A comparative analysis of biogas upgrading technologies: Photosynthetic vs physical/chemical processes

1 Photosynthetic biogas upgrading to bio-methane: boosting nutrient 1 recovery via biomass productivity control. 2 Alma Toledo-Cervantes1, Mayara L. Serejo2, Saúl Blanco3, Rebeca Pérez1, Raquel 3 Lebrero1, Raúl Muñoz1* 4 5 1- Department of Chemical Engineering and Environmental Technology, University of 6 Valladolid, Dr. Mergelina s/n., Valladolid 47011, Spain. 7 2- Faculty of Engineering, Architecture and Urbanism and Geography, Federal 8 University of Mato Grosso do Sul, Campo Grande 79090-900, Brazil. 9 3- Department of Biodiversity and Environmental Management, University of León, 10 León 24071, Spain. Current address: The Institute of the Environment, La Serna, 58, 11 24007 León, Spain. 12 13 *Corresponding author: [email protected]s 14 15 *Manuscript with continuous line numbering Click here to view linked References 2 Abstract. 16 A pilot high rate algal pond (HRAP) interconnected to an external CO2−H2S absorption 17 column via settled broth recirculation was used to simultaneously treat a synthetic 18 digestate and to upgrade biogas to a bio-methane with sufficient quality to be injected 19 into natural gas grids. An innovative HRAP operational strategy with biomass 20 recirculation based on the control of algal-bacterial biomass productivity (2.2, 4.4 and 21 7.5 g m-2 d-1) via settled biomass wastage was evaluated in order to enhance nutrient 22 recovery from digestate at a constant hydraulic retention time. The influence of the 23 recycling liquid to biogas (L/G) ratio on the quality of the upgraded biogas was 24 assessed. The bio-methane composition under a L/G ratio of 1 (0.4 ± 0.1% CO2, 0.03 ± 25 0.04% O2, 2.4 ± 0.2% N2 and 97.2 ± 0.2% CH4) complied with the technical 26 specifications of most European bio-methane legislations regardless of the biomass 27 productivity established. The HRAP operational strategy applied allowed increasing the 28 N and P recovery from 19 and 22% to 83 and 100%, respectively, when the biomass 29 productivity was increased from 2.2 to 7.5 g m-2 d-1. Finally, the dynamics of 30 microalgae and bacteria population structure were characterized by morphological 31 identification and DGGE analysis. 32 33 Keywords: Biogas upgrading; bio-methane; microalgae-based processes; nutrients 34 recovery; wastewater treatment. 35 36 3 Highlights: 37  A removal of CO2 and H2S from biogas higher than 99% was achieved. 38  A low L/G ratio prevented O2 and N2 contamination of the upgraded biogas. 39  The bio-methane complied with EU legislation for injection into natural gas 40 grids. 41  A novel HRAP operation based on biomass productivity control was developed. 42  This operation strategy allowed maximizing nutrient recovery from digestate. 43 44 4 Introduction. 45 Anaerobic digestion offers a cost-effective and environmentally feasible solution for 46 organic waste management while contributing to satisfy the global demand for 47 renewable energy via biogas production. In this context, the annual biogas production in 48 the European Union accounted for ~13.4 Mtoe in 2013 [1]. Biogas is composed mainly 49 of methane (CH4) (40-75%), carbon dioxide (CO2) (25-50%), hydrogen sulfide (H2S) 50 (0.005-2%) and ammonia (NH3) (˂1%). Other gases such as hydrogen (H2), nitrogen 51 (N2), oxygen (O2) and halogenated hydrocarbons are also present in raw biogas at lower 52 concentrations [2]. The concentration of these biogas pollutants depends on the 53 composition of the initial organic substrate and the type of anaerobic digestion process. 