphorylation in PPB [43].

PPB are a unique group of microorganisms capable of absorbing radiation of wavelengths in the near-infrared (NIR) spectrum (Figure 4). Indeed, PPB exhibit a special spectral niche among photosynthetic microorganisms due to the presence of bacteriochlorophyll a and b, which present a maximum absorption at 804 nm and 1040 nm, respectively [51]. PPB contain carotenoids that absorb radiation below 500 nm and provide its characteristic purple-red colour. In addition, carotenoids are able to reduce the photodegradation of bacteriochlorophyll under high irradiance conditions [16].

On the other hand, PPB can grow aerobically under chemoheterotrophic or chemoautotrophic metabolisms, obtaining energy via oxidative phosphorylation (Figure 2) by degrading macromolecules for ATP synthesis [52,53]. Molecular oxygen is used an electron acceptor under these growth conditions. However, this O₂ causes a repression and inhibition of bacteriochlorophylls synthesis, which harms the photosynthetic capacity of PPB [54].

R. palustris is a PPB that holds multiple metabolic pathways like the tricarboxylic acid cycle, Embden-Meyerhof pathway, pentose phosphate route, fatty acids metabolism and even can fix CO₂ via the Calvin-Benson-Bassham (CBB) cycle [45], which confer this microorganism an efficient metabolism for the assimilation of alcohols, amino acids, carbohydrates, fatty acids, organic acid and even recalcitrant pollutants in wastewater [16,38]. It has been recently described that R. palustris can re-assimilate the carbon produced in the catabolic pathways and strive for maximum carbon efficiency as most CO₂ produced from acetate oxidation is fixed and used for biomass production [55]. In addition, PPB can assimilate all forms inorganic of nitrogen such as NO₃^-, NO₂^-, N₂ and NH₄⁺ [56]. This ability to fix N₂ is remarkable due to the high energy contained in the triple bond of this molecule. In this context, PPBs are able to synthesize the enzyme nitrogenase, which breaks the triple bond present in the N₂ and generates NH₃ using the energy contained in ATP. PPB can also assimilate the organic nitrogen present in amino acids such as aspartate, glutamate and glutamine. Certain species of PPB can even assimilate aromatic compounds like cyclohexane carboxylate, toluene, benzoate and their derivatives [39]. These versatile metabolic pathways provide PPB a great potential for the effective treatment of different types of wastewaters.

PPB can treat a wide variety of wastewaters from domestic, industrial and agro-industrial sources with high pollutants removal efficiencies [38,56]. For instance, concomitant removal efficiencies of 63% for COD, 99% for NH₄-N and 88% PO₄-P have been recorded during domestic wastewater treatment using PPB in batch tests with acetate supplementation [13]. In addition, domestic wastewater treatment by PPB under low radiation intensities (< 3 W m⁻²) with high removal efficiencies over 90% for COD and up to 86% and 91% for TN and TP removal, respectively, has been reported in literature [57]. In addition, an effective domestic wastewater was achieved in a novel continuous photoanaerobic membrane bioreactor with a PPB dominance in the culture broth over 60% [49]. Similarly, high COD removal efficiencies have been reported during industrial wastewater treatments with PPBs: COD removal of 96% in brewery wastewater [58], 90% in acidic food wastewater [59] and 89% in VFA-rich food industry wastewater [60]. On the other hand, PPB can also support satisfactory organic matter and nutrient removals during agro-industrial wastewaters treatment. For instance, PPB-based treatment provided removal efficiencies higher than 90% for COD and TN during poultry processing wastewater treatment [36], >83% for COD during digested piggery wastewater treatment [61], >87%, 83% and 89% for TOC, TN and TP removal during PWW treatment [34], >78% for TOC during piggery wastewater treatment coupled with biogas upgrading [35].

