Elucidating the role of nanoparticles on photosynthetic biogas upgrading: Influence of biogas type, nanoparticle concentration and light source

Three different nanoparticles, namely Fe 2 O 3 , carbon coated zero valent iron (CACOI) and SiO 2 , were added to a mixed microalgae culture in order to improve photosynthetic biogas upgrading. Fe 2 O 3 and CACOI nanoparticles at 10 mg/L supported higher CO 2 consumptions compared to their respective controls. The addition of Fe 2 O 3 nanoparticles at 70 mg/L resulted in a 38% enhanced biomass productivity, and 20% higher CO 2 consumption but delayed exponential growth. The CACOI nanoparticles at 70 mg/L resulted in a shorter lag phase, enhanced CO 2 consumption by 13%, and carbohydrate content enhancement by 64%, while the addition of SiO 2 nanoparticles at this concentration induced an enhanced lipid and carbohydrates production by 47% and 68%, respectively. Interestingly, UV light exposure reduced the beneficial effects of nanoparticles, although CACOI nanoparticles still supported a shorter lag phase and higher carbohydrates production at 70 mg/L. In brief, CACOI nanoparticles hold an untapped potential to promote the metabolism of microalgae during photosynthetic biogas upgrading.

mandatory prior to its use as a vehicle fuel or to its injection to the natural gas grid [2].
Today, several physical and chemical methods are commercially available for biogas upgrading, including membrane separation, cryogenic separation, pressure swing adsorption, water scrubbing, organic solvent scrubbing, chemical absorption [3]. The high operating costs and environmental impacts of these physical/chemical technologies have triggered research in biological methods such hydrogenotrophic and photosynthetic biogas upgrading.
Photosynthetic biogas upgrading in microalgae-bacteria photobioreactors has been validated at pilot and demo-scale, and widely reported to be an economically and environmentally friendly option to upgrade biogas into biomethane coupled to nutrient recovery from the liquid fraction of digestates [4]. Despite algal-bacterial photobioreactors interconnected to external biogas absorption columns have reached CO 2 removals of up to 98.6% at pilot [5] and demo scale [4], there are still some challenges that need to be addressed to maintain a robust biogas upgrading performance. Recently, Bose et al [6] compared seven different factors affecting the bubble column performance in photosynthetic biogas upgrading. The main process limitations identified to date are i) the low CO 2 mass transfer to the culture medium, ii) the high sensitivity of biomethane quality to variations in the gas and liquid flow rates and pH, and iii) the diurnal and seasonal variability of environmental parameters influencing photosynthetic activity [7]. Indeed, this process requires the development of innovative operational strategies to enhance CO 2 absorption and fixation.
During the past years, the use of nanomaterials in environmental applications is gaining attention due to their unique physicochemical properties such as size, morphology, high reactivity, chemical stabilization, high surface area-to-volume ratio, abundant active J o u r n a l P r e -p r o o f Journal Pre-proof sites and high adsorption capacity [8]. Indeed, nanomaterials and nanoparticles can play an key role in CO 2 capture technologies, biogas production and biogas upgrading processes [9]. To date, many solid adsorbents, mainly porous materials, have been effectively used to remove CO 2 from biogas, such as activated carbons and metal oxides [10]. Moreover, it is kwon that the use of metal oxide NPs mediates the formation of carbonates, bicarbonates and carboxylates when CO 2 interacts with the NPs surfaces [11]. In this context, the addition of nanoporous materials to microalgae cultures devoted to biogas upgrading can create a symbiosis where the materials adsorb CO 2 to form carbonates and bicarbonate species that can be further fixed via photosynthesis by microalgae. This would result in enhancements in biomass production and in the performance of biogas purification. In addition, it has been recently demonstrated that the supplementation of graphene oxide quantum dots under UV-light exposure stimulated the CO 2 capture and lipid production in Chlorella pyrenoidosa [12]. Thus, NPs can be also used as a strategy to scavenge the damage of solar UV radiation to microalgae.
The addition of metal oxide NPs to microalgae culture is still a controversial topic, since NPs can be toxic to some microalgae species or stimulate their growth and lipid production ( Table 1). Even if there is very little information on how the NPs addition to microalgae culture can improve the CO 2 adsorption, the reported studies present promising results [13]. In this way, the physico-chemical properties of the NPs can represent an advantage to improve CO2 adsorption since they can act as electron donors/acceptors and light conversion aids, or form carbonates when CO 2 interacts with their surface, among others. Jeon et al. [14] reported that SiO 2 NPs enhanced the gas-liquid mass transfer rate of CO 2 in C. vulgaris cultures, resulting in an enhanced growth and lipid production.
Similarly, the use of polymeric nanofibers containing Fe 2 O 3 NPs has been reported as a J o u r n a l P r e -p r o o f Journal Pre-proof promising technique to enhance CO 2 fixation of Chlorella fusca LEB 111 cultures [13]. On the other hand, the addition of zero-valent iron NPs have proved to have beneficial effects on microalgae species like Pavlova lutheri, Isochrysis galbana, Tetraselmis suecica, [15] Desmodesmus subcapicatus, Dunaliella salina, Parachlorella kessleri and Trachydiscus minutus [16]. However, to the best of our knowledge, little is known about the effect of carbon NPs on microalgae culture. The literature on the effect of carbon-coated zero-valent iron NPs on microalgae and the number studies devoted to investigate the potential of NPs during photosynthetic biogas upgrading is scarce.
This study aimed at assessing the effect of two metal oxides NPs (Fe 2 O 3 and SiO 2 ) and one magnetic NP (carbon coated zero-valent iron) on microalgae growth and photosynthetic biogas upgrading efficiency at laboratory scale in batch enclosed photobioreactors. Additionally, the influence of NPs concentration and light source (visible versus visible+UV) on the parameters above mentioned were also investigated. (JEOL JSM-6490LV) and energy-dispersive spectroscopy (EDS) (EDX-700/800, Hitachi, Japan) were carried out to determine the surface morphology and elemental composition of the target NPs.

