microorganisms

Article Wastewater Biofilm in Photobioreactors

Antonella Guzzon 1 , Francesca Di Pippo 2 and Roberta Congestri 1,*

1 LBA-Laboratory of Biology of , Department of Biology, University of Rome “Tor Vergata”, 00133 Rome, Italy 2 CNR-IRSA, National Research Council, Water Research Institute, Area della Ricerca di Roma 1, 00010 Montelibretti (RM), Italy * Correspondence: [email protected]; Tel.: +39-06-7259-5989

 Received: 26 June 2019; Accepted: 8 August 2019; Published: 10 August 2019 

Abstract: Photosynthetic performance of algal-bacterial biofilms from an Italian plant was studied in a flow-lane photobioreactor at different irradiances, temperatures, and flow regime to evaluate the effects of these environmental parameters on biofilms’ functioning, in view of application of these communities in wastewater biological treatment. Pulse amplitude modulated fluorescence was used to estimate the effective quantum yield of PSII (∆F/Fm’) of the light-acclimated biofilms and to perform rapid light curves (RLCs) for the determination of the photosynthetic parameters (rel.ETRmax, α,Ik). Chl a, ash free dry weight (AFDW), and dry weight (DW) were measured to assess phototrophic and whole biofilm biomass development over time. From the analysis of photosynthetic parameter variation with light intensity, temperature and flow rate, it was possible to identify the set of experimental values favoring biofilm photosynthetic activity. Biomass increased over time, especially at the highest irradiances, where substrata were fastly colonized and mature biofilms developed at all temperatures and flow conditions tested.

Keywords: phototrophic biofilm; wastewater; photosynthesis; photobioreactor; Pulse Amplitude Modulated fluorescence; biofilm biomass

1. Introduction Biological treatment is an important and integral part of wastewater treatment (WWT) procedures. Currently, the activated sludge process is the most commonly applied biological WWT technology. The activated sludge treatment utilizes dense bacterial suspended communities, forming biological flocs, to degrade organic material under aerobic conditions [1]. Although this technology provides satisfactory pollutant removal, it requires complex and multistep recycles of activated sludge to meet the limits imposed by environmental regulations. The overall increase of process costs, complexity, and energy input has led, in the last years, to explore more sustainable approaches [2,3]. Thanks to the synergistic relationship between photosynthetic and heterotrophic microorganisms, the use of microalgal-bacterial consortia in WWT is now considered a cost-effective, efficient, and sustainable alternative to conventional activated sludge treatment, especially for municipal wastewaters, due to the potential for cost-free oxygenation, efficient nutrient removal, and carbon dioxide sequestration provided by metabolism [4,5]. In this way, microalgal-bacterial combinations have been employed for treating secondary wastewater effluents in the form of suspensions in the enhanced pond and wetland (EPW) system, a multiple step process including high-rate algal ponds (HRAPs) [6], and gravity algal harvesters (AHs) [7]. Alternatively, microalgal-bacterial consortia have been grown as attached communities in algal turf scrubbers (ATSs) [8], coupled to constructed wetlands [9] or as