54 The H2S present in biogas corrodes metal parts, reduces the durability of the motors and 55 generates hazardous sulfur dioxide when biogas is combusted for the generation of heat 56 and electricity. Likewise, CO2 reduces the specific calorific value of biogas and 57 increases carbon monoxide and hydrocarbon emissions during combustion. Therefore, 58 these biogas pollutants must be previously removed in order to comply with the 59 technical specifications for biogas to be used as a transport fuel or injected into natural 60 gas grids. Most international legislations for bio-methane, which is the most common 61 term to refer to the upgraded biogas, require concentrations of CH4 ≥95%, CO2 ≤2%, O2 62 ≤0.3 % and negligible amounts of H2S [3]. 63 64 Conventional physical-chemical technologies such as water scrubbing, chemical 65 scrubbing and membrane separation are commonly applied for CO2 removal from 66 biogas. However, these technologies often require a previous H2S cleaning step such as 67 activated carbon adsorption or chemical scrubbing [4]. On the contrary, biological H2S 68 removal technologies such as anoxic and aerobic biotrickling filters are not able to 69 5 remove CO2 and present operational problems such as elemental sulfur accumulation 70 (and subsequent clogging of the packed-bed) and biogas contamination with O2 and N2 71 [5, 6]. In addition, the physical-chemical technologies capable of simultaneously 72 removing CO2 and H2S (for example chemical scrubbing with alkali aqueous solutions) 73 exhibit high operating costs and a significant environmental impact [7]. 74 75 In this regard, microalgae-based processes have emerged as a competitive and 76 environmentally sustainable alternative for the simultaneous removal of CO2 and H2S 77 from biogas [8]. These processes are based on the fixation of CO2 via photosynthesis by 78 microalgae and the oxidation of H2S to sulfate by sulfur oxidizing bacteria using the 79 oxygen photosynthetically produced. Moreover, the anaerobic effluents produced on-80 site can eventually support microalgae growth, thus reducing their associated treatment 81 costs and eutrophication potential [9]. In addition, the algal biomass generated during 82 the photosynthetic biogas upgrading process can be used as a feedstock for bio-fuel or 83 bio-fertilizer production [10, 11], provided that biomass production has been properly 84 maximized. However, the increase in pH and modification of metal ion speciation (e.g. 85 Ca2+, Mg2+ and Fe2+) in the cultivation broth induced by microalgae growth can 86 promote the abiotic removal of N and P by volatilization and precipitation, respectively 87 [12]. This abiotic nutrient removal mechanism contributes to a detrimental loss of 88 nutrients and causes a severe environmental impact derived from the indirect N2O 89 emissions associated to NH4+ stripping [13]. 90 91 Several proof of concept studies of this innovative photosynthetic biogas upgrading 92 process coupled with nutrient removal from digestate have been recently conducted by 93 Bahr et al. [8], Serejo et al. [14] and Posadas et al., [15] in a HRAP interconnected to an 94 6 external CO2−H2S absorption column (AC). However, while a complete H2S removal 95 was always observed, CO2 removal was low (<80%) and the upgraded biogas was 96 contaminated with N2 and O2 (stripped out from the cultivation broth), the latter 97 decreasing the CH4 content in the upgraded biogas to ~80%. Therefore, the O2 and N2 98 content in the upgraded biogas represents nowadays the main limitation of this 99 technology to achieve a high quality bio-methane, which entails the need to explore new 100 operational strategies to minimize the desorption of these bio-methane pollutants from 101 the algal-bacterial broth. In addition, little attention has been also paid to the 102 optimization of nutrient recovery from digestates, which would enhance the 103 environmental sustainability of the photosynthetic biogas upgrading process. 