Microalgae culture and biogas
The microalgae culture used in our study consisted of a consortium of microalgae and  W/m 2 ). The UV light exposure was added to simulate real solar radiation. A control containing only algal biomass and biogas A was conducted. Each condition was run in triplicate.

Analytical procedures
The biogas composition in the headspace of the bottles was determined two times per day by gas chromatography-TCD (Bruker) to quantify the gas concentration of CH 4 , CO 2 , H 2 S and O 2 in the headspace according to [26]. Microalgae growth was daily determined by optical density at 750 nm using a Shimadzu spectrophotometer (Japan), microalgae samples were daily taken and properly diluted to obtain an absorbance under 1, then the obtained absorbance was multiplied by the dilution factor. The initial CO 2 and O 2 content in the headspace along with the OD 750 were normalized and are presented as cumulative values.
pH was determined at the beginning and at the end of the experiment (SensION TM + PH3 pHmeter, HACH, Spain). TSS concentrations were determined according to standard methods [27]. The biomass obtained from test series II and II was harvested (10000 rpm, 4 ºC) and freeze-dried for further macromolecular characterization. The carbohydrate content was determined according to [28], while the lipid content was determined gravimetrically following biomass extraction with chloroform:methanol (2:1 v/v) as described elsewhere [29].

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The results are presented as mean values  standard deviation. An analysis of variance (ANOVA) followed by Tuckey's test considering =0.05 was performed to assess the influence of nanoparticles on microalgae growth.

Nanoparticle characterization
The SEM micrographs show the morphology of the three NPs used in this study (Fig. 1).
The Fe 2 O 3 NPs presented a nanorod morphology, which has been previously reported to exhibit a high specific surface area and better electrochemical and magnetic properties compared to other Fe 2 O 3 morphologies [30]. The CACOI NPs were characterized by agglomerated NPs in accordance with [31]. Finally, the SiO 2 NPs presented the smallest particle size among the three NPs tested. Moreover, the chemical composition of each NPs is presented in Table 2. The presence of Na in the Fe 2 O 3 NPs was attributed to trace levels of the catalyst used for their synthesis. Interestingly, the CACOI NPs exhibited low levels of essential minerals for microalgae growth, which could serve as nutrients and promote microalgae growth.  [33]reported that the CO 2 adsorption capacity of Fe 2 O 3 increased up to four times when the particle size decreased from 160.5 nm (bulk form) to 24.5-56 nm. In our particular study, the synthesized Fe 2 O 3 NPs exhibited an average particle size of 24 nm, which are in the range reported by [33].  [34] and [35], suggesting that SiO 2 NPs could support superior adsorption properties than CACOI and Fe 2 O 3 NPs. Thus, the results of the adsorption/desorption analysis confirmed that nanopowders exhibited a high surface area [36], but the nature of the CACOI and Fe 2 O 3 NPs, inherently containing essential trace metals for microalgae growth, could play a key role in microalgae metabolism.