Microorganisms 2019, 7, 252; doi:10.3390/microorganisms7080252 www.mdpi.com/journal/microorganisms Microorganisms 2019, 7, 252 2 of 18 microalgal-integrated fixed film activated sludge (MAIFAS), an advanced biological WWT process that integrates distinct algal and nitrifying biofilm carriers within conventional activated sludge [10]. Although the complex interactions between microalgae and in WWT are not yet fully elucidated, outputs of many studies conducted to date have suggested that microalgae, through photosynthesis, provide to heterotrophic bacteria to reduce chemical oxygen demand (COD). On the other side, bacteria release carbon dioxide and mineral nutrients from respiration and organic matter degradation which are efficiently converted into biomass by microalgae when exposed to solar light in outdoor systems [2,4,11,12]. Simultaneously, the exchange of metabolites results in an overall increase in biomass productivity and hence, pollutant removal efficiency. Moreover, WWT based on microalgae-bacterial consortia has the added benefits to allow for nutrient recover and biomass recycle into valued compounds as , growth enhancers, aquaculture feeds, and/or bioenergy, also implementing a biorefinery approach of the biomass produced starting from wastewaters and up-cycling of the effluent water [13–15]. The use of municipal wastewaters as a source of nitrogen and phosphorus to feed the algae is also important for reducing the cost of culturing biomass destined for commercial purposes, such the extraction of biocompounds [16]. In this context, the use of phototrophic biofilms, attached matrix-enclosed microbial communities comprising both phototrophs and chemotrophs, can offer an additional advantage over HRAPs, in terms of time and cost savings involved in biomass accumulation and harvesting. Biofilm indeed is easily removed by scraping it off from the growth support. Moreover, an effluent with a lower total suspended solids concentration is produced with respect to suspended growth systems [12,17–20]. Despite the favorable outlook of wastewater fed biomass production, routine large-scale application of benthic photosynthetic communities in WWT treatment systems is not efficiently exploited, with much effort put on research only at the laboratory and/or pilot scale [2,21]. Phototrophic biofilm efficiency in wastewater treatment depends on biomass development and physiology, including photosynthetic activity [22]. Although studies have addressed the potential of nutrient removal from wastewaters [23–26] and the intracellular accumulation by aquatic phototrophic biofilms [27], few have investigated photosynthetic performance and evaluated the environmental factors influencing the process, i.e., geographical location, temperature and oxygen accumulation, light availability, light–dark cycle, as well as pH variation with photosynthesis. Therefore, understanding the overall community photosynthesis and photoacclimation capability is pivotal to the exploitation of phototrophic microbial consortia in biological treatment of wastewaters. Different approaches have been applied to measure photosynthesis of aquatic phototrophic biofilms, such as oxygen microelectrodes, photosynthetic chambers in combination with oxygen probes, and pulse amplitude modulated (PAM) fluorimetry [28–35]. The fluorescence method is advantageous for estimating the rates of photosynthesis for intact biofilms being rapid and non-destructive, as measurements are performed in a shorter time than conventional methods and without the need to remove the phototrophs from the substrata [32]. Moreover, the parameters measured allow insight into key aspects of photosynthetic light capture and electron transport [36]. Accordingly, the aim of this study was to investigate the photosynthetic behavior, biomass development, and production of phototrophic biofilms collected from an Italian WWT plant and cultured in a flow-lane incubator system at different abiotic conditions. Species composition, matrix characterization and nutrient removal capability of these autotrophic-heterotrophic cultured biofilms have been previously investigated [27,37–40]. Therefore, the present work provides additional useful insights into phototrophic biofilms functioning, thus increasing the knowledge of how these complex autotrophic–heterotrophic consortia behave at different environmental factors, in view of their exploitation in wastewater . Microorganisms 2019, 7, 252 3 of 18

