Microalgae impact on inactivation of indicator virus in a large-scale wastewater treatment system using microalgae

doi:10.21203/rs.3.rs-5004931/v1

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Microalgae impact on inactivation of indicator virus in a large-scale wastewater treatment system using microalgae

https://doi.org/10.21203/rs.3.rs-5004931/v1

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Wastewater treatment systems using microalgae called High Rate Algal Ponds (HRAP) have been increasingly considered as alternative solutions to classical activated sludge systems. In these paddlewheel-mixed shallow raceways (30 cm depth), oxygenation of the HRAP by microalgae replaces artificial aeration for aerobic degradation of organic matter. In HRAP, pathogen removal mainly relies on ultraviolet (UV) radiation from the sun. UV radiation induces photochemical modifications of DNA and RNA, leading to pathogen inactivation. However, high turbidity due to microalgae and detritus from the wastewater reduces UV penetration in HRAP. Paddlewheel mixing has then a profound impact on the treated water quality by exposing microbial pathogens to higher UV irradiation at the pond surface. Microalgae are expected to contribute significantly to turbidity in HRAP, however, they are also responsible for high oxygen concentration, high pH and, in the presence of UVA, production of Reactive Oxygen Species (ROS) favoring disinfection, questioning the relative impact of microalgae on pathogen inactivation. The purpose of this study was to investigate, in a laboratory UVA cabinet, the impact of microalgae on indicator viruses’ inactivation, in terms of UVA attenuation (inhibition of inactivation) and production of ROS (enhancement of inactivation). This study highlighted a significant negative impact of microalgae due to UVA attenuation over 30 cm depth together with a strong inherent capacity to produce ROS for virus inactivation, confirming the relevance of vertical mixing for disinfection in Peterborough HRAP.

high rate algal ponds

disinfection

ROS

ultraviolets

turbidity

The contribution of ROS from wastewater to MS2 inactivation was very low.
When UVA penetration is sufficient, microalgae play a major role in MS2 inactivation by ROS.
Thin-film systems could be considered as tertiary treatments targeting pathogens removal.

Wastewater treatment systems using microalgae called High Rate Algal Ponds (HRAP) have been increasingly considered as alternative solutions to classical activated sludge systems. In these paddlewheel-mixed shallow raceways (30 cm depth), oxygenation of the HRAP by microalgae replaces artificial aeration for aerobic degradation of organic matter. The removal of pathogens also constitutes a crucial challenge for these alternative wastewater treatment systems. Pathogens conveyed by wastewater are responsible for diseases such as cholera, dysentery, typhoid, intestinal worm infections and polio. Poor wastewater pathogen removal contributes both to spreading those diseases and to enhancing antibiotic resistance (World Health Organisation 2022). In classical wastewater treatment systems, pathogens are removed by tertiary treatments such as chlorination, UV irradiation or ozonation, when necessary (Lian et al. 2018). In HRAPs, solar disinfection contributes to pathogen removal. Sun radiations include UVB (280–315 nm), UVA (315–400 nm) and visible light (400–800 nm). While visible light generally show negligible effect on pathogens inactivation (Bolton 2012), ultraviolet radiation from the sun generate photochemical modifications of microorganisms and viruses DNA and RNA, causing the inhibition of DNA replication and transcription and the interruption of cell division. UVB, that cause direct damage to RNA, was reported to be the predominant mechanism associated with disinfection by sunlight (Lian et al. 2018). In the presence of UVA, the molecules present in the water can act as photosensitizers and contribute to disinfection process. Photosensitizers are light absorbing compounds that transfer their energy to other molecules leading to the formation of Reactive Oxygen Species (ROS) that can damage microorganisms, virus membranes and capsid proteins. Photosensitizers are either exogenous, including humic substances, photosynthetic pigments and dissolved organic matter, or endogenous, including cells able to absorb wavelengths between 290 and 750 nm (Bolton et al. 2011).

Microalgae are generally highly resilient to UV damage due to efficient biosynthesis of photoprotectants and antioxidant molecules, DNA repair mechanisms, migration through the water column and biofilm formation capacities (Rastogi et al. 2020). In contrast, solar radiation enhance inactivation of E. coli, MS2 virus, echovirus and norovirus. Park et al. (2021) reported that, in an outdoor pond, the inactivation of those pathogens was 10 times slower in darkness than when exposed to sunlight. However, microorganisms and virus DNA can be repaired by a photolyase enzyme after irradiation, causing their regrowth (Putois 2012). Pathogen inactivation in wastewater irradiated with natural UV may be influenced by other light dependent processes mediated by algal photosynthesis. Bolton et al. (2012) reported synergistic effects of photosensitizers, high dissolved oxygen concentration, high pH and sunlight on pathogen inactivation in Waste Stabilization Ponds (WSP). Further, noting that while HRAPs presented higher turbidity and lower sunlight penetration through the pond depth than WSPs, they also had higher pH and photosynthetically produced dissolved oxygen concentration due to higher algal biomass and photosynthetic activity. Furthermore, the contribution of photosensitizers from microalgae compared to photosensitizers from wastewater is still poorly studied.

