Wastewater Substrate Disinfection for Application of Synechococcus Elongatus PCC 7942 Cultivation as Tertiary Treatment

The cultivation of microalgae or/and cyanobacteria in nutrient-rich wastewaters presents a signicant opportunity for enhancing sustainability of tertiary wastewater treatment processes via resources/energy recovery/production. However, maintaining a monoculture in wastewater-media constitutes a signicant challenge to be addressed, as a plethora of antagonistic and predating microorganisms exist is such media. In this regard, the present work assesses the eciency of the low-cost wastewater substrate disinfection techniques of ltration, use of NaClO, H 2 O 2 or Fe(VI), in terms of antagonistic or/and predating microbial species growth inhibition in Synechococcus elongatus PCC 7942 cultivations. Nitrates and phosphates removal rates were also experimentally assessed. The results showed that lter thickness has a greater effect on disinfection eciency than that of lter’s pore size. Furthermore, the disinfection eciency of Fe(VI), which was produced on-site by electrosynthesis via a Fe 0 /Fe 0 cell, was greater than that of NaClO and H 2 O 2 . Filtration at ≤ 1.2 µm pore size coupled with chemical disinfection leads to unhindered S7942 growth and ecient nitrates and phosphates removal rates, at dosages of CT ≥ 270 mg min L −1 for NaClO and CT ≥ 157 mg min L −1 for Fe(VI). The coagulation action of Fe(III) species that result from Fe(VI) reduction and the oxidation action of Fe(VI) can assist in turbidity, organic compounds and phosphorous removal from wastewater-media. Moreover, the residual iron species can assist in S7942 harvesting and may enhance photosynthesis rate. Thus, the utilization of wastewaters for S7942 cultivation as tertiary treatment seems a promising and novel alternative to common nutrient removal processes that can reduce environmental footprint and operational costs of wastewater treatment plants.


Introduction
Preservation of freshwater quality and inversion of climate change are inextricably linked to the development and full-scale application of sustainable technologies that align with the concept of circular economy i.e., reduce-reuse-recycle (Sakai et al. 2011) and respond to the challenge of a carbon neutral future, as set in Paris Agreement (2016). Thus, the implementation of the best available technologies, practices and techniques (BAT) for the protection of the environment, the recovery of resources and the mitigation of greenhouse gases emissions must be the response to the challenge of sustainability.
Due to the high nutrient and energy content of wastewaters, the wastewater treatment sector offers signi cant opportunities for the application of BAT that align with the concept of circular economy (Hoek et al., 2016). Wastewater treatment processes that are based on phyceae or/and cyanobacteria for the removal/recovery of nutrients suggest the evolution of activated sludge (AS) systems. In particular, the utilization of cyanobacteria for wastewater treatment coupled with resources recovery have received much attention as a novel and alternative resource for carbon emission mitigation and the production of third generation biofuels and/or biochemicals (Vassilev and Vassileva 2016;Maurya et al. 2021).