104 105 This research aimed at optimizing both the photosynthetic biogas upgrading process and 106 nutrient recovery from digestate in an algal-bacterial HRAP interconnected to a biogas 107 absorption column via recirculation of the settled broth. A preliminary optimization of 108 the recycling liquid to biogas ratio was conducted in order to obtain a bio-methane with 109 sufficient quality to be injected into natural grids. Then, an innovative HRAP 110 operational strategy based on the control of algal-bacterial biomass productivity via 111 settled biomass wastage was evaluated in order to enhance nutrient recovery from a 112 synthetic digestate while producing a high quality bio-methane. 113 114 2. Materials and methods. 115 2.1 Experimental setup and operational conditions. 116 The experimental setup, located at the Dept. of Chemical Engineering and 117 Environmental Technology at Valladolid University (Spain), consisted of a 180 L high 118 rate algal pond (170 cm length × 82 cm width × 15 cm depth) with an illuminated area 119 7 of 1.21 m2, interconnected to a 8 L conical settler and to a 2.2 L absorption column (4.4 120 cm diameter, 165 cm height) via recirculation of the settled algal cultivation broth 121 (Figure 1). The HRAP was fed with a synthetic digestate at an influent flow rate of 1.3 122 ± 0.2 L m-2 d-1, continuously agitated at an internal liquid recirculation velocity of ≈20 123 cm s−1, and illuminated with fluorescent lamps at 420 ± 105 μmol m−2 s−1 using 16:8 h 124 light:dark cycles. Tap water was supplied to compensate evaporation losses. The 125 composition of the synthetic digestate was (mg L-1): ammonium (NH4+) = 526 ± 132, 126 total nitrogen (TN) = 646 ± 61, total phosphorous (TP) as P-PO43‑= 53 ± 11, inorganic 127 carbon (IC) = 4458 ± 106 and sulfate (SO42-) = 317 ± 83. Digestates are characterized 128 by a high alkalinity and nutrient concentrations [16]. The effluent from the HRAP was 129 collected in the settler and the clarified effluent was then pumped to the bottom of the 130 AC at 1.6 m3 m−2 h−1 (flow rates referred to the AC cross sectional area) co-currently 131 with the biogas sparged (70% CH4, 29.5% CO2, 0.5% H2S, Abello Linde (Barcelona, 132 Spain)) through a metallic diffuser at 1.6 m3 m-2 h-1. The liquid phase exiting the AC 133 was returned to the HRAP, while the excess of effluent from the system was removed 134 by overflow from the settling tank. This innovative photobioreactor configuration 135 allowed decoupling the hydraulic retention time from the algal bacterial biomass 136 productivity by controlling the rate of settled biomass wasted and returned to the HRAP. 137 138 8 139 Figure 1. Schematic diagram of the experimental setup used for the continuous 140 upgrading of biogas coupled to digestate treatment. 141 142 2.2 Influence of the recycling liquid to biogas ratio on the quality of the upgraded 143 bio-methane. 144 L/G ratios ranging from 0.5 to 60 were tested in order to maximize CO2 and H2S 145 removal while minimizing O2 and N2 desorption from the recycling liquid to the 146 upgraded biogas. The synthetic biogas was sparged into the AC at 5.3, 16.0, and 31.5 147 mL min-1, while the external liquid recirculation rate was set at 15, 60, 120, 203, and 148 315 mL min-1 for each biogas flow rate tested. The AC was constantly fed with the 149 algal-bacterial broth at a pH of 10 ± 0.3. The absorption system was allowed to stabilize 150 for two times the AC hydraulic retention time (HRT) prior to the monitoring of the 151 upgraded biogas composition by GC-TCD. 152 153 2.3 Influence of biomass productivity on biogas upgrading and nutrient recovery. 