Microorganisms2. Materials 2019 and, 7, Methodsx FOR PEER REVIEW 3 of 19

2.1. Biofilm Sampling and Setup of Biofilm Cultures samples were collected by scraping off the overflow system of the sedimentation tank (ST) ofBiofilm an Italian samples wastewater were collectedtreatment by plant scraping (WWTP) off thelocated overflow in Fiumicino system (Rome, of the sedimentation Italy). Taxonomic tank assessme(ST) of annt Italianof in situ wastewater consortia treatment can be found plant in (WWTP) [41–45] located. The plant in Fiumicino is designed (Rome, to serve Italy). the Taxonomic Airport “Leonardoassessment da of Vinci” in situ and consortia receives can municipal be found in wastewater [41–45]. The with plant an isestimated designed capacity to serve of the 6000 Airport m3 sewage“Leonardo per day. da Vinci” Physico and-chemical receives municipalcharacteristics wastewater of the plant with have an estimated been previously capacity described of 6000 m 3[41,46sewage]. perAfter day. Physico-chemicalsampling, ST biofilm characteristics biomass was of subjected the plant haveto a pre been-treatment previously to obtain described safe- [handling41,46]. and homogeneousAfter sampling, inocula STaccording biofilm biomassto [43]. Briefly, was subjected scrapings to awere pre-treatment washed in to 2% obtain sodium safe-handling hypochlorite and forhomogeneous 15 min to eliminate inocula macro according- and tomicrozoobenthos [43]. Briefly, scrapings and then were rinsed washed in BG11 in 2% medium sodium [47 hypochlorite] modified forfor ensuring 15 min to balanced eliminate algal macro- growth and microzoobenthosby adding vitamins and and then silicates rinsed infor BG11 diatoms. medium Moreover, [47] modified NO3− − andfor PO4 ensuring concentration balanced algalwas modified growth by to adding approximate vitamins wastewater and silicates levels. for diatoms.Thereafter, Moreover, were NO3− hoandmogeni PO4−zedconcentration and frozen at was −20 modified °C for 24 to h approximate to remove grazers wastewater [43]. To levels. start Thereafter,the cultures, biofilms 100 mL were of cellhomogenized suspension andwas frozen made atup to20 4 CL with for 24 the h tomodified remove grazersBG11 and [43 filtered]. To start through the cultures, a 300 μm 100 net. mL of cell − ◦ suspensionThe resulting was made mixture up to was 4 L inoculated with the modified in a semi BG11-continuous and filtered flow through-lane incubator a 300 µm system net. (PBI) [48]. The resulting mixture was inoculated in a semi-continuous flow-lane incubator system (PBI) [48]. PBIPBI contained contained four four separate separate light light chambers chambers (LCs) (LCs) (120 (120 x 1010 cm cm each) each) (Figure (Figure 11aa).). ×

Figure 1. Schematic view of the prototype incubator: In and outlet, light chambers with separator walls Figurefor medium 1. Schematic guidance, view illuminated of the prototype at four di incubator:fferent irradiances In and outlet, (a). Experimental light chambers conditions with separator of the four wallsruns for performed medium (guidance,b). MINI-PAM illuminat useded to at perform four different fluorescence irradiances measurements (a). Experimental on biofilms conditions colonizing of thethe four slides runs in theperformed incubator (b with). MINI the- fiberopticPAM used positioned to perform above fluorescence biofilm surface measurements (c). on biofilms colonizing the slides in the incubator with the fiberoptic positioned above biofilm surface (c). Medium flowed above polycarbonate slides (76 25 1), used as substratum for biofilm growth, × × andMe wasdium changed flowed at above regular polycarbonate intervals (twice slides per (76 week). x 25 x Flow 1), used rate as was substratum valve-regulated. for biofilm “True growth, light” and(T-8, was Auralight, changed Sweden) at regular lamps intervals were (twice used as per the week). light source. Flow rate was valve-regulated. “True light” 2 1 (T-8, Auralight,Biofilms were Sweden) cultured lamps at were four used photon as the flux light densities source. (PFDs): 120 µmol photon m− s− (LC1), 2 1 2 1 2 −2 1−1 60 µBiofilmsmol photon were m cultured− s− (LC2), at four 30 µphotonmol photon flux densities m− s− (LC3),(PFDs): 15 120µmol μmol photon photon m− ms−s (LC4). (LC1), Two 60 −2 −1 −2 −1 1 1 −2 −1 μmoltemperatures photon m (20 ◦sC and(LC2 30), ◦ 30C) andμmol two photon flow conditions m s (LC3 (25), L 15 h− μmoland 100 photon L h− m) were s tested.(LC4). These Two temperaturesfour incubator (20 experiments °C and 30 °C) were and referred two flow to conditions as “Runs” (25 (Figure L h−11 andb). Transmittance100 L h−1) were was tested. measured These fourin each incubator chamber experiments by nine subsurface were referred light to sensors as “Runs” located (Figure under 1b). the Transmittance growth surface. was Growthmeasured of thein eachbiofilm chamber could beby accurately nine subsurface tracked light by the sensors reduction located in light under transmittance. the growth The surface. incubator Grow designth of the was biofilm could be accurately tracked by the reduction in light transmittance. The incubator design was previously described [48]. All light and temperature sensors were connected to a computer through a serial port for continuous data monitoring and acquisition [48].