This study follows on from previous work on the 3D characterization of a large-scale HRAP operated in Peterborough, in the mid-north of South Australia (Jahan et al. 2024). In the Peterborough HRAP, penetration of UV radiation was very low since turbidity (absorbance of 2 at 750 nm), chlorophyll a concentration (6 mg.L^− 1) and suspended solids (1 g.L^− 1), composed of approximately 30% of microalgae, were high. These results imply that microalgae were a contributor to sunlight attenuation throughout the pond depth. However, Peterborough HRAP also had very high organic detritus fraction similarly influencing sunlight attenuation. Moreover, inactivation by UV radiations was reported to be less efficient at higher suspended solids concentrations (Lazarova et al. 1999). While organic detritus may adversely affect pathogen inactivation by increasing UV attenuation in the pond, suspended solids may also contribute to the adsorption of viruses, potentially shading them from the UV irradiation.

In winter 2020, Peterborough HRAP exhibited a log reduction value of 1 for coliphage viruses and 2.26 for E. coli along with an E. coli concentration of 3.68 log₁₀MPN.100 mL^− 1 (MPN, Most Probable Number) in the treated water (Butterworth and Fallowfield 2024), which complies with Australian regulations for irrigation of non-food crops but not for irrigation of food crops or public gardens.

The contribution of both wastewater and microalgae to sunlight attenuation in Peterborough HRAP remains unclear, as well as its direct effect on pathogen inactivation. Evaluating the impact of microalgae on pathogen inactivation would be useful for the design of a tertiary treatment system targeting pathogens and would contribute to the optimization of pathogens removal in HRAP. This study aims to determine the impact of microalgae and wastewater on inactivation process in terms of UVA attenuation and ROS production.

The present study focuses on the effect of UVA, that were detected in the volume of Peterborough HRAP and reported to enhance pathogens inactivation (Bolton 2012), of which role through ROS production is less well understood than UVB, especially in systems with algal biomass adversely affecting UVA and UVB penetration. Note that while UVB causes most inactivation, UVB measurements in Peterborough HRAP showed that this range of radiation does not penetrate the pond surface due to high turbidity.

Viruses were found in very large quantities in a HRAP. Hisee et al. (2020) reported a viral load of approximately 10^9.5.mL^− 1 of virus-like particles and 10^8.5.mL^− 1 of large virus-like particle in Kingston-on-Murray HRAP (South Australia). Viruses also constitute a major issue for public health, notably because only one of few infective, viral, particles can lead to a human infection. Furthermore, viruses are usually more resistant than bacteria to disinfecting agents. For these reasons, this study will focus on the inactivation of viruses.

The coliphage virus MS2, a widely used indicator for the presence of coliphages in wastewater, was chosen as the model pathogen. It is an icosahedral virus that belongs to the Leviviridae family. It is a non-enveloped and single stranded RNA virus of very small size (27 nm, Dedeo et al. 2011). 32 pores of 2 nm each on the virus capsid allow the diffusion of small molecules (Dedeo et al. 2011). MS2 also present a protein A for binding to E. coli F-pilus (Zhong et al. 2016). MS2 infection of E. coli down regulates TCA cycle, altering bacterial cell growth and biosynthesis of the cell wall (Jain and Srivastava 2009). This F-RNA coliphage is used extensively to determine likely virus inactivation rates when validating disinfection rates of wastewater treatment systems. MS2 virus is of simple composition (Kuzmanovic et al. 2003), is non pathogenic, has similar resistance as human pathogenic viruses (e.g., poliovirus, influenza A, and rhinovirus) to antimicrobial agents and enumeration methods are robust and repeatable (Woo et al. 2010). This virus constitutes an interesting model microorganism for studying inactivation in wastewater.

In this study, irradiation of samples with UVA was completed in sterile 250 ml quartz-capped bottles in controlled, sterile conditions in a UVA cabinet. Firstly, the contribution of both microalgae and wastewater to UVA attenuation through water column (30 cm depth) was determined. Secondly, the impact of exogenous photosensitizers from a microalgae extract, wastewater and of microalgal cells as photosensitizers on MS2 inactivation under UVA irradiation was measured. Here, incubations with and without L-histidine, used to inhibit the virus inactivation effects of singlet oxygen produced from photosensitizers exposed to UVA radiation (Méndez-Hurtado et al. 2012), were compared to determine the effect of ROS. The impact of each factor (UVA attenuation and the presence of photosensitizers) was compared to consider the net effect of microalgae on coliphage inactivation in wastewater. Thirdly, this statement was verified by measuring MS2 inactivation in 30 cm depth wastewater columns in the presence or absence of microalgae.