154 9 The HRAP was inoculated with a consortium of cyanobacteria/microalgae composed of 155 Geitlerinema sp. (61.5%), Staurosira sp. (1.5%) and Stigeoclonium tenue (37%) from a 156 previous culture grown in diluted centrate wastewater. The consortium was then 157 acclimated to the digestate for 40 days prior to the experiment start-up. A biomass 158 productivity of 2.2 g m-2 d-1 was set during stage I (days 0-77) by controlling the rate of 159 withdrawal of settled biomass based on the total suspended solids (TSS) concentration 160 in the settler. The biomass productivity was increased to 4.4 g m-2 d-1 during stage II 161 (days 78-159) and to 7.5 g m-2 d-1 during stage III (days 160-202). The latter 162 productivity was selected based on the maximum biomass productivity expected from 163 the TP daily fed into the HRAP (assuming a P biomass content of 1 % according to 164 Alcántara et al., [17]). The experimental system was operated indoors for 202 days. 165 Liquid samples (100 mL) were collected twice a week from the digestate influent, the 166 treated digestate and the cultivation broth of the HRAP to monitor the pH and 167 concentration of IC, TN, NH4+, nitrite (NO2-), nitrate (NO3-), phosphate (PO43-), SO42- 168 and TSS. The TSS concentration of the settled biomass was also determined twice a 169 week to control biomass productivity. The temperature and dissolved O2 concentration 170 (DO) were monitored in-situ. Gas samples from the inlet and outlet of the biogas 171 absorption column were periodically drawn to monitor the concentrations of CO2, H2S, 172 O2, N2, and CH4. The inlet and outlet gas flow rates in the AC were also measured. An 173 aliquot of 50 ml of algal-bacterial biomass was taken in each steady state to characterize 174 the populations of microalgae and bacteria. 175 176 2.4 Analytical procedures. 177 Dissolved IC and TN concentrations were determined using a Shimadzu TOC-VCSH 178 analyzer (Japan) equipped with a TNM-1 chemiluminescence module. NH4+ was 179 16 The biological oxidation of CH4 resulted in average CH4 losses of 4.9 ± 2.4% (on a 280 mass basis) during stage I, no methane losses being recorded afterwards. The CH4 281 content in the upgraded biogas was 95.8 ± 0.8%, 96.9 ± 0.7% and 97.2 ±0.2% in stages 282 I, II and III, respectively (Figure 3a). These values are comparable to those achieved by 283 water scrubbing technologies, where CH4 losses by dissolution in the pressurized water 284 of 3-5% result in CH4 purities of 80-99%, depending on the N2 and O2 content of the 285 upgraded biogas [23]. 286 287 The O2 demand in the absorption column resulting from the biological oxidation of H2S 288 caused an oxygen content in the upgraded biogas of 0.1 ± 0.2% in stages I and II and 289 0.03 ± 0.04% in stage III (Figure 3b). These O2 concentrations recorded in the bio-290 methane were lower than the values obtained by Meier et al. [20] (1.2%) and Posadas et 291 al., [15] (0.7-1.2%), and remained significantly below those reported in literature during 292 biogas upgrading in algal photobioreactors (10-24%) [21, 24]. Finally, the N2 stripped 293 out from the recycling cultivation broth resulted in average concentrations of 2.6 ± 294 0.9%, 2.4 ± 0.5% and 2.4 ± 0.2% during stages I, II and III, respectively (Figure 3b), 295 due to the low L/G ratio applied in this study (which limited the amount of N2 296 potentially desorbed). Higher N2 concentrations in the upgraded biogas of 6-8% were 297 recorded by Posadas et al., [15] and Serejo et al., [14] during photosynthetic biogas 298 upgrading at a L/G of 10. In this context, the optimum bio-methane composition was 299 obtained at the highest microalgae productivity evaluated (0.4 ± 0.1% CO2, 0.03 ± 300 0.04% O2, 2.4 ± 0.2% N2 and 97.2 ± 0.2% CH4), which complied with the regulatory 301 limits of most European legislations for bio-methane injection in natural gas grids 302 (Figures 3a and 3b). For instance, the injection of bio-methane into the Spanish network 303 17 allows up to 0.3% of O2 provided that CO2 concentration does not exceed 2% and CH4 304 concentration remains over 95% [25]. 305 306 3.3 Influence of biomass productivity on nutrient removal and nutrient recovery. 