Microorganisms 2019, 7, 252 4 of 18 previously described [48]. All light and temperature sensors were connected to a computer through a serial port for continuous data monitoring and acquisition [48]. Biofilms were sampled at three stages of development as function of biofilm light absorbance values (Absorbance = 100%—percent transmitted light value): (1) Initial (in), when the average value of absorption was 10%; (2) Active (ac), when mean value was 50%; (3) Mature (ma), at 90–95%. Each run lasted ~30 days. On the last day, biofilm was collected without regard of light absorption and labelled as last sampling (ls).

2.2. PAM Measurements of Photosynthetic Parameters Photosynthetic parameters of cultured biofilms were assessed at each sampling stage using a miniaturized pulse amplitude modulated (Mini-PAM) fluorometer, coupled to WinControl Software (Walz GmbH, Effeltrich, Germany) for computer control and data analysis. In the Mini-PAM, fluorescence was excited by pulses (3 µs width) of measuring red light from a light-emitting-diode (LED) with the peak excitation band at 650 nm. Fluorescence measurements were performed on light-acclimated biofilms to assess the effective quantum yield of charge separation in photosystem II PSII, either under the incubator light conditions or during the measurement of electron transport rate versus irradiance (ETR/I) curves. Three slides were sampled in the middle of light period (8 h ca.) at the initial stage of biofilm development, immersed in culture medium to avoid desiccation, inhibition of photosynthesis, and/or damage to photosystems, and positioned on a specifically designed grid so that biofilm surface could be virtually divided into 27 spots (8 8 mm each). The fiberoptic was kept at × 10 mm above biofilm surface using a holder to obtain a homogeneous field of illumination (Figure1c). After PAM measurements, the slides were put back in the same position in the light chamber and then re-used for fluorescence assessment in the following samplings, i.e., at the active and mature stage of biofilm development. The effective quantum yield of charge separation in PSII (∆F/Fm’) of the light-acclimated biofilms was calculated according to [49] the following:

∆F/Fm’ = (Fm’ F)/Fm’, (1) − where F is the steady-state fluorescence of the light-adapted sample and Fm’ is the maximum light-adapted fluorescence after the application of a single saturating light pulse (800 ms, 3000 µmol -2 -1 photon m s )[49,50]. ∆F/Fm’ was measured on 27 spots of three slides (81 replicates) to take into account the heterogeneity of microalgal distribution. Thereafter, ETR/I curves were recorded on three spots on each slide providing nine replicates for each sampling. The position of the spots was chosen randomly at the initial sampling and then kept constant in the active and mature phase. ETR/I curves were measured by exposing the sample to eight increasing PFDs, up to 720 µmol photon m-2 s-1. Each period of actinic light lasted 10 s before the saturation pulse was applied to determine ∆F/Fm’. The internal halogen lamp (20 W) of the Mini-PAM served as light source for saturation pulse and for continuous actinic illumination. The photosynthetic electron transport rate (ETR) between PSII and PSI was calculated from the estimate of ∆F/Fm’ at different PFDs in actinic light according to the expression:

ETR = ∆F/Fm’ PFD 0.5 a* , (2) · · · PSII where 0.5 accounts for the excitation of both PSII and PSI and a*PSII is the optical cross section of PSII. Since we could not measure a* , relative ETR was calculated as ∆F/Fm’ PFD 0.5. PSII · · The ETR/I curves were fitted by means of an automatic spreadsheet based on linear regression for estimating ETR per light intensity and a chi-square minimization for the exponential function ETR α I ETRmax = ETRmax (1 -e − )[51]. From the fit of the maximum rate of relative ETR (rel.ETRmax), the Microorganisms 2019, 7, 252 5 of 18

initial slope, i.e., photosynthetic efficiency (α) and the light saturation parameter (Ik = rel.ETRmax/α) Microorganisms 2019, 7, x FOR PEER REVIEW 3 of 19 were calculated.