4.2. Microalgae-Based Treatment

Microalgae are photosynthetic microorganisms similar to plants on the fact that they carry out the oxygenic photosynthesis process and both present similar pigments belonging to chlorophyll and carotenoids group. Microalgae are the main photosynthetic microorganisms studied for wastewater treatment since the pioneer investigations of Oswald (1988) [62]. In addition, in the past decades a huge research effort was devoted to microalgae-based technologies for biofuel production, wastewater treatment and production of high value-added bioproducts from microalgae biomass [63,64,65,66]. The main microalgae species investigated were Chlorella vulgaris, Chlamydomonas reinhardtii, Phaeodactylum tricornutum and Arthrospira platensis among others. Microalgae can grow photoautotrophically fixing CO₂ or photoheterotrophically by aerobically metabolizing organic compounds. Both metabolisms utilize energy from sunlight as the main energy source. Microalgae use H₂O as electron donor during photoautotrophic growth to produce O₂ as a by-product of the reaction [43]. Indeed, this photosynthetic oxygen production is crucial to sustain life in the planet since microalgae produce massive amounts of oxygen in the vast oceans and surface freshwater bodies. This ability to produce oxygen has been successfully used for wastewater treatment, where microalgae grow in symbiosis with bacteria. These bacteria use oxygen as electron acceptor for organic matter and ammonium oxidation, and are able to assimilate the ammonium and phosphorous present in wastewater. In addition, microalgae benefit from essential vitamins and CO₂ released by pollutant degrading bacteria (Figure 5) [15,67].

Photosynthesis in microalgae in comparison to PPB differs mainly on the fact that microalgae have two different photosystems for harvesting photons from solar irradiation, photosystem I (PSI or P700) and the photosystem II (PSII or P680) [68]. P680 catalysed the main step in oxygenic photosynthesis, where H₂O is split into O₂ and electrons (Figure 3). This step starts when light energy is absorbed by P680 which donates an electron to pheophytin a (Ph), turning P680 strongly electropositive and with the capacity that accept electrons from H₂O. The oxidation of H₂O is produced in a water-oxidizing complex catalysed by a Mn₄Ca cluster. Electrons are transported through different quinones (Q_A and Q_Be) within P680. Those electrons are used to reduce plastoquinone (PQ) to PQH₂. Electrons from PQH₂ are transferred to cytochrome b₆f. From cytochrome b₆f, electrons are transferred to P700 through a copper-containing plastocyanin (PC). The absorption of photons by P700 allows to accept electrons from PC. These different steps generate a proton motive force that is used by ATP synthase to produce ATP [68]. This photosynthesis process is noncyclic since the electrons do not return to P680 and are used for the generation of NADPH from NADP⁺. Nevertheless, electrons can return from PSI to PSII in some species of cyanobacteria via an electron transport chain linked (dashed line in Figure 3) [43].

The main pigments present in the photosystems of microalgae are chlorophylls, which provide them with their characteristic green colour. Chlorophyll a exhibits maximum absorption at 430 nm and 680 nm, while chlorophyll b absorbs mainly at 450 nm and 640 nm [43,51]. In addition, microalgae present a variety of carotenoids such as astaxanthin, β-carotene and lutein that absorb mainly at wavelengths between 400 nm and 520 nm (Figure 4). Carotenoids mainly act as accessory pigments in photon absorption. However, they also have a photoprotective function, preventing damage to the photosystems as a result of microalgae exposure to high irradiations [39,69]. On the other hand, cyanobacteria also contain accessory pigments belonging to the phycobiliprotein group, mainly phycocyanin, phycoerythrin and allophycocyanin. Phycocyanin and allophycocyanin are a blue pigments that absorb mainly at 620 nm and 650 nm [12]. Phycoerythrin is a red pigment that absorbs mainly in the range of 550 nm. Overall, most pigments present in microalgae and cyanobacteria absorb radiation in the visible spectrum (Figure 4).