307 Most recent life cycle analyses have shown that the use of wastewater as a low-cost 308 nutrients and water source can reduce the overall energy requirements and improve the 309 environmental sustainability of microalgae mass production [26, 27]. In our particular 310 case, microalgae production using the N and P present in anaerobically digested 311 wastewaters can significantly decrease the operating costs of the biogas upgrading 312 process, while preserving fresh water resources and recovering these nutrients in the 313 form of a microalgae biomass that can be further valorized as a bio-fertilizer. Despite 314 the potential of microalgal biotechnology to fix nutrients from digestates, abiotic 315 removal still represents an important mechanism for nutrient removal from wastewater 316 in algal-bacterial processes. Thus, N removal by stripping can account for up to 82% 317 [28] and P removal by precipitation for up to 63% [29] of the total nutrients supplied. 318 Nonetheless, the monitoring of this abiotic nutrient removal in HRAPs is often 319 disregarded [12]. 320 321 322 Table 1. Average dissolved oxygen concentration, pH, temperature, total suspended solid concentration and biomass productivity recorded during the three operational stages. Stage T HRAP (ºC) PHHRAP DO (mg O2 L-1) TSS HRAP (g L-1) Productivity (g m-2 d-1) I 22 ± 3 9.1 ± 0.1 5.4 ± 0.8 1.6 ± 0.1 2.2 ± 1.4 II 25 ± 2 9.6 ± 0.3 7.5 ± 1.4 1.2 ± 0.4 4.4 ± 1.5 III 28 ± 1 10.6 ± 0.1 9.6 ± 0.4 0.9 ± 0.1 7.5 ± 0.1 18 The high buffer capacity of the cultivation broth as a result of the high IC 323 concentrations present in the digestate and the high water evaporation losses, together 324 with the high photosynthetic activity in the system, maintained high pH values during 325 the three operational stages without an automatic pH control (Table 1). The temperature 326 of the algal–bacterial broth slightly increased concomitantly with the seasonal variation 327 of the ambient temperature, but remained close to optimum values for microalgae and 328 bacteria cultivation. Apart from the impinging radiation, other variables such as the 329 nutrients load (determined by the flow rate and nutrients concentration of the target 330 wastewater) and biomass concentration in the cultivation broth (determining light 331 penetration) influence microalgae productivity in HRAPs devoted to wastewater 332 treatment. For instance, low biomass concentrations (~0.5 g L-1) are typically 333 encountered in open ponds treating domestic wastewaters at HRTs of 5-10 days. 334 Biomass productivity can be thus boosted by increasing the nutrients load into the 335 HRAPs, provided that light supply does not limit the process. However, while an 336 increase in wastewater flow rate might induce microalgae washout, the use of 337 wastewaters with high nutrient concentrations (such as digestates) would entail very 338 dense microalgae cultures, which would ultimately limit microalgae productivity as a 339 result of an excessive mutual shading. In this context, the decoupling between the 340 hydraulic retention and biomass retention time (inversely related to microalgae 341 productivity) represents an innovative strategy for maximizing biomass productivity 342 during microalgae cultivation in high-strength wastewaters. The control of biomass 343 productivity via regulation of the settled biomass wastage rate would allow maximizing 344 nutrient recovery from wastewaters. A TSS concentration of 1.6 ± 0.1 g L-1 was 345 recorded in the HRAP when operating at an average productivity of 2.2 g m-2 d-1 in 346 stage I. This TSS concentration decreased to 1.2 ± 0.4 g L-1 and 0.9 ± 0.1 g L-1 under 347 19 operation at 4.4 g m-2 d-1 and 7.5 g m-2 d-1, respectively. The results clearly showed that 348 an increase in the rate of biomass wastage from the settler resulted in lower TSS 349 concentrations, which likely improved the overall photosynthetic efficiency as a result 350 of an enhanced light penetration. In addition, the control of biomass productivity was 351 supported by the good settling properties of the algal-bacterial biomass present in the 352 HRAP. However, a decrease in the TSS removal efficiency of the settler from 95 ± 3% 353 in stage I to 84 ± 4% in stage III was recorded, which was attributed to the shift in 354 microalgae population observed in stage II (see section 3.4). Unfortunately, the effluent 355 TSS concentrations (70 ± 50 mg L-1) remained always over the maximum discharge 356 limit in European Union legislation (35 mg L-1) [30]. 