2.1. UVA incubation cabinet

UVA LED panel (Bergquist, Reliance Laboratories) was suspended in an enclosed in a light-tight cabinet. Samples were placed under the panel in a chilled, shaking water bath (Ratek Ltd.) to maintain a constant temperature of 20°C. The location of each bottle sample in the water bath was determined to minimize the deviation of incident UVA irradiance between the samples. The six samples were irradiated with a combination of 5 LEDs (365 nm, 370-375 nm, 380-385 nm, 385-390 nm, 395-400 nm), covering the range of wavelengths in the UVA spectrum. The intensity of the UV LEDs was calibrated using a Solar Light UVA weatherproof detector PMA2110-WP connected to Solar Light PMA2100 meter.

2.2. Preparation of culture media

In order to investigate the effects of UV attenuation and ROS on MS2 inactivation, MS2 was incubated in six different types of media: reverse osmosis (RO) water (Millipore Q), Bold Basal Medium (BBM), wastewater, filtered wastewater, microalgae and microalgae extract. RO water and BBM were both considered optically clear.

BBM, classically used in microalgae culture, was prepared according to the composition provided in the Supplementary material.

Microalgae were isolated from Peterborough HRAP (S 32°58’24.143’’ E 138°48’5.958’’) by successive spread-plating on BBM agar plates. Ten isolated algal colonies were first resuspended in 25 mL conical flasks containing BBM and grown, illuminated at 80 µmol.m^-².s^-1 PAR (400-800 nm). The microalgae culture was then subcultured in to larger volumes of BBM until reaching approximately 6 mg.L^-1 of chlorophyll a, as measured in Peterborough HRAP.

To produce a cell-free organic extract, a proportion of the microalgal stock culture was heated at 80°C for 30 min and filtered through 0.45 µm GF/C filter (Whatman Ltd).

Anaerobically pre-treated wastewater was sampled at the surface of the Peterborough anaerobic pond and stored at 4°C. Part of the wastewater was filtered through 0.45 µm GF/C filter (Whatman Ltd) and stored at 4°C.

The total organic carbon (TOC) concentration in GF/C (Whatman) filtrates was determined for each medium using Shimadzu TOC-L carbon analyser.

2.3. MS2 and E. coli stock and MS2 quantification

2.3.1. Stock

The F-RNA coliphage virus MS2 was used as a non-pathogenic surrogate for the behavior of human pathogenic viruses in the environment. E. coli ATCC 700891, resistant to streptomycin and ampicillin, was used as the host coliform and kept in a microtube at -20°C in 50% glycerol/50% tryptone water.

The MS2 stock was prepared 24 hours before inoculation of the samples. Then, 500 µL of TSB broth containing E. coli were transferred into 5 mL of TSA containing antibiotics streptomycin (1.5 g.L^-1) and ampicillin (1.5 g.L^-1). This top layer agar was poured over a bottom layer of prepoured TSA agar plates. Two drops of MS2 15597-B1 stock solution were poured in the middle of the plate. The plate was incubated overnight at 37°C. If MS2 effectively infected E. coli, a stain appeared on the centre of the plate after a few hours. Subsequently, 9 mL of reverse osmosed water were poured onto the plate. The plate was then placed in the incubator at 37°C for 45 min and swirled every 10 min. The water was then transferred on to a new plate where MS2 effectively infected E. coli. Again, the plate was placed in the incubator for 45 min and swirled every 10 min. This step was repeated for 4 successive MS2 plates. The liquid recovered on the last plate was then syringe filtered at 0.45 µm into 100 mL reverse osmosis water to remove E. coli particles. This new MS2 stock at a concentration of 10⁶-10⁸ Plaque Forming Unit (PFU).mL^-1 was kept at 4°C. The samples destined to be incubated in the UVA cabinet were inoculated with 1 mL of this stock at the start of the experiment (t₀).

2.3.2. Quantification of MS2 virus

The double layer agar method (Noble et al. 2004) was used to quantify MS2 in the samples after incubation in the UVA cabinet. 500 µL of TSB broth containing E. coli and 200 µL of the respective, suitably diluted MS2 sample were transferred into 5 mL of TSA containing the antibiotics streptomycin (1.5 g.L^-1) and ampicillin (1.5 g.L^-1). This TSA was poured over a bottom layer of a pre-poured TSA agar plate. The required dilutions of the MS2 sample were similarly prepared in triplicate. After 24 hours incubation at 37°C, MS2 was quantified from the number of plaques on the plate, corresponding to locations where E. coli was infected by MS2 and was unable to grow. All the plates with a MS2 count between 15 and 300 were considered for the calculation of MS2 concentration, expressed as plaque forming units (PFU).mL^-1.