According to this, many studies have been carried out where cyanobacteria are used as a bioremediation tool in order to treat wastewater e uents of various origins including pig slurries, domestic activities, aqua culture systems e.t.c (Ansari et al. 2017; Morillas-España et al. 2021; Winayu et al. 2021;Samiotis et al. 2021). Worth mentioning that some cyanobacteria strains may produce cyanotoxins, while other species such as Synechococcus elongatus PCC 7942 (hereafter S7942) do not produce cyanotoxins and under saline stress can increase its intracellular sucrose accumulation leading to increased yields of biofuels (biodiesel, bioethanol, biogas, bio-hydrogen) or/and relevant industrial products from S7942 (Stamatakis et al. 1999;Vayenos et al. 2000;Nowruzi et al. 2021). The cultivation of cyanobacteria in wastewater streams is considered a sustainable alternative to common nutrient removal processes and most promising for reducing the additional costs associated with cyanobacteria cultivation in full-scale (Arias et al. 2017; Samiotis et al. 2021). Cyanobacteria xate signi cant quantities of CO 2 and produce O 2 and high-value biomass that can be used for the production biofuels, hydrocarbons, proteins, pigments and biopolymers for pharmaceutical, chemical and food industry (Trivedi et al. 2015;Pathak et al., 2018). The cultivation of cyanobacteria for the production of biofuels outperforms, in terms of sustainability, the conventional technologies that utilize sugar, starch, vegetable or animal fats due to the competitive consumption of food resources and the associated commitment of arable land, especially if it is combined with wastewater and/or ue gases treatment (Rosegrant et  It is important to highlight that many studies on cultivation of microalgae have been adapted to laboratory settings and thereby somewhat have been detached from real world conditions. This included nutrient addition (Ansari et al. 2017), use of synthetic wastewater (Barnharst et al. 2018), or very short cultivation periods (Halfhide et al. 2014). Wastewater quality may directly affect the growth rate and nutrient removal e ciency of microalgae. This has been con rmed by the study of Tejido-Nuñez et al. (2019) that used Chlorella vulgaris and Tetradesmus obliquus to treat e uents from sh aquaculture systems. Their results showed that the e ciency of nitrate and phosphate removal was highly depended on the choice of the species involved and the pretreatment of wastewater using sterile ltration prior to cultivation, which enhanced the growth of the aforementioned species.
In addition, there are many limitations resulting from microalgae implementation including the use of wastewater as a culture medium and the generation of biomass in the presence of other microorganisms that compete for nutrients. The co-presence of other microorganisms needs to be limited by the application of several common disinfection methods including ltration or the addition of chemical disinfectants comparing to simple lab techniques such as autoclave that cannot be applied due to high cost and energy demands. Sodium hypochlorite (NaClO) in concentrations of 0.2 mg L −1 to 2 mg L −1 has shown good disinfection, with 1.9 mg L −1 being recommended. This dose allows disinfection and at the same time chlorine residues of less than 0.2 mg L −1 , after 24 hours still remain in the wastewater media (Medrano-Barboza et al. 2021).
Besides disinfection with NaClO, novel and considered environmentally friendly disinfection techniques have been developed, with those based on hexavalent iron (ferrates) production showing a particular interest. The merits of using hexavalent iron for disinfection is that it presents high oxidative ability and it is transformed completely to the non-toxic trivalent form, simultaneously acting as a coagulant (Al Umairi et al. 2021). Due to their high oxidative and aggregation ability, ferrates can oxidize organic compounds from wastewaters, as well as microorganisms and their spores leading to clearer growth media, where photosynthetically active radiation (PAR) can travel unhindered by turbidity (Škulcová et al. 2021). Furthermore, the aggregative action of ferrates can assist on the collection of microalgae or/and cyanobacteria from a photobioreactor, a process that has been criticized due to its low e ciency and/or the high associated cost It is well known that ultra ltration and sand ltration are simple techniques that have been used in water and wastewater treatment as low-cost particle and pathogen removal lters ( sand lters for the removal of Cryptosporidium in commercial swimming pools. Their review clearly explains that sand lters e ciency is not only depended on pore size but also on lter depth. Particles which are too small to be screened could be retained by the lter media as a result of weak intermolecular binding forces that come into play if the particles can get very close to the surface of the sand grains. Young-Rojanschi and Madramootoo (2014) also observed that increased lter media depths resulted in improvements of performance for E. coli reduction.
Aim of this study is to assess the possibility of utilizing biologically treated wastewaters as substrate for cultivation of S7942 monoculture in a photobioreactor, which can be used for tertiary treatment applications. More speci cally, the e ciency of low-cost wastewater substrate disinfection techniques for antagonistic or/and predating microbial species growth inhibition are evaluated. For this aim, S7942 growth rate and nutrients removal in properly disinfected wastewater-media were studied. This work is considered of great importance for the implementation of cyanobacteria-based wastewater treatment processes, as sustainable alternative or supplementary treatment stage to conventional biological nutrient removal processes.