357 358 Table 2. Average removal efficiencies of total nitrogen, ammonium, phosphorus, inorganic carbon and total suspended solids recorded during the three operational stages. Stage Removal efficiencies (%) TN N-NH4+ P-PO43- IC TSS I 91 ± 4 100 77 ± 16 86 ± 6 95 ± 3 II 92 ± 4 100 63 ± 18 78 ± 10 91 ± 10 III 98 ± 2 100 73 ± 19 70 ± 9 84 ± 4 359 A complete removal of ammonium was observed during all stages, while TN-REs 360 increased from 91 ± 4% up to 98 ± 2% when biomass productivity increased from 2.2 to 361 7.5 g m-2 d-1 (Table 2). Despite the slight influence of biomass productivity on TN-REs, 362 the share of the inlet TN assimilated into biomass varied from 19 ± 13 % at the lowest 363 microalgae productivity to 83 ± 9% at the highest productivity (Table 3). In this context, 364 the low nitrification activity recorded along with the high pH value supported a 365 significant N-NH4+ removal by stripping, which decreased from 75 ± 12% in stage I to 366 13 ± 9% in stage III. On the other hand, phosphorus removal remained stable regardless 367 of the biomass productivity set, with REs of 77 ± 16%, 63 ± 18% and 73 ± 19% in 368 20 stages I, II and III, respectively. Serejo et al. [14] recorded similar phosphorous REs (71 369 ± 3%) at a comparable biomass productivity (7.1 ± 0.8 g m-2 d-1) during the treatment of 370 anaerobically digested vinasse coupled to biogas upgrading. Similar to the share of TN 371 assimilated, the increase in biomass productivity resulted in an increase in the 372 contribution of P assimilation to the TP removal from 22 ± 12% to 100%. The absence 373 of PO43- volatilization, together with the high pH prevailing in the cultivation broth 374 throughout the entire experimental period, suggested that precipitation was the main 375 phosphorous removal mechanism under low biomass productivities. Therefore, the 376 control of biomass productivity via regulation of the biomass wastage rates allowed 377 maximizing nutrient recovery in the form of algal biomass in detriment of the abiotic 378 nutrients removal mechanisms. 379 380 Table 3. Nutrient recovery via biomass assimilation estimated from the nutrient removed and the elemental composition and mass flow rate of the biomass harvested during the three operational stages. Stage Nutrient recovery as biomass (%) Biomass elemental composition (%) C P N C P N I 6 ± 3 22 ± 12 19 ± 13 43.6 0.7 6.5 II 16 ± 5 50 ± 19 36 ± 18 46.5 0.8 7.2 III 30 ± 1 100 83 ± 9 48.0 0.9 6.7 381 3.4 Consortia of cyanobacteria/microalgae and bacteria. 382 The microalgae and cyanobacteria species initially present in the inoculum were 383 gradually replaced along the three operational stages. The cyanobacterium prevailing in 384 the inoculum (Geitlerinema sp.) was not observed under steady state conditions in 385 stages I, II and III. Thus, the cyanobacteria/microalgae consortium was mainly 386 composed of Limnothrix planktonica (32.9%), Acutodesmus obliquus (2.6%), Chlorella 387 vulgaris (2.6%), Mychonastes homosphaera (5.9%), Navicula sp. (0.7%), Phormidium 388 sp. (19.7%) and Stigeoclonium tenue (35.5%) during stage I. This high diversity was 389 21 similar to that reported in indoor HRAP treating digestates [14, 15]. Surprisingly, this 390 high microalgae diversity disappeared in stage II with the establishment of an unialgal 391 culture of the Chlorophyta Mychonastes homosphaera. This unialgal culture remained 392 dominant