Microalgae have a powerful photoautotrophic metabolism that allow them to grow using solar energy and assimilating inorganic compounds. Microalgae can efficiently fix CO₂ via the Calvin cycle as the main carbon source [70], although are also capable of using NaHCO₃ or Na₂CO₃ under photoautotrophic mode. The preferred forms of nitrogen used by microalgae under photoautotrophic mode are ammonium (NH₄⁺) and nitrate (NO₃^-) [71], which are used for the synthesis of amino acids and proteins. Some cyanobacteria species are able to fix molecular nitrogen (N₂) from the atmosphere as the main nitrogen source. On the other hand, microalgae are able to grow photoheterotrophically using organic compounds, such as carboxylic acid, glucose and glycerol, as a carbon source, and amino acids, peptone and urea as a nitrogen source [72]. However, PPB are more efficient than microalgae under photoheterotrophic growth and biomass production photoheterotrophically is more expensive when synthetic organic compounds are used.

A part from nitrogen assimilation, the symbiotic association between microalgae and bacteria (Figure 5) can support a sequential nitrification and denitrification, which can further boost nitrogen removal during domestic and agro-industrial wastewater treatment. The nitrification process is performed by ammonia oxidizing and nitrite oxidizing bacteria which can transform ammonium (NH₄⁺) into nitrite (NO₂⁻) and nitrite into nitrate (NO₃^-), respectively [73]. Heterotrophic denitrification is an anoxic process performed by heterotrophic bacteria that converts NO₃⁻ to NO₂⁻ and finally to nitrogen gas (N₂).

Microalgae in association with bacteria represent the most widely studied group of photosynthetic microorganisms for wastewater treatment. In this context, an efficient treatment of domestic wastewater with TOC removals of 88% and TN of 82% was recorded in anoxic-aerobic photobioreactors [74], while the treatment of textile wastewater using this innovative configuration achieved TOC removals of 48%, TN removals of 87% and TP removals 57%, along with an efficient decolourization of this wastewater [75]. An efficient treatment of synthetic food waste digestate, with removal efficiencies up to 96% and 84% for TOC and TN, respectively, was recently reported [76]. In addition, multiple studies of agro-industrial wastewater treatment reporting an efficient carbon and nitrogen removal have been reported for PWW [15,19,34,35,37].

5. Photobioreactors for Wastewater Treatment

The photobioreactors used for the cultivation of photosynthetic microorganisms are typically classified according to their contact with the atmosphere in open or closed photobioreactors. Photobioreactor design criteria involves a high irradiated surface area, effective mixing and good scalability [65]. Multiple shapes and configurations have been described and investigated for the design of these photobioreactors. In this context, the most common configurations for PPB and microalgae culture are open ponds, open raceways, closed tubular and photo-anaerobic membrane bioreactor (Figure 6) [77]. Open configurations exhibit low investment and operational costs compared to closed photobioreactors. However, they are susceptible to contamination by unwanted microorganisms, the conditions inside the photobioreactor are difficult to control and they have high-water losses by evaporation due to their open configuration [21]. On the other hand, the main disadvantage of closed photobioreactors is their high investment costs. Nevertheless, the limited contact with the open atmosphere avoids microbial contamination of the culture broth, enhances the control of physical-chemical parameters and minimizes water losses.

However, both open and closed photobioreactors must maintain appropriate physical-chemical conditions to support an efficient growth of photosynthetic microorganisms. The key environmental parameter for the cultivation of these microorganisms is radiation, which is necessary for the photosynthetic process but requires extensive surface areas for an effective collection. On the other hand, an optimal control of temperature, pH and dissolved oxygen inside photobioreactors favours PPB and microalgae growth.