Influence of type of biogas and nanoparticle addition on microalgae growth
The CO 2 consumption and O 2 production recorded in the headspace of the bottles served as indicators of microalgae growth, along with the optical density of the culture broth (Fig. 2).
The addition of biogas B resulted in microalgae inhibition regardless of the presence of NPs, and can be mainly attributed to the absence of sulfur-oxidizing bacteria responsible for the rapid oxidation of H 2 S to SO 4 2- [37].
J o u r n a l P r e -p r o o f  NPs did not support any significant improvement in CO 2 cumulative consumptions but a slight enhancement of 5% in O 2 production was observed.
The addition of NPs at 10 mg/L did not enhance Px under biogas A atmosphere in any of the conditions tested. This can be attributed to the microalgae species used in this study, since the effect of the NPs is species specific (  CACOI NPs are covered with carbon, and this particular material has been widely used for CO 2 capture [9]. The mechanism of interaction between the NPs and the CO 2 capture is not well understood yet, but one of the main mechanism of interaction is the "shuttle effect", which can be described as follows: CO 2 is adsorbed by the NPs and then J o u r n a l P r e -p r o o f the loaded NPs release the adsorbed CO 2 into the aqueous medium, or the algal broth in this case [39]. Thus, the reduced lag phase achieved in the presence of CACOI NPs could be explained by the adsorption capacity of activated carbon [31]. Hence, the CO 2 present in the biogas atmosphere was adsorbed to the surface of the NPs and rapidly released in the aqueous broth for microalgae consumption, thus stimulating an early algal metabolism.