2.4. Incubation of the samples

2.4.1. Effect of UVA attenuation

As illustrated in the Fig. 1, the depth averaged UVA irradiances, G_{30cm_RO}, G_{30cm_WW}and G_{30cm_ALG}were obtained by measuring UVA irradiance throughout a 30 cm-depth column of RO water, anaerobically pretreated wastewater and microalgal suspension at a concentration close to microalgal biomass concentration reported in the Peterborough HRAP, respectively. The incident irradiance was 22 W.m^-2, which corresponds to the average UVA irradiance measured over 3 days per months in winter between 2008 and 2011 in Old Reynella, South Australia (S 35°5’56.3’’ E 138°32’25.9’’) (Bolton 2012). This was considered as a reference value typical of winter UVA exposure of HRAPs, relevant since Australian regulatory validations are based on winter performance (NRMMC 2006).

To determine disinfection rate in optically clear water, the respective depth averaged irradiances, G_{30cm_RO}, G_{30cm_WW}and G_{30cm_ALG}were applied as incident UVA irradiances to 250 mL RO water inoculated with MS2 in quartz capped bottles. A control experiment in darkness was also completed. A sample was taken aseptically every 12 h during 50 h for MS2 concentration determination and calculation of inactivation rates. Each condition was incubated in triplicate.

2.4.2. Effect of photosensitizers

As illustrated in Fig. 3, MS2 was incubated in 250 mL of RO water, BBM, 0.45 µm filtered wastewater, microalgal extract and microalgal suspension. The objective was incubating all substrates at a constant depth averaged irradiance i.e. G_{4cm_RO/BBM}≈ G_{4cm_WW}≈ G_{4cm_EXTR}≈ G_{4cm_ALG}. This required varying the incident UVA irradiance and in situ measurement of attenuation within a 4 cm depth of substrate above the UVA probe (as shown in Fig. 2). Table 1 synthesizes the UVA irradiance values measured over the sample depth after determining the incident irradiance (in red) so the average UVA intensity (in bold) is approximately equal to 9 ± 0.5 W.m^-2 in RO water, BBM, filtered wastewater, microalgae and microalgae extract.

Table 1: UVA irradiance (in W.m^-2) over 4 cm-depth RO water and BBM, microalgae, microalgal extract and filtered wastewater after adjusting incident UVA irradiance (at depth = 0 cm, in red) to obtain similar average irradiance through the 4 cm depth (in bold)

Depth	RO water and BBM	Microalgae	Microalgae extract	Filtered wastewater
0 cm	8.8	27.7	12.4	16.8
1 cm	8.7	10.9	11.1	12.5
2 cm	8.3	3.8	8.6	8.8
3 cm	8.8	1.4	7.3	5.4
4 cm	8.8	0.3	6.4	3.9
Average	8.7	8.8	9.2	9.5

MS2 incubated in RO water, BBM, 0.45 µm filtered wastewater, microalgae extract and microalgae were irradiated (Fig. 3) respectively with the previously obtained (Fig. 2) depth averaged irradiances G_{0_RO}, G_{0_BBM}, G_{0_WW}, G_{0_EXTR}and G_{0_ALG}. Each experiment was completed in triplicate, with and without L-histidine. Added at a concentration of 20 mmol per 250 mL sample, L-histidine inhibits the effects of the ROS singlet oxygen produced by photosensitizers when irradiated with UV. Singlet oxygen binds to L-histidine and is consequently ineffective for MS2 inactivation.

A sample was taken aseptically every 12 h during 50 h for MS2 concentration determination and calculation of inactivation rates. Singlet oxygen effect on MS2 inactivation was then deduced by difference from the inactivation rate obtained with and without L-histidine.

2.4.3. MS2 inactivation in 30 cm wastewater in presence and absence of microalgae

The third series of experiments aims at evaluating the global impact of microalgal cells on MS2 inactivation in a 30 cm depth wastewater column. As illustrated in the Fig. 4, MS2 was inoculated in 30 cm depth anaerobically pretreated wastewater columns with and without microalgae and irradiated with UVA at G₀ = 22 W.m^-2. A sample was taken aseptically every 12 h during 50 h for MS2 concentration determination and calculation of inactivation rates. Each condition was tested in triplicates, with and without L-histidine.

2.5. Calculation of MS2 inactivation

The MS2 inactivation rate K h^-1was calculated according to the equation below, where t is the time in h from the beginning of the incubation, n_t is the number of viable viruses (PFU.mL^-1) remaining at time t and n₀ the number of viable MS2 (PFU.mL^-1) at the beginning of the incubation:

$$\:K=\:\frac{{-log}_{10}.\frac{{n}_{t}}{{n}_{0}}}{t}$$

The Figures were created with MATLAB 2022b.