Light intensity is the main parameter for the cultivation of photosynthetic microorganisms via phototrophy. The electromagnetic spectrum of solar light provides the energy source for the photosynthetic process. In this regard, photosynthetically active radiation (PAR) is the wavelength range suitable to support the growth of photosynthetic organisms (mainly plants and microalgae), corresponding to wavelengths between 400 nm and 780 nm (visible spectrum), where light absorption by chlorophylls and carotenoids occurs (Figure 4). On the other hand, PPB can absorb radiation in both visible and near-infrared (NIR) spectrum (Figure 7). At high radiation intensities and low culture densities, inhibition of photosynthetic activity can occur, resulting in an increase in intracellular carotenoid concentrations or inhibiting growth totally. However, process operation with dense microalgae cultures can be sustained under light intensities of up to 1300 µmol m⁻² s⁻¹, which represents the average solar intensity on a clear day [34,35].

Temperature is also a key parameter for cultivation of photosynthetic microorganisms. Psychrophiles are microorganisms that grow in the temperature range between 0 to 15 °C, mesophiles in temperature range between 15 to 45 °C and thermophiles are microorganisms whose growth optimum temperature exceeds 45 °C [43]. PPB and microalgae species corresponding to these three groups have been consistently reported in literature. For instance, PPB species such as Rhodoferax antarcticus have been found in the Antarctic [78], while the microalga Chlamydomonas nivalis is known to be responsible for the red colour in the snow surface [43]. In addition, an effective treatment of domestic wastewater at low temperatures (10-13 °C) using PPB has been observed [16,48,49]. Most common species of PPB and microalgae, such as Rhodopseudomonas palustris or Chlorella vulgaris, grow at moderated temperatures. Finally, PPB such as Thermochromatium tepidum and the cyanobacterium Oscillatoria terebriformis have been found in hot springs growing at temperatures above 55 °C [42]. Most studies in literature were carried out using photosynthetic microorganisms at ambient temperatures between 20 to 30 °C.

Microorganisms can be classified in terms of their oxygen tolerance in three groups: (i) aerobic microorganisms that can grow and tolerate high oxygen concentrations, (ii) microaerophiles that can grow in the presence of low concentrations of oxygen and (iii) anaerobes that cannot grow in the presence of oxygen. The latter can be classified as strict or obligate anaerobes, when oxygen is a growth inhibitor that can even killing them, and oxygen tolerant, when oxygen has no biological function in their metabolism [43]. For instance, microaerophilic and anaerobic PPB can tolerate dissolved oxygen concentrations up to 1 mg L⁻¹, which are mainly used in the electron transport chain as an electron acceptor in the oxidative phosphorylation process [53]. However, oxygen can be harmful to PPB during wastewater treatment processes since the growth of aerobic chemotrophic bacteria is favoured [79,80]. On the other hand, while microalgae can grow in the presence of oxygen, very high dissolved oxygen concentrations (>25 mg L⁻¹) can cause damage in microalgae cells and inhibit their growth [65]. Fortunately, the oxygen produced by microalgae is rapidly utilized (as electron acceptor) by bacteria for the oxidation of organic matter and NH₄ during wastewater treatment (Figure 5) [37].

5.1. Open Photobioreactors

Open ponds were the oldest configurations proposed for photosynthetic microorganisms cultivation at industrial scale for microalgae biomass production [81]. This configuration was proposed to mimic the most common growth of microalgae in nature, using natural or artificial lakes for microalgae culture with different depths. These ponds are less expensive to operate and build compared to other photobioreactor configurations at large-scale. However, their open configuration render them more sensitive to the variations in the weather conditions, to microbial contamination and exhibit high water evaporation levels [82]. This first configuration was limited by the small photic zone when microalgae concentration is high and the high costs associated to nutrient homogenization.