Influence of nanoparticle concentration under visible light
The addition of 20, 40 and 70 mg/L of Fe 2 O 3 NPs resulted in higher cumulative CO 2 consumption and higher Px during the exponential growth phase (Table 5) (Fig. 3). Indeed, the addition of Fe 2 O 3 NPs resulted in up to 38% Px enhancement. The higher Px values herein obtained can be mainly attributed to: 1) the culture media composition, which is a synthetic centrate supporting high biomass productions due to its rich nutrients content compared to other types of wastewater [40]; 2) the high CO 2 concentration in the headspace of the bottle, which resulted in accelerated microalgae growth; 3) the biostimulant nature of the nanoparticles used and 4) the high photosynthetic active radiation and the high illuminated surface to volume ratio. However, the higher the Fe 2 O 3 NPs concentration, the higher the lag phase in the assays (Fig 3a, d,  NPs were added, which is similar to the biomass enhancements obtained in our study.    Compared to the assays with Fe 2 O 3 NPs, the addition of CACOI NPs did not increase the lag phase of algal metabolism. Indeed, the addition of 70 mg/L of CACOI NPs enhanced both Px and the rates of CO 2 consumption, and reduced the lag phase (Fig. 3b, 3e,   3h). The cumulative CO 2 consumption in the 70 mg/L CACOI assays was 13% higher than in the control tests. The latter confirms the fact that CACOI NPs stimulated the CO 2 adsorption mainly by the nature of the material, and the increased CO 2 consumption could be mainly attributed to the higher CO 2 availability in the headspace of the bottles.
Notwithstanding, the increasing concentrations of CACOI NPs did not impact significantly on the cumulative O 2 production and OD 750 .. However the lag phase was significantly increased by the addition of the CACOI NPs, and was related to the increasing concentration. On the other hand, the carbohydrate content of the algal biomass increased by a factor of 2.6 when the cultivation broth was supplemented with 70 mg/L, which can be explained by the superior CO 2 biofixation mediated by the NPs [8]. Thus, the higher CO 2 availability mediated by CACOI NPs at 70 mg/L stimulated the activity of the RuBisCO enzyme, which is widely known to be the catalyst of CO 2 biofixation [13]. Therefore, an enhanced CO 2 biofixation resulted in an increased biomass productivity during the logarithmic growth phase, and in the accumulation of high-value products [13]. Hence, the improved logarithmic phase coupled to the accumulation of carbohydrates confirms that the biofixation capacity of microalgae was significantly improved by the addition of 70 mg/L of CACOI NPs.
Finally, the addition of 70 mg/L of SiO 2 NPs led to a cumulative CO 2 consumption 11.50% higher than that of the control, thus confirming that SiO 2 NPs acted as CO 2 adsorbents mediating a faster CO 2 dissolution in the algal broth. Notwithstanding, the addition of 40 and 70 mg/L of SiO 2 NPs resulted in a longer lag phase while no statistical J o u r n a l P r e -p r o o f difference was observed on the Px. Even if the addition of SiO 2 NPs induced higher cumulative CO 2 consumptions, no statistical difference was observed neither in the cumulative O 2 production nor in OD 750 , which did not agree with the observations of Jeon and co-workers [14]. The latter confirms the fact that the exposure to NPs is species specific and even if the addition of SiO 2 NPs has been reported to enhanced biomass and lipid production in Chlorella vulgaris, the mixed culture used in our study did not experience the same beneficial effects. Interestingly, the addition of SiO 2 NPs enhanced both the carbohydrate and lipid content of the final biomass regardless of the NPs concentration tested. For instance, the addition of 40 mg/L SiO 2 NPs supported the highest carbohydrate content, which was 1.91-fold higher than that in the control assays. Similarly, enhancements in the lipid content ranging by a factor 1.3-1.5 were obtained when SiO 2 NPs were supplemented to the algal broth. Thus, the superior accumulation of carbohydrates and lipids can be explained by the fact that the addition of SiO 2 NPs created a stress environment to the microalgae and, as a response, carbohydrates were accumulated as an energy reserve [43]. On the other hand, lipid accumulation can be explained by the fact that the SiO 2 NPs induced an oxidative stress to microalgae, resulting in a lipid accumulation as a nonenzymatic antioxidant mechanism of defense to scavenge the excessive ROS [44]. Similar findings have been reported by Jeon et al [14], who reported lipid enhancements of up to 340% compared to the control mainly attributed to the environmental stress created by the NPs. Thus, our results suggest that the SiO 2 NPs served as CO 2 adsorbents, however the nature of the NPs to created stress conditions that limited the growth of microalgae. Nonetheless, the SiO 2 NPs can be used as a technique to improve the value of the produced biomass.
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Influence of nanoparticle concentration under UV-visible light
Solar UV radiation can seriously affect microalgae integrity and biological function, since it induces the production of ROS [45]. However, Yang et al. [12] recently proved that UVlight mediated a positive effect on CO 2 capture and lipid production in C. pyrenoidosa when graphene oxide quantum dots were added. In this context, NPs could act as UV protectors and/or spectrum converters to promote microalgae growth and macromolecule accumulation.
In our study, the addition of Fe 2 O 3 NPs supported an enhancement of up to 50% in Px compared to the control (Table 5). However, no statistical difference was observed in the cumulative CO 2 consumption between the assays (Fig. 5) However, no statistical difference in term of lipid content was observed among the assays (Fig. 6). Moreover, the supply of UV light in this test series led to a higher lipid production compared to the assays conducted exclusively with visible light regardless of the addition of NPs. Thus, our results are in agreement with the observations of Yang et al. [12], who reported that the addition of graphene quantum dots enhanced the photosynthetic activity and the CO 2 fixation of Chlorella pyrenoidosa when exposed to UV-light. Additionally, Dinc et al [46] has also observed beneficial effects on C. vulgaris growth by the addition of J o u r n a l P r e -p r o o f   J o u r n a l P r e -p r o o f peroxidation in this particular test series was induced by ROS formation mediated by the interaction of the zero valent iron contained in the NPs and the UV light exposure [12].
However, the addition of 70 mg/L induced a carbohydrate content enhancement of up to 22% and an OD 750 15% higher than the control. The results herein obtained confirm the fact that CACOI NPs effect of microalgae are mainly as CO 2 adsorbents, resulting in enhanced CO 2 availability in the headspace of the bottles. Moreover, the increased CO 2 led to an activation of microalgae metabolism and storage as carbohydrates under both visible and visible + UV light.
Finally, the addition of SiO 2 NPs did not support an enhancement in Px despite the addition of 70 mg/L SiO 2 NPs led to a CO 2 consumption 18% higher than that recorded in the control tests. Moreover, the addition of 20 and 40 mg/L of SiO 2 NPs led to 11% and 6% higher O 2 cumulative productions compared to the control tests, respectively. Additionally, OD 750 increased as the NPs concentration increased, and the addition of 70 mg/L SiO 2 induced a OD 750 enhancement of 16%. Furthermore, the addition of 70 mg/L SiO 2 mediated a 69% enhancement in the lipid content compared to the control. Finally, our results indicate that the UV-light exposure induce a higher lipid accumulation in microalgae likely due to a mechanism of defense against the generation of ROS [47]. Interestingly, SiO 2 NPs and UV radiation can induce an oxidative stress on microalgae [14]. However, in our particular study, neither biomass loss nor lipid peroxidation by the combination of SiO 2 NPs and UV radiation was observed. Therefore, our results suggest that the oxidative stress caused by SiO 2 NPs did not increase with UV exposure. Thus, even if no biomass enhancements were observed with the addition of SiO 2 NPs neither under visible and visible + UV light exposure, contrary to the observed by Jeon et al [14], these NPs still can be used as a strategy to produce high-value biomass even under UV radiation.
J o u r n a l P r e -p r o o f Finally, the addition of SiO 2 NPs supported lipid production enhancements. Promising results to improve the CO 2 adsorption couple to biomass production and high-value products were obtained in the present study. However, it is important to highlight that these particular results were obtained under controlled conditions. In this regard, future studies should be directed to assessed the effect of the NPs on microalgae under uncontrolled conditions, i.e. real centrate and environmental conditions. Moreover, life cycle assessments, exergy analyses, techno-economic analyses and energy analyses as described in [48], should be considered to assess the sustainability of the process. Finally, the results herein obtained can be scaled-up to pilot plants to boost CO 2 consumption coupled to microalgae growth. The later could represent a feasible technique to improve the performance of the stablished technology for photosynthetic biogas upgrading.