3.1. Effect of UVA attenuation by wastewater and microalgae on MS2 inactivation

3.1.1. UVA attenuation by RO water, wastewater and microalgae

Fig. 5 shows the impact of depth on UVA irradiance in RO water, wastewater and microalgae culture at an incident irradiance of 22 W.m^-2. The strongest attenuation of UVA radiation was observed in the microalgae culture, where UVA radiation was completely absorbed below 6 cm depth. In wastewater, UVA radiations were completely attenuated below 9 cm depth, while in RO water UVA radiation was still detected (9 W.m^-2) at the base of the column. The depth averaged UVA irradiances were 12.9 W.m^-2, 2.5 W.m^-2 and 2.1 W.m^-2 in the 30 cm depth RO water, wastewater and microalgae columns respectively. Those values correspond to the average UVA intensities at which viruses would be exposed to in a 30 cm depth column of RO water, wastewater and microalgae culture respectively.

3.1.2. MS2 inactivation rates under different UVA irradiances

The UVA intensities G_{30cm_RO}= 12.9 W.m^-2, G_{30cm_WW}= 2.5 W.m^-2 and G_{30cm_ALG}= 2.1 W.m^-2, determined experimentally as described in the Section 3.1.1, were applied to RO water samples inoculated with MS2 to determine the equivalent MS2 inactivation in optically clear RO water at the respective depth averaged UVA irradiances for wastewater and microalgae. The inferred inactivation rates in the respective substrates are presented in Fig. 6.

The control experiment in darkness revealed a very low MS2 decay rate (0.0068 h^-1) in the absence of UVA radiations in RO water. This value is of the same order of magnitude as the MS2 inactivation rate of 0.005 h^-1 measured in dark conditions in RO water by Bolton (2012). In comparison, the MS2 inactivation rate at UVA irradiances of G_{30cm_RO}was 0.084 h^-1, 0.043 h^-1 for G_{30cm_WW}and 0.023 h^-1 for G_{30cm_ALG}. The turbidity attributable to wastewater or microalgae induced a reduction of 48.8 % and 72.6 % respectively in the inactivation rate of MS2 compared to an optically clear medium such as RO water.

3.2. Effect of ROS from wastewater and microalgae on MS2 inactivation

MS2 inactivation rates (K in h^-1) in the presence of ROS (incubations without L-histidine) and in the absence of ROS (incubations with the addition of L-histidine) from wastewater and microalgae while irradiated with the respective depth averaged UVA irradiances (Section 2.4.2) are shown in Fig. 7.

In RO water, the inactivation rate K was 0.0042 h^-1in the presence of L-histidine and 0.0082 h^-1in the absence of L-histidine. RO water was considered free of photosensitizers, consequently the inactivation rates with and without L-histidine were expected to be similar. However, traces of organic molecules from agar plates may be in the MS2 inoculum and could have acted as photosensitizers in this experiment. The contribution of ROS to MS2 inactivation in RO water was, however, very low (0.0040 h^-1) and comparable with the dark inactivation rate (0.0068 h^-1), suggesting that UVA per se did not extensively inactivate viruses and that a significant amount of photosensitizer was needed to produce ROS and damage the viral particles.

In addition, as illustrated in Fig. 7, inactivation rates with L-histidine in RO water, wastewater, BBM, microalgae and microalgae extract were all similarly low compared to the inactivation rates obtained without L-histidine, highlighting the major contribution of ROS to MS2 inactivation regardless of the medium.

The Fig. 7 shows that the MS2 inactivation rate in filtered wastewater (0.022 h^-1) was higher than in RO water (0.0082 h^-1). Filtered wastewater contained a significant amount of organic carbon (65 mgC_org.L^-1) compared to RO water (Fig. 8), suggesting that the higher inactivation rate in filtered wastewater was due to a higher production of ROS induced by the presence of organic molecules acting as photosensitizers in filtered wastewater.

The highest MS2 inactivation rate (0.176 h^-1)was obtained in the presence of microalgal cells without L-histidine, even while the dissolved TOC in the microalgae medium was only 37 mgC_org.L^-1 (lower than in filtered wastewater). Note that this TOC concentration is equivalent to the organic carbon concentration attributable to EDTA in BBM, extracellular TOC derived from the algae would then be negligible here. MS2 inactivation by ROS was not correlated with dissolved organic carbon concentration, suggesting that microalgal cells themselves induce the production of singlet oxygen that significantly impact MS2 inactivation. The MS2 inactivation rate in microalgae extract, obtained by heating then filtering at 0.45 µm a microalgae culture grown in BBM, reached 0.088 h^-1. This inactivation rate is half that measured in the presence of microalgal cells but four times higher than in filtered wastewater. This result suggests a significant impact of organic compounds contained inside the microalgal cells (pigments, carbohydrates...) on MS2 inactivation. The MS2 inactivation in BBM reached only 0.020 h^-1, meaning that nitrates, EDTA and traces of organic molecules from the MS2 inoculum has negligible impact on MS2 inactivation in the samples with microalgal cells and microalgae extract. This result confirms that the high MS2 inactivation rates obtained in the presence of microalgal cells and in microalgae extract was mainly due to photosensitizers from microalgae and only little to the BBM in which they were suspended during the experiment. In the end, ROS mediated disinfection was the highest in the microalgal suspension, followed by the microalgae extract, the wastewater, the BBM and the RO water.