The design of open ponds was improved in order to optimize microalgal activity by reducing their depth to favour photosynthetic activity and reducing energy costs for mixing, which resulted in the so called open raceways or high rate algal ponds. This type of photobioreactor consists of a shallow lagoon (deep of 0.2–0.4 m) with multiple channels and mixing via a paddle wheel motorized (Figure 6) [63]. The continuous mixing by a paddle wheel favours the agitation of the culture broth, exposing the cells to constant radiation during the day (under solar irradiation), which favours the photosynthetic process. This agitation mode is simple and requires a low electricity consumption. Numerous studies describing the cultivation of microalgae and even wastewater treatment in open raceways, with moderated-to-high microalgae productivities are available in literature [63,64,65,66,83,84]. On the other hand, PPB growth in 10 cm deep open raceways using aerobic PPB cultures (dominated by Rhodobacter capsulatus) in synthetic media has been reported [85]. The main disadvantage of open raceways and open photobioreactors are the high evaporation rate and the associated consumption of water to compensate these evaporation losses [64,77].

5.2. Closed Photobioreactors

Closed photobioreactors design involve a limited contact of the cultivation broth with the atmosphere. This configuration exhibits a superior performance in term of control of microorganisms growth, high surface area to volume ratio, low water evaporation losses, higher light availability and easy control of operational parameters, which increase photosynthetic biomass productivity compared to their open counterparts (Table 3) [77,81].

The most studied closed photobioreactors for cultivation of photosynthetic microorganisms are horizontal tubular and flat-panel photobioreactors due to their easier scalability. These photobioreactors need colourless material for their construction, such as glass, polymethyl-methacrylate or polyvinyl chloride (PVC), which increases their investment cost [65]. In this sense, flat-panels photobioreactors using bags of colourless high-density polyethylene supported in a metal cage have been proposed as one of the most economical enclosed photobioreactors for the cultivation of microalgae. On the other hand, tubular configurations are easily scalable by increasing the number and length of tubes, and exhibit higher light utilization efficiencies compared to flat-panel photobioreactors. Both types of closed configurations favour the availability of solar radiation, enhance photosynthesis and reduce the footprint required for their construction.

In recent years, the most studied photobioreactor configuration for the cultivation of PPB is the photo-anaerobic membrane bioreactor [36,49,88]. This configuration is composed of a rectangular polymethyl-methacrylate photobioreactor with a submerged flat sheet membrane (0.45 µm pore size) and operated under anaerobic conditions (air-tight system) with internal gas recycling as agitation strategy [88]. This type of photobioreactor enables an excellent biomass concentration inside of the photobioreactor and provides an effluent with a low concentration of total solids. However, photo-anaerobic membrane bioreactors also present disadvantages such as the formation of biofilms on the internal walls of the photobioreactor and on the membrane, which hinders an adequate suspension of the PPB cultures. Additionally, the membrane module increases both the investment and operating cost of this technology.

6. Biomass Valorization

The potential high productivities of photosynthetic microorganisms (PPB and microalgae) and the inherent presence of high value-added products represent a unique opportunity to turn wastewater treatment based on photosynthetic microorganism into a profitable process [38,46,89]. PPB biomass contains a high content of pigments (bacteriochlorophylls and carotenoids), single cell protein, coenzyme Q₁₀, pantothenic acid, amino acid 5-ALA and polyhydroxyalkanoates [56]. On the other hand, microalgae can synthetize high concentrations of carbohydrates, proteins, lipids and pigments (chlorophylls and carotenoids) [30,70]. In addition, the biomass of photosynthetic microorganisms can be used as a feedstock for the production of biofuels like biodiesel and bioethanol [90], while some species of PPB and cyanobacteria are capable of producing biohydrogen. PPB and microalgae biomass can be also used as a biofertilizer for plants as an N₂-fixing rhizosphere and are known to accumulate compounds that are beneficial for plant growth [76,86] (Figure 8).