Materials And Methods
For the assessment of biologically treated wastewaters adequacy as S7942 growth-media in terms of growth inhibition by the presence of antagonistic or/and predating microbial species, a series of cultivation photobioreactors were set up using sterilized 500 ml Erlenmeyer asks. Sterile cotton caps were used for the uninhibited air transfer inside the photobioreactors avoiding the risk of air borne contamination. Moreover, nutrients removal rate was evaluated. A culture of S7942 in BG-11 growthmedium (Rippka et al. 1979) was used as inoculum for control and test setups. The inoculums were separated from media via centrifugation at 5000 rpm for 10 minutes. The photobioreactors were kept under continuous agitation and arti cial lighting ( uorescent lamps at light intensities of 5 -30 µmolphotons m −2 s −1 ), at controlled temperatures of 20 o C to 26 o C.
The control setups contained BG-11 media, while the test setups contained biologically treated industrial wastewater (wastewater-media) that had been subjected or not to a single or a combination of low-cost disinfection techniques. The wastewater-media was obtained from the nitri cation tank of a dairy industry's activated sludge (AS) wastewater treatment plant (WWTP), after suspended solids removal via sedimentation. Both control and test setups had an initial S7942 biomass content, in terms of chlorophyll a concentration, of approximately 1 mg L −1 .
The disinfection performance of ltration (at pore size of 0.45 µm up to 5-13 µm), the use of NaClO (chlorination), hydrogen peroxide (H 2 O 2 ) and ferrates (Fe(VI)) in wastewater-media were evaluated prior the S7942 growth assessment. The disinfection e ciency of each technique was determined by microbiological examination (total viable count -TVC at 22 o C). The effectiveness of the disinfectants was evaluated in terms of CT calculation and TVC measurement (ISO 6222/99). CT is de ned as the disinfectant residual concentration (C) multiplied by the effective contact time (T), expressed as mg min L −1 . The inhibitory action of different concentration disinfectants on S7942 growth rate was also studied. The growth of antagonistic or/and predating microbial species in the test setups was checked via microscopic examination using a Leica phase contrast microscope (x40 up to x1000 magni cation).
The disinfection with ferrate, which is considered a novel and environmentally friendly disinfection technique (Al Umairi et al. 2021) was evaluated additionally in terms of its coagulation ability by assessing organic compounds (chemical oxygen demand, COD), phosphates and turbidity removal.

Assessment of ltration
The porosity and the thickness of a lter-media are two key parameters that dictate ltration performance. The small pore size of a lter introduces higher retention rate, whereas the high porosity is de ned by a higher permeability capability. Furthermore, lter thickness affects the duration of ltration and subsequently ltration e ciency (Fahimirad et al. 2021). In our study, lters of different pore size and thickness were evaluated regarding their ability to remove the existing microbial load of the wastewatermedia for S7942 cultivation. More speci cally, lters with pore size of 0.45 µm, 0.7 µm, 1 µm, 1.2 µm 2-4 µm, 3-5 µm, 5-7 µm and 5-13 µm were tested for TVC reduction in the ltrates. Their thickness and respective material are presented in Table 1, along with the results from TVC measurements. As shown in Table 1, the highest disinfection e ciency was achieved with the ber glass lters, which are characterized by increased thickness (0.26 mm to 0.70 mm). The 0.45 µm pore size cellulose lters, which had signi cantly smaller thickness than of the ber-glass, presented lower disinfection e ciency, despite having the smallest lter pore size. The paper lters with pore sizes greater than 2 µm presented worst disinfection e ciency than the ber-glass and cellulose lters. Among these paper lters, the thickest one (0.32 mm) presented the highest disinfection e ciency, in contrast to the two smaller pore sizes paper lters (2-4 µm and 3-5 µm).