The Fig. 8 shows that TOC content in the microalgae extract (41 mgC_org.L^-1) was not significantly higher than in BBM (40 mgC_org.L^-1) or microalgae culture (37 mgC_org.L^-1), despite microalgae were heated at 80°C. However, the very low quantity of organic molecules extracted from microalgae induced a higher MS2 inactivation rate than the organic molecules from wastewater, even though the TOC content was higher in wastewater (65 mgC_org.L^-1). A different TOC composition in the microalgae extract may then produce more active photosensitizers.

3.3. Balance between positive and negative impact of microalgae and wastewater on MS2 inactivation

The two series of experiments presented above aimed at discriminating and evaluating the impact of microalgae and wastewater on MS2 inactivation via UVA attenuation and ROS production. Fig. 9 shows that the reduction of UVA penetration through the water column due to microalgae induced a reduction in K of -0.061 h^-1 on the MS2 inactivation rate compared to the control with RO water. However, the production of ROS by microalgal cells induced an increase of +0.153 h^-1on the MS2 inactivation rate compared to the control with RO water. The negative impact of microalgae on MS2 inactivation rate due to attenuation of UVA could then be significantly compensated by its inherent capacity to produce ROS that enhances MS2 inactivation.

However, it was not the case for wastewater. The reduction of UVA penetration through the water column due to wastewater turbidity induced a reduction of -0.041 h^-1on the MS2 inactivation rate compared to the control with RO water. The production of ROS by photosensitizers found in wastewater induced an increase of only +0.005 h^-1on the MS2 inactivation rate compared to the control with RO water. The negative impact of wastewater on MS2 inactivation rate due to the reduced UVA penetration through the water column would then not be compensated by the production of ROS attributable to wastewater organic molecules.

3.4. MS2 inactivation in a 30 cm depth wastewater column

The results presented in the previous section suggest that the presence of microalgae enhance MS2 inactivation in wastewater, despite their strong contribution to UVA attenuation over a 30 cm depth water column. In order to confirm this hypothesis, 30 cm depth wastewater columns were inoculated with MS2, in the presence and in the absence of microalgae and in the presence and in the absence of L-histidine and placed under UVA radiation.

As illustrated in Fig. 10, MS2 inactivation rate was surprisingly slightly higher in the wastewater column that did not contain microalgae (0.014 h^-1) than in the column with microalgae (0.010 h^-1). In addition, MS2 inactivation rates with and without L-histidine were very similar in both experiments (0.013 and 0.014 h^-1 without microalgae; 0.012 and 0.010 h^-1 with microalgae), suggesting a very low impact of ROS in MS2 inactivation in those experiments.

Measurement of UVA intensity along the depth of the columns in the presence and in the absence of microalgae revealed that, in wastewater alone, UVA was completely attenuated below 9 cm depth, against 1 cm in wastewater added with microalgae. Consequently, 97 % of the volume of the column containing microalgae was not irradiated by UVA. This observation could explain why the inactivation rates obtained in the UVA-irradiated 30 cm depth columns containing microalgae were only slightly higher than the inactivation rate obtained in darkness (0.0068 h^-1).

4.1. Effect of UVA attenuation

The experiment conducted in darkness confirmed that MS2 exhibited negligible decay rate over 50 h at 20°C in RO water. This confirms that significant MS2 decay observed when samples were irradiated with UVA was due to UVA exposure. UVA attenuation attributable to both microalgae and wastewater highly impacted MS2 inactivation, with 40 % of the reduction in inactivation rate attributable to wastewater turbidity and 60 % to microalgae turbidity.

4.2. Effect of ROS production

In RO water, filtered wastewater, BBM, microalgae and microalgae extract, the inactivation rates of viruses with L-histidine, that inhibits the effects of ROS, were close to inactivation rates measured in darkness. This result confirms that exposure to UVA per se has minimal effect on virus inactivation. Rattanakul and Oguma (2017) also reported that UVA treatment alone did not induce damage in the target genome and that MS2 was not removed. Indeed, Davies (2003) reported that viruses lack a bound chromophore within their protein structures that would act as an endogenous photosensitizer and permit inactivation in the absence of exogenous photosensitizers, explaining why MS2 would survive exposure to UVA. However, the viral capside of MS2 is permeable and MS2 is sensitive to damages by ROS that cross the capsid (Majiya et al. 2018). This was confirmed by the present study since the MS2 inactivation rate was higher in filtered wastewater, BBM, microalgae and microalgae extract, all containing photosensitizers, than in RO water. However, inactivation rates in the media containing photosensitizers were also slightly higher than in RO water when ROS inhibitor L-histidine was added to the media. L-histidine, however, inhibits only the most commonly formed species of ROS, singlet oxygen. However, other forms such as hydroxyl radical HO• can also be present depending on the media and enhance MS2 inactivation in the presence of L-histidine, which might explain the small differences in MS2 inactivation between the different media even in the presence of L-histidine.