Pigments: Photosynthetic microorganisms inherently present high concentrations and a broad portfolio of natural pigments since they are essential molecules for obtaining energy from solar radiation [68]. PPB synthesize a variety of pigments mainly of the group of bacteriochlorophylls and carotenoids, which conform light harvesting complexes [38]. Bacteriochlorophyll a and b are pigments found mainly PPB, both pigments with a characteristic green colour. Carotenoids provide the characteristic orange to purple colours to the group of PPB [13]. Thus, the major carotenoids synthesized by PPB are lycopene, rhodopsin, spheroidene, spirilloxanthin and their derivatives [39,91]. On the other hand, microalgae are an important source of commercial high value compounds and can synthetize mostly pigments of the group of chlorophyll and carotenoids [69,92]. This last group of pigments include β-carotene, astaxanthin and lutein [92,93]. In addition, cyanobacteria can synthesize pigments belonging to the group of phycobiliproteins. An important and rare pigment of this last group is phycocyanin, a pigment with a striking blue colour that can absorb at wavelengths of ~620 nm and present promising antibacterial, anticancer, anti-inflammatory and antioxidant activities [12,94]. All those pigments are nowadays commercialized for the production of cosmetics, pharmaceuticals and functional foods.

Single-cell protein: Single-cell proteins, a term coined to refer to proteins from microbial origin, have been proposed as a sustainable substitute of traditional animal or vegetable proteins. Photosynthetic microorganisms can achieve a fast growth compared to plants and animals, and could potentially support high protein productivities, even when growing in low nutrient media or wastewater. This PPB and microalgae biomass valorization strategies is attracting and increasing attention due to the high commercial value of proteins for the production of animal feed or supplements for human nutrition [38]. PPB typically exhibits a protein content between 41% and 86% [95], while microalgae protein a content ranges between 28% and 71% [96]. Spirulina platensis is the most cultivated microalgae (cyanobacteria) for commercial purposes due to its high content of protein (up to 70%) and essential amino acids supporting a high nutritional value. Indeed, S. platensis has been authorized for human consumption as a protein supplement [97], and as a feed supplement in livestock and aquaculture [98]. Single-cell protein can be produced independently of the climatic conditions and availability of arable lands, although nowadays their high costs of production limit the widespread commercialization of single cell protein. In this context, photosynthetic microorganisms can obtain the energy for growth from sunlight, use wastewater as a nutrient and water source and fix nutrient from atmosphere (CO₂ or N₂), which represents a unique combination to reduce the production costs of single-cell protein [99].

Coenzyme Q₁₀: PPB can synthesize coenzyme Q₁₀ under phototrophic conditions and accumulate similar concentrations to other microorganisms such as Escherichia coli and Rhizobium radiobater [38]. The main function of this coenzyme in PPB is the transport of electrons in the electron transport chain, playing an essential role to produce ATP during oxidative phosphorylation. The coenzyme Q₁₀ has antioxidant properties and is widely used in the cosmetic and medical industry [100]. Coenzyme Q₁₀ supplementation in the skin can reduce the wrinkle formation. In addition, this compound prevents and causes beneficial effects in patients with diseases such as Parkinson, cardiovascular and hypertension, thus improving cellular bioenergetics and reducing the occurrence of free radicals due to its antioxidant properties [101]. These benefits for human health entail a high economical value of the coenzyme Q₁₀, with current market prices of ~€500 per kilogram and increasing demand worldwide in the last years [38]. This coenzyme can be efficiently produced in non-toxic wastewater treatment using PPB [102].