2.3. Biomass Biomass E Estimationstimation Phototrophic biomass biomass was was assessed assessed by by determining determining chlorophyll chlorophyll a concentrationa concentration per unit per area unit (mg m−2). Chlorophyll2 a was extracted overnight in 90% acetone and then quantified area (mg m− ). Chlorophyll a was extracted overnight in 90% acetone and then quantified spectrophotometrically [52 [52]].. Biomass productivity was determined as dry weight (DW g m−22 d−11) by oven drying samples at Biomass productivity was determined as dry weight (DW g m− d− ) by oven drying samples at 60 °C for 72 h. Samples were then burnt at 500 °C for 1 h to determine organic biomass (phototrophs 60 ◦C for 72 h. Samples were then burnt at 500 ◦C for 1 h to determine organic biomass (phototrophs + + heterotrophs) as ash-free dry weight (AFDW) per unit area (mg m2−2) [53]. heterotrophs) as ash-free dry weight (AFDW) per unit area (mg m− )[53].

2.4. Data Data E Elaborationlaboration To test the influenceinfluence of the experimental conditions and their interactions on all biofilmbiofilm photosynthetic parameters, thethe standardstandard ANOVAANOVA provided provided by by the the Past Past software software (v (v 3.2.5) 3.2.5) was was used used in incombination combination with with the Tukey the Tukey Honestly Honestly Significant Significant Difference Difference (HSD) test (HSD) for multipletest for multiplepost-hoc testing post-hoc or testingMann–Whitney or Mann test.–Whitney The eff test.ect of The these effect environmental of these environmental conditions on initial conditions and latest on initial phases and of biofilm latest phasesdevelopment of biofilm was development evaluated by repeatedwas evaluated ANOVA. by Correlationrepeated ANOVA. between Correlation Chl a and AFDW between was Chl assessed a and AFDWby applying was assessed the Spearman’s by applying rho-statistic the Spearman’s [54]. rho-statistic [54].

3. Results Results and and D Discussioniscussion

3.1. Biofilm Biofilm G Growthrowth BiofilmBiofilm development in the microcosm was monitored through an online and automatic system thatthat continuously continuously recorded recorded the the light light transmitted transmitted through through biofilm biofilm thickness thickness over over experimental experimental time, time, allowing the the calculation calculation of of absorbance absorbance and and the the subsequent subsequent construct constructionion of of growth growth curves curves [40,55 [40,55]].. Growth of biofilms biofilms was witnessed by the increasing light absorbance (Figure(Figure2 2).).

Figure 2. GrowthGrowth curves curves of of LC1, LC1, LC2, LC2, LC3 LC3,, and and LC4 LC4 biofilms biofilms in in Run1 Run1 ( (aa),), Run2 Run2 ( (bb),), Run3 Run3 ( (cc),), Run4 Run4 ( (dd)) obtained by t transformingransforming the on on-line-line recorded light transmitted values in light absorbance.absorbance.

A sigmoidal trendtrend waswas observedobserved forfor biofilms biofilms grown grown at at the the higher higher irradiances irradiances (LC1 (LC1 and and LC2) LC2) in allin allruns, runs, diversely diversely from from most most of theof the cultures cultures under under lower lower irradiances irradiances (LC3 (LC3 and and LC4) LC4) that that reached reached only only the theinitial initial and and active active phase. phase. Initial Initial phase phase lasted lasted between between 7 (LC1 7 (LC1 Run3) Run3) and 14and days 14 days (LC2 (LC2 Run1) Run1) for high for high light cultures and between 18 (LC3 Run1, LC3 Run4) and 30 days (LC4 Run4) for the low light ones. Irradiance appeared to influence biofilm development, in particular the duration of the initial phase, i.e., the higher the irradiance, the shorter resulted the lag phase. This is more evident in LC1 cultures, likely due to the dominance of unicellular green algae which have higher growth rates [40].