Photosensitizers in wastewater induced only a slight increase in MS2 inactivation rate compared to the control experiment in RO water. In contrast, a previous study reported that wastewater could produce significant concentrations of ROS (Dong and Rosario-Ortiz 2012), the main type of ROS produced by organic matter being singlet oxygen (¹O₂) and hydroxyl radical (HO•) (Niu et al. 2014). In wastewater, both organic carbon and nitrate can act as photosensitizers. Dong and Rosario-Ortiz (2012) reported that nitrates and organic carbon from wastewater presented similar capacity to form ROS by photolysis. In Peterborough HRAP, nitrates represent only 9.2 mgN.L^-1, against 36.7 mgC.L^-1 for organic carbon. The main contributor to photosensitizers in Peterborough HRAP is then most likely organic carbon.

In contrast, in the presence of microalgal cells a significantly higher MS2 inactivation rate was recorded compared to wastewater. This finding is consistent with a previous study (Niu and Croué 2019) that demonstrated that organic matter from algae (AOM) was an efficient photosensitizer that produced significantly more ROS than terrestrial sourced organic matter due to aromatic protein-like and soluble microbial substances found in AOM. In the present study, AOM implied in the production of ROS could possibly be exopolysaccharides (EPS) that are usually formed around the microalgal cell wall.

In the experiment with microalgae extract, the amount of TOC extracted from microalgae was very low, even while a first extraction test by heating microalgae at 80°C increased the dissolved TOC by more than 40 %. The first extraction test was performed on a microalgae sample in exponential phase of growth, while in this study microalgae were in stationary phase, which might have increased the resistance of the cell wall to extraction by heating. However, MS2 inactivation rate in the microalgae extract was still much higher than in BBM and filtered wastewater, showing that the effect of ROS on inactivation was not necessarily correlated with the TOC content. The specific capacity of the molecules to act as photosensitizers might be of more relevance. As well, molecules extracted from microalgae other than TOC could have acted as photosensitizers in this experiment (nitrogen species for example). A potential release of carotenoids with a protector effect against the oxidation of MS2 by ROS is also possible, but probably marginal regarding the low carotenoids content in microalgae (less than 1 % of the total dry weight, Bonnanfant et al. 2021). Further investigation should include a more efficient method for extracting TOC from microalgae, notably by using microalgae in their exponential phase.

Furthermore, one of the purposes of the present study was to investigate the impact of microalgae as a photosensitizer on MS2 inactivation. However, the presence of microalgal cells is often associated with high dissolved oxygen concentrations. Bolton (2012) demonstrated that increasing DO from 0 to 8.5 mg.L^-1 did not have any effect on MS2 inactivation. While increasing the range of dissolved oxygen to 25 mg.L^-1 would be relevant regarding the conditions observed in HRAP, dissolved oxygen was consequently not controlled in the samples in the present study. Moreover, dissolved oxygen was expected low as UVA radiations are not photosynthetically active radiations. Nevertheless, for future research, the significant capacity of microalgal cells to produce ROS should be confirmed by ensuring similar dissolved oxygen concentration than in the control.

Moreover, in the experiments aiming at exploring the effect of ROS from microalgae and wastewater on MS2 inactivation, the applied UVA irradiance was depth averaged according to the turbidity of the medium. Consequently, higher incident UVA intensity was applied on the samples with microalgae than on the samples with microalgae extract, filtered wastewater, BBM or RO water to compensate for the higher turbidity. Adjusting incident UVA irradiances to compensate for medium turbidity was necessary to decorrelate the effects of UVA attenuation and ROS production on viruses’ inactivation. This method was based on exposure to an average UVA irradiance over the depth of the sample; however, mixing efficiency may influence UVA attenuation and MS2 exposure to UVA. Notwithstanding, samples with microalgae extract presented a higher MS2 inactivation rate than filtered wastewater, while the incident UVA irradiance applied to the microalgae extract (12.4 W.m^-2) was lower than the incident UVA irradiance applied to the filtered wastewater (16.8 W.m^-2).

Finally, the constant exposure of samples to UVA radiations raises the question of the stability of the microalgae sample throughout the experiment. In the sample containing microalgae extract, there is a possibility that the chlorophyll and other pigments or organic compounds progressively degraded during the 50 h experiment. For further investigation, the main factors affecting viruses’ inactivation such as turbidity, chlorophyll, TOC, dissolved oxygen and pH should be measured along the experiment to ensure for consistency and conservation of the experimental conditions.