Polyhydroxyalkanoates: Polyhydroxyalkanoates are intracellular biopolymers accumulated by multiple photosynthetic microorganisms and bacteria as energy and carbon reserves [103]. These molecules are a type of biopolymer with biodegradable properties and a high potential for substituting traditional petrochemical plastics such as polypropylene or polyethylene (produced from petroleum derivatives), highly harmful to terrestrial and marine biodiversity [104]. The production of polyhydroxyalkanoates in microorganisms using monocultures or mixed cultures requires a limitation of nitrogen or other essential nutrients to promote the accumulation of these biopolymers in PPB and microalgae [38,46]. Short feast periods or nutrient limitations favour the adaptation of microorganisms to accumulate polyhydroxyalkanoates over time [103]. The production of polyhydroxyalkanoates from wastewaters by photosynthetic microorganisms requires low concentration of nutrients to favour the intracellular accumulation of this biomolecule under excess of carbon supply. The main PPB described in literature for their high polyhydroxyalkanoates accumulation capacity are Rhodobacter sphaeroides and Rhodobacter capsulatus [38]. Likewise, cyanobacteria such as Synechococcus sp., Nostoc muscorum and Spirulina platensis have also been described as potential candidates to produce polyhydroxyalkanoates [105]. Although more research is still needed to enhance the accumulation and production of polyhydroxyalkanoates in photosynthetic microorganisms, this solar driven technology platform represents an economic alternative to produce bioplastics.

Biofuels: Photosynthetic microorganisms can produce different types of biofuels, either directly synthesized such as biohydrogen or by additional processes utilizing their biomass such as biodiesel, bioethanol and biogas [38]. Biofuels represent a unique opportunity to boost the development of renewable energy technologies and to reduce CO₂ emissions from anthropogenic activity [70].

Photosynthetic microorganisms have emerged as potential candidates for biohydrogen production due to its high conversion efficiency and the broad portfolio of substrates used to sustain their growth. Biohydrogen (H₂) can be produced directly by PPB (R. palustris, R. sphaeroides and R. rubrum) mainly under a photoheterotrophic growth mode. Biohydrogen is a clean fuel since during its combustion (H₂ + O₂) only energy and water (H₂O) are produced [106]. This gas can be also used in fuel cells providing a very efficient alternative to combustion engines in transportation [107]. The production of H₂ in PPB occurs under illumination, anoxygenic conditions and nitrogen limitations due to the synthesis of the enzyme responsible for hydrogen production (nitrogenase) is inhibited in the presence of ammonium [38,108]. Likewise, microalgae and cyanobacteria species such as Chlamydomonas reinhardtii, Chlorella sorokiniana, Synechocystis sp. or Anabaena sp. can produce directly biohydrogen [109].

Biodiesel was proposed as a potential substitute for conventional petroleum-based fuels. Biodiesel can be produced from plant and animal oils or recently from microorganisms, with a special emphasis on the production from microalgae oil. Biodiesel is a mixture of fatty acid alkyl esters produced by a transesterification process [110]. This process includes three sequential steps, where triglycerides are converted in diglycerides, monoglycerides and finally converted into esters (biodiesel) and glycerol (by-product). The transesterification process involves a triglyceride and a short chain alcohol (e.g. methanol) in presence of a catalyst (e.g. sodium hydroxide) for synthesis. The triglyceride to biodiesel ratio is 1:1 (e.g. 100 g of biodiesel can be obtained from 100 g of triglycerides). The ability of PPB to produce transesterificable oils to produced biodiesel has not been yet investigated. However, the ability of microalgae to synthesize high lipid contents under nutrient limitation (40–75% for the species with the highest lipid production) has attracted a great interest and research efforts in the mass cultivation of this type of photosynthetic organisms to produce biofuel in the past 15 years [63,82].

Bioethanol is a biofuel produced from the carbohydrates contained in plant biomass or microorganisms [111]. This biofuel is produced through alcoholic fermentation processes of hydrolyzed biomass, which produce an ethanol with an identical chemical composition to that of chemical origin, and that also requires a distillation step for its purification [90]. Bioethanol can be used as a biofuel directly or mixed with conventional gasoline. In the past, ethanol production from microalgae biomass has been proposed based on its high carbohydrate content in terms of cellulose and starch, which can be fermented for bioethanol production [112]. In addition, the CO₂ produced from ethanol combustion can be recovered since microalgae are capable of fixing CO₂, thus neutralizing carbon emissions to the atmosphere and creating a cycling process for carbon utilization [64,113].