In the test setups, the growth of antagonistic and predating microbial species suppressed to the decline S7942 growth leading to almost complete extinction of S7942 population in terms of chlorophyll a ( Figure 1). Chlorophyll a concentrations dropped from the initial concentration of 1 mg L −1 to 0.03 mg L −1 up to 0.19 mg L −1 after 20 days. The lters that presented the highest disinfection e ciency (0.7 µm and 1 µm pore size ber-glass lters) presented a 5-days delay in the decline of chlorophyll a concentration, attributed to the low initial population of antagonistic or/and predating microbial species in the ltrates.
It is concluded that none of the applied ltration techniques is e cient to sustain a monoculture of S7942 in the photobioreactors. It is evident from Table 1 and Figure 1 that both pore size and lter thickness have a signi cant effect on disinfection e ciency. However, the thickness of the lter, i.e. the duration of ltration, seems to have a greater effect on disinfection e ciency than that of lter's pore size. This is based on the observations that (a) the smaller TVC count was obtained using the thickest lter (0.70 mm thickness, 1 µm pore size) and not when using the smallest pore size lters of 0.70 µm or 0.45 µm and (b) the thickest paper lter (0.32 mm, 5-7 µm pore size) presented the highest disinfection e ciency when compared to lters with >2 µm pore size. Thus, in real scale applications, an ultra ltration con guration or a slow sand ltration technique could be used as a low-cost preliminary disinfection process for the signi cant minimization of viable microorganisms in a wastewater-media, but further polishing would be required for complete disinfection.

Assessment of chemical disinfection by Sodium hypochlorite, Hydrogen peroxide, Hexavalent iron
Similarly to ltration, chemical disinfection alone, using NaClO or H 2 O 2 or Fe(VI), is proven to not be able to alleviate the problem of S7942 culture contamination. While higher dosages of these disinfectants, in terms of CT, led to lower TVC at 22 o C, complete disinfection could not be achieved. Even at considerably high NaClO or H 2 O 2 or Fe(VI) dosages of 5530 mg min L −1 , 12000 mg min L −1 , 3105 mg min L −1 respectively, viable microorganisms are still accounted in the wastewater-medias (Figure 2). The effect of the suggested NaClO solution for wastewater disinfection has been also studied by Medrano-Barboza et al. 2021. According to their study and similarly to our results, total inactivation of bacterial load could not be achieved at NaOCl concentrations up to 60 mg L −1 , despite the observed signi cant decrease in TVC.
In our study high e ciency disinfection was achieved at NaOCl concentrations of 92 mg L −1 and 184 mg L −1 for 30 min and 60 min application respectively. Sodium hypochlorite solution presented approximately 2.2 times higher disinfection e ciency compared to H 2 O 2 , thus the use of NaClO is suggested.
The highest disinfection e ciency was observed using the freshly prepared via electrosynthesis Fe(VI) solution ( Figure 2). Moreover, the resulting Fe(III) species from ferrates reduction proved to be a potent coagulant for the removal of particular matter and colloidal organic compounds that may be present in wastewater-media, which has a signi cant impact on light transfer e cacy in a photobioreactor (Škulcová et al. 2021). For Fe(VI) concentrations of 4.2 mg L −1 to 51.7 mg L −1 , the process e ciency, in terms of turbidity and COD removal, ranged from 89-97% and 71-84% respectively. Worth mentioning that COD removal can be also attributed to organic compounds oxidation by Fe(VI) .
Our results are in accordance with the ndings of other researchers. More speci cally, the dual-function of ferrate as a coagulant and disinfectant for chemically-enhanced primary treatment has been also reported by Al Umairi et al. (2021). They found that a ferrate dose of 0.5 mg L −1 Fe removed 80% of total suspended solids (TSS), 57% of chemical oxygen demand (COD), whereas higher concentrations of ferrate at 15 mg L −1 as a disinfectant were necessary in order to achieve a 5-log removal of E. coli. Zhang et al. (2020) also demonstrated that ferrate (VI) addition at 6 mg L −1 can effectively serve as a core treatment process removing simultaneously turbidity (98%) and inactivating 100% total coliforms in one single dose.