throughout stage III likely due to the extreme environmental conditions 393 prevailing in this study (high pH and salinity as a result of the high water evaporation 394 losses). In addition, the control of biomass productivity via regulation of the settled 395 biomass wastage rate applied might have also influenced the dominant species and 396 algal/bacterial ratio since the increase in biomass productivity likely induced the 397 development of fast growing microorganisms. Mychonastes homosphaera (Skuja) 398 Kalina & Puncochárová is currently regarded as a taxonomic synonym of Chlorella 399 minutissima Fott & Nováková. The potential of this microalga for wastewater treatment 400 [31], heavy metal removal [32] and biodiesel production has been consistently 401 demonstrated, Mychonastes homosphaera being capable of storing a desirable fatty acid 402 profile under nitrogen starvation [33]. The valorization of this microalga into high-403 added value chemicals or biofuels such as syngas, bioethanol or bio-oil using a 404 biorefinery approach will certainly enhance the sustainability and economic viability of 405 microalgae-based biogas upgrading [34]. 406 The high diversity revealed by microscopic observation was confirmed by the Shannon-407 Wiener diversity indexes obtained, which ranged from 1.5 to 3.5 (Figure 1, 408 supplementary material). The slight decrease of this index from 3.2 in stage I to 2.9 in 409 stage II also confirmed the shift in algae diversity microscopically observed. Likewise, 410 the analysis of the Pearson similarity coefficients showed a high similarity between the 411 microbial communities present in stages II and III (99%), which was in agreement with 412 the above mentioned establishment of a dominant microalga specie. The DGGE analysis 413 (Figure 1, supplementary material) showed 24 bands, which were sequenced. Six 414 22 different phyla were retrieved from the RDP database: Cyanobacteria/Chloroplast (10 415 bands), Acidobacteria (4 bands), Proteobacteria (4 bands), Deinococcus-thermus (1 416 band), Chloroflexi (1 band), Actinobacteria (1 band) (Table 1, supplementary material). 417 The morphological identification of Mychonastes homosphaera was confirmed by 418 bands 8, 9 and 10, which belonged to the genus Chlorophyta and were related to 419 Chlorella species. The phyla Acidobacteria (bands 12 and 13), Proteobacteria (bands 420 15 and 16) and Actinobacteria (band 21) were found in the three operational stages, 421 while the phylum Chloroflexi was detected in the inoculum and stages II and III. 422 Bacteria from the genus Blastocatella (band 11) and the Gammaproteobacteria class 423 (band 15), which have been identified in activated sludge [35] and HRAPs treating 424 piggery wastewater [36], respectively, likely supported the aerobic biodegradation of 425 the organic matter and ammonia contained in the digestate. Finally, the identification of 426 the genus Thioalbus (band 16) confirmed the biological nature of H2S oxidation [37]. 427 To the best of our knowledge, this is the first time that sulfur-oxidizing bacteria 428 (facultative microorganisms that can use O2 or NO3- as electron acceptors) have been 429 found in these photosynthetic biogas upgrading processes. 430 431 3.5 Conclusions. 432 This study confirmed the potential of photosynthetic biogas upgrading to support a cost-433 efficient bio-methane production coupled to nutrient recovery from digestate. To the 434 best of our knowledge, this is the first experimental study reporting biological biogas 435 upgrading to a bio-methane complying with most European legislations for biogas 436 injection into natural gas grids. An almost complete removal of H2S and CO2, and 437 concentrations of O2 and CH4 in the upgraded biogas <0.1% and >95%, respectively, 438 were achieved regardless of the biomass productivity set. 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