Finally, the most studied biofuel produced from photosynthetic microorganisms biomass is methane. Methane can be produced from anaerobic digestion of PPB or microalgae biomass, where the degradation of the organic matter contain in these photosynthetic microorganisms occurs under anaerobic conditions in different steps catalyzed by anaerobic bacteria and archaea [11]. The first process in anaerobic digestion is a hydrolysis step, where complex organic matter is converted into simpler molecules such as amino acids, fatty acids and monosaccharides by different microorganisms. These simpler compounds are converted in a second acidogenesis step into volatile fatty acids and alcohols. The latter compounds are biotransformed by acetogenic bacteria during the acetogenic step into acetic acid, hydrogen and carbon dioxide as the main metabolites. Finally, methanogenic archaea transform the acetic acid, carbon dioxide and H₂ into biogas during the methanogenic step [114]. Biogas is a mixture of gases mainly composed of high concentrations of methane (40–75%), carbon dioxide (15–60%), hydrogen sulfide (0.005–3%), nitrogen (0–2%) and oxygen (0–1%) [30]. Biogas upgrading to biomethane is necessary to remove unwanted gases such as CO₂ and H₂S if biogas is used as vehicle fuel or injected into natural gas grids. This biogas upgrading step can be carried out using PPB [31,35] and microalgae [115,116,117] based on the ability of these microorganisms to fix CO₂ and support H₂S oxidation.

Biofertilizers: Biofertilizers are fertilizer biological origin capable of promoting growth and the nutritional quality of plants. PPB can be directly used as biofertilizers (active benefit) since these photosynthetic microorganisms can grow in symbiosis with plants as rhizosphere and are capable to synthesize and excrete compounds promoting plant growth. For instance, PPB can synthesize the amino acid 5-ALA, beneficial to plant growth [56]. 5-ALA can be used as plant growth enhancer and even possesses insecticide and herbicide properties [38,95]. In addition, when PPB are associated with plant roots, they can contribute to heavy metal remediation in soils via accumulation into PPB biomass, which reduces adverse effects of these metals inside plant [118]. In addition, PPB biomass can be used as dried biomass (passive benefit) due to its high nutrient (N and P) content [38,86]. PPB can accumulate phosphate in form of polyphosphates and fix nitrogen from the atmosphere, nutrients that are necessary for plant growth and limited in soils used for intensive agriculture [86]. On the other hand, the merits of microalgae biomass as slow-release biofertilizers have been consistently reported in literature [119]. Microalgae biomass is also rich in nitrogen, phosphorous and trace elements such as potassium, calcium, magnesium and iron, that can be absorbed by plants when microalgal biomass is supplemented and even increase the nutritional quality of biofertilized fruits [12].

7. Conclusions and Future Prospects

PWW management represents a serious challenge to the sustainability of pig farming due to its high production and pollutant concentrations. Conventional PWW treatments are only partially able to treat this type of wastewater at the expenses of a high energy consumption, prohibitive greenhouse gas emissions and a waste of resources. Photosynthetic microorganisms such as PPB and microalgae represent a promising solution for PWW valorization due to their ability to obtain energy from solar radiation, fast growth and high nutrients assimilation. The high versatility of photosynthetic metabolism supports PWW treatment under aerobic and anaerobic conditions in a wide variety of photobioreactor configurations. Added value bioproducts such as pigments, single-cell protein, biopolymers, biofuels and biofertilizers can be obtained from both PPB and microalgae biomass. However, despite the promising advances achieved in PWW treatment using photosynthetic microorganisms, the future research needed to move this platform technology to commercial scale should focus on its optimization under outdoor conditions, which will allow evaluating the effects of direct solar irradiation, daily and seasonal changes in light intensities and temperature, changes in PWW composition and microbial contamination on process performance. In addition, the scale-up of continuous photobioreactors treating PWW using photosynthetic microorganisms in relevant environments and the potential of the photosynthetic biomass generated during PWW treatment must be evaluated.