The results of our study suggest the use of ferrate solution as a disinfectant/coagulant is a novel and effective treatment approach for disinfection of wastewaters meant to be used as substrate for cyanobacteria monoculture, offering the additional bene t of collectively removing TSS, turbidity and microbial load. Worth mentioning that the evident coagulative action of ferrates can assist on the emerging problem of microalgae or/and cyanobacteria harvesting from the photobioreactors (Addison et al. 2021). An additional merit of a ferrate-based disinfection technique is that the resulting Fe(III) from ferrates reduction could enhance photosynthesis in the photobioreactor. Fe(III) species have been shown to enhance biomass production (Rana and Prajapati 2021). Thus, a ferrate solution could replace NaClO or H 2 O 2 as disinfectant, offering a more sustainable solution to wastewater-media disinfection.
Nevertheless, further study regarding optimal Fe 0 /Fe 0 cell con guration and current density is considered essential towards energy consumption minimization for Fe(VI) production.

Synergy of studied disinfection techniques
As previously presented, each of the studied disinfection technique alone could not address the challenge of maintaining a growing S7942 monoculture in the photobioreactor. Hence, the suggested low-cost disinfection technique of ltration was evaluated as synergetic couple with the use of NaClO or Fe(VI), in terms of disinfection e ciency and S7942 growth in the experimental setups ( Figure 3).
The assessment of disinfection e ciency showed that complete wastewater-media disinfection, in terms of TVC, can be achieved with minimized NaClO or Fe(VI) dosages, if a preliminary ltration stage is applied. No viable microorganisms were accounted at all wastewaters ltrated with ≤1.2 µm pore size lters (Table 1) and at disinfectant dosages of CT ≥ 270 mg min L −1 and CT ≥ 157 mg min L −1 for NaClO and Fe(VI) respectively. The obtained wastewater-media with this procedure are considered properly disinfected. In the case of NaClO disinfection, dechlorination of the wastewater-media with sodium thiosulphate is necessary prior its use, in order to prevent the inhibition of S7942 growth by residual chlorine.
The properly disinfected wastewater-media were used for the study of S7942 growth in 8 test setups (Figure 3). The results showed that S7942 cultures in test setups present comparable to the control cultures growth rates. This implies that ltration coupled with disinfection using NaClO or Fe(VI) is effective. The ber-glass lters with pore size 0.7 µm and 0.45 mm thickness, as well as with pore size 1 µm and 0.70 mm thickness were proved more effective. It should be noted that at the applied dosages of Fe(VI) solution, neither the quantities of residual Fe(VI) nor of total iron had an impact on S7942 growth rate. There was no residual action of NaClO, due to the applied dechlorination of wastewater-media. Thus, low-cost ltration coupled with NaClO or Fe(VI) disinfection is an e cient procedure prior the use of wastewaters as S7942 cultivation substrate and tertiary treatment.
Antagonistic and predating species growth in S7942 culture setups Microscopic examination of S7942 cultures setups with different non-properly disinfected wastewater media, one from a dairy industry and another from a salty-snack industry, were conducted. As shown in Figure 4 -inset 2, yeasts were the dominant antagonistic species to S7942 in the dairy wastewater test setups. On the other hand, protozoan (mainly ciliates) and metazoan (mostly rotifers) dominated in in the salty-snack wastewater test setups (Figure 4 -inset 3). Beside the growth of non-phototrophic microorganisms, antagonistic to S7942 phototrophic species were also developed, especially in experimental setups with non-ltrated wastewater-media (Figure 4 -inset 4). In Figure 4 -inset 1, an uninfected S7942 culture is depicted.
The results indicate the necessity of biological contaminants control, since they pose a serious threat to microalgae or cyanobacteria cultivation. According to Day et al. (2017) even small numbers of herbivorous protozoa can rapidly multiply in and thus destroy a culture of microalgae. Tejido-Nuñez et al.