Microorganisms 2019, 7, 252 6 of 18 light cultures and between 18 (LC3 Run1, LC3 Run4) and 30 days (LC4 Run4) for the low light ones. MicroorganismsIrradiance appeared 2019, 7, x FOR to influencePEER REVIEW biofilm development, in particular the duration of the initial phase,3 of 19 i.e., the higher the irradiance, the shorter resulted the lag phase. This is more evident in LC1 cultures, Thelikely slope due of to t thehe curves, dominance reflecting of unicellular the active green phase algae of biofilm which havedevelopment, higher growth was more rates pronounced [40]. The slope in LC1of the and curves, LC2 cultures reflecting irrespective the active phase of the of different biofilm development,temperature and was flow more conditions pronounced tested, in LC1 reflecting and LC2 thecultures fast growth irrespective of phototrophs. of the different In LC3 temperature and LC4, andthe active flow conditions phase was tested, not completed reflecting except the fast for growth LC3 biofilmsof phototrophs. in Run3In (Figure LC3 and 2c) LC4,. Complete the active development phase was of not a completedmature community except for was LC3 obtained biofilms inonly Run3 in cultures(Figure2 atc). 120 Complete μmol photons development m−2 s−1 of (LC1) a mature in all community runs when wasbiofilms obtained were only dominated in cultures by filamentous at 120 µmol 2 1 cyanobacteriaphotons m− s−[40(LC1)] able in toall runsproduce when high biofilms amount weres dominatedof exopolysaccharides by filamentous [38 ], allowing [the40] establishmentable to produce of high complex amounts and of structured exopolysaccharides mature communities. [38], allowing The the highest establishment light intensity of complex tested and appearedstructured to mature be the communities. most favorable The for highest biofilm light formation intensity and tested development appeared to as be the the lag most phase favorable was shortefor biofilmr, the formationactive growth and developmentphase was fast as, theand lag mature phase communities was shorter, thewere active formed growth in all phase runs was within fast, theand experimental mature communities time. were formed in all runs within the experimental time. WhenWhen observed observed at at the the macroscopic macroscopic level, level, biofilm biofilm biomass biomass accumulation accumulation on on the the slides slides was was patchy patchy atat the the initial initial phase phase of of development development reflecting reflecting the the adhesion adhesion of of the the pioneer pioneer microorganisms microorganisms during during the the colonicolonizationzation process process [ [3737]].. The The following following active active phase phase was was characteri characterizedzed by by an an increasing increasing and and more more homogeneoushomogeneous coverage coverage of of the the substrata substrata which which ended ended to to be be completely completely coloni colonizedzed in in the the mature mature stage stage (Figure(Figure 33))..

FigureFigure 3. 3. DetailsDetails of of LC1 LC1 biofilms biofilms growing growing in in the the incubator incubator at at the the mature mature phase phase of of development development ( (aa, –bc,). cLight). Light sensor sensor positioned positioned under under the the lamp lamp (d ,(ed)., e).

BiofilmBiofilm detachment detachment from from the the slides slides occurred occurred in in a a number number of of cases, cases, visible visible from from the the concomitant concomitant aappearanceppearance of of bare bare areas areas on on the the slides slides and and biofilm biofilm flocs flocs in in the the circulating circulating medium, medium, these these representing representing thethe dispersion, dispersion, i.e. i.e.,, the the last last phase phase of of biofilms biofilms development. Beyond Beyond self self-detachment,-detachment, which which occurs occurs whenwhen losses, losses, due due for for example example to to cell cell death, death, grazing, grazing, pa pathogens,thogens, etc., etc., become become equal equal to to growth, growth, other other factorsfactors (e.g.,(e.g., flowflow disturbance, disturbance, temperature, temperature, light light and and nutrient nutrient availability) availability) may have may an have impact an impact [20,56]. [20,56Nutrient]. Nutrient concentration concentration does not does represent not represent a limitation, a limitation, as long as N:P as long ratio isas maintained, N:P ratio is when maintained, biofilms whenare grown biofilms on wastewaterare grown on in bothwastewater indoor andin both outdoor indoor photobioreactors. and outdoor photobioreactors. Therefore, optimization Therefore of, optimiflow regime,zation of temperature flow regime, and temperature light intensity and light are the intensity main parametersare the main to parameters focus on for to highfocus qualityon for highbiomass quality production biomass production when the process when th ise scaledprocess up is toscaled real up plant to real operations. plant operations. Additionally, Additionally, biomass biomassharvesting harvesting frequency frequency has to be has assessed to be assessed in order in to order ensure to an ensure effective an effective treatment treatment of WWs. of WWs.

3.