4.3. Impact of microalgae in 30 cm depth columns

Adding microalgae to a 30 cm deep column of wastewater from an anaerobic pond did not improve MS2 inactivation, even while the two first sets of experiments suggest a strong inherent capacity of microalgae to produce ROS. Yet, UVA measurement inside the column containing wastewater and microalgae revealed that UVA was completely attenuated below 1 cm depth only. It is important to note that ROS production is enhanced by UVA radiations, yet microalgal capacity to form ROS cannot be expressed while UVA does not penetrate the column. Regarding the low MS2 inactivation whether in the presence of the absence of microalgae, strong attenuation in 30-cm deep columns leads to insufficient UVA for ROS production. A balance between UV availability (relying on attenuation), subsequent ROS production and inactivation needs to be achieved in HRAPs. Note that UVA attenuation in Peterborough HRAP was probably higher than in most HRAP, this being due to higher suspended solids (1 g.L^-1against 0.2-0.5 g.L^-1in Kingston-on-Murray HRAP, in South Australia according to Buchanan et al. 2018). A review (Bolton et al. 2010) reported a UVA penetration of 14 cm in a HRAP, much greater than the 1 cm UVA penetration measured in Peterborough HRAP.

In addition, even while UVA measurements in the field in Peterborough HRAP showed similar conditions as in the columns (UVA radiations were fully attenuated below 0.5-1 cm depth), the columns were not stirred (except every 12 h before sampling) while Peterborough is well-mixed (Jahan et al. 2024). Inactivation might have been underestimated in the columns compared to HRAP. However, note that the reverse phenomenon was reported by Park et al. (2021), where higher removal rates were obtained in the presence of suspended solids due to the absorption of viruses on solid particles.

To conclude, the strong inherent capacity of microalgae to produce ROS that successfully improves the inactivation of viruses suggests that the implementation of a thin-film system as a tertiary treatment downstream the HRAP could enhance the penetration of sun radiations and the production of ROS by microalgae for improving pathogens disinfection. Hawley and Fallowfield (2016) reported encouraging results using an inclined pond wall with a culture depth of a few millimeters only. While the model system permitted a significant enhancement of MS2 inactivation compared to normal pond conditions, the operation of the system on the field did not observe a significant improvement of pathogens removal. Further research should then focus on the constraints raised by the operation of those thin-film systems targeting pathogens removal in real conditions. Moreover, thin film systems also increase the MRPA associated with high oxygen production that could possibly favor disinfection through oxidative stress. The number of viral particles decreased from 10⁵ to 0 in only 30 h in the presence of microalgal cells in the experiments in the UVA cabinet, which means that the retention time in the thin film system could be relatively short, from a few hours to 2 days depending on the level of disinfection wanted. This constitutes an important point regarding implementation of such a system at large scale. HRT in thin film systems should indeed be short in order to treat significant volumes of wastewater despite the low depth.

The present study demonstrated that when UVA penetration was sufficient the presence of microalgae per se and their extracts acted as photosensitizers, which contributed significantly to UVA induced ROS production and greatly enhanced MS2 inactivation by ROS. MS2 inactivation by UVA-induced ROS production was dependent upon UVA exposure, which was mediated by attenuation by suspended solids and algal biomass within the wastewater. This study confirms the importance of efficient vertical mixing, for ensuring adequate exposure of viruses to UVA radiation. Thin-layer systems including microalgae for tertiary treatment of wastewater may provide an opportunity to ensure UV penetration and ROS production by microalgae thereby improving pathogen inactivation and enabling reuse of the treated wastewater for the irrigation of a wider range of crops with greater economic value, including food crops.

AOM

Algal Organic Matter

BBM

Bold Basal Medium

DNA

deoxyribonucleic acid

Dissolved Oxygen

EPS

Exopolysaccharides

HRAP

high rate algal pond

MPN

Most Probable Number

PFU

Plaque Forming Unit

RNA

Ribonucleic acid

Reverse osmosis water

ROS

Reactive Oxygen Species

TOC

Total Organic Carbon

TSA

Tryptone Soy Agar

TSB

Tryptone Soya Broth

Ultraviolet

WSP

Waste Stabilisation Pond

Competing interests:

The authors have no relevant financial or non-financial interests to disclose.

CRediT authorship contribution statement:

Solène Jahan: Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Conceptualization. Jérémy Pruvost: Writing – review & editing, Supervision. Guillaume Cogne: Writing – review & editing, Supervision. Mariana Titica: Writing – review & editing, Supervision. Howard Fallowfield: Writing – review & editing, Supervision, Methodology.

Funding:

This study was funded by College of Science and Engineering, Flinders University and French « Ministère de l’enseignement supérieur, de la recherche et de l’innovation

Author Contribution

SJ: Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Conceptualization. HF: Writing – review & editing, Supervision, Methodology. JP, GC, MT: Supervision. All authors reviewed the manuscript.

Acknowledgement

The authors acknowledge Andreana Shakallis and Raj Indela of Flinders University Environmental Health laboratory for training and technical support and Peterborough District Council, South Australia, for access to their wastewater treatment facility.

Data Availability

Data will be made available on request.

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No competing interests reported.

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Microalgae impact on inactivation of indicator virus in a large-scale wastewater treatment system using microalgae

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