(2019) who studied the effect of sterile ltration as a pretreatment on Chlorella vulgaris and Tetradesmus obliquus growth, found that protozoa were observed in all non-sterile water samples resulting in the decline of C. vulgaris biomass and therefore reducing the nutrients removal e ciency of the process.
It is noteworthy that in properly (coupled) disinfected wastewater-media with ltration using cellulose lters with pore size 0.45 µm and 0.135 mm thickness, as well as using ber-glass lters with pore size 1.2 µm and 0.26 mm thickness, limited growth of yeast and small-size ciliates (smaller or even-sized to S7942) was observed after the rst week of cultivation. However, the growth of these microbial species did not hinder S7942 growth.
S 7942's growth rate and nutrients removal rate in disinfected wastewater-media The properly disinfected wastewater-media, presented in Figure 3, were used for the study of S7942 growth. Based on equation (1), the % speci c growth rate of S7942, as well as the % speci c nitrates and phosphates removal rates were evaluated in control and test setups ( Figure 5). For this purpose, 2 control setups, 4 test setups with wastewater-media ltrated at ≤1.2 µm pore size and chlorinated/dechlorinated and 4 test setups with wastewater-media ltrated at ≤1.2 µm pore size and disinfected with Fe(VI) were monitored for a 20 days period. The average values of % relative S7942 growth rate, as well as the average values of nitrates and phosphates removal rates of each setup group are presented in Figure 5. Nutrients removal rates in the test setups were similar or even greater to those obtained from control setups, with signi cantly higher phosphates removal rate observed when Fe(VI) is used, attributed to the coagulative action of the resulting Fe(III).
According to Figure 5, the average relative growth rates (RGR (chl a) ) in the test setups with properly disinfected wastewater-media was 19.7% when ltration/chlorination was applied and 22.4% when ltration/Fe(VI) chemical disinfection was applied. These values indicate unhindered growth of S7942 in wastewater media, since they are similar to the RGR (chl a) average value of 21.9% obtained in the control setups with BG-11 medium. The su cient growth of S7942 is also evident from the average values of nitrates relative reduction rate (RRR NO3_N ) and phosphates relative reduction rate (RRR PO4_P ), which ranged from 2.51-3.07% and 11.33-14.40% respectively, compared to those obtained in the control setups (RRR NO3_N = 2.84% and RRR PO4_P 0.72% respectively). It is evident that RRR NO3_N of control and test setups is analogous to their respective growth rate, having a relatively constant RRR NO3_N /RGR (chl a) ratio of approximately 0.13. Thus, it can be concluded that nitrates removal is attributed solely to assimilation of nitrate-nitrogen into the S7942 biomass and not on additional physicochemical processes. On the other hand, the RRR PO4_P /RGR (chl a) ratio is signi cantly different in the control setups and the test setups, which indicate that apart from phosphates assimilation, parallel physicochemical processes occur in the photobioreactors. The RRR PO4_P /RGR (chl a) ratio of the control setups was 0.49, while of the test setups with ltrated/chlorinated wastewater-media or ltrated/chemically disinfected with Fe(VI) wastewater-media, were 0.58 and 0.64 respectively. The higher phosphates removal rates of test setups is attributed to increased coagulation induced by elevated pΗ levels (da Silva Cerozi and Fitzsimmor 2016), since both NaClO solution and Fe(VI) solution were highly alkaline, as well as to the coagulation action of Fe(III) species resulting from Fe(VI) reduction (Zhang et al. 2020

Conclusions
Synechococcus elongatus PCC 7942 (S7942) can be cultivated in properly disinfected wastewaters, thus used for tertiary treatment applications. E cient disinfection of wastewater-media can be achieved by coupling ltration with chemical disinfection using NaClO or the more environmentally friendly Fe(VI).  Indicative S7942 growth curves in uninfected and infected experimental culture setups (Microscopic depiction of S7942 cultures at x1000: Inset1 = uninfected; Inset 2-4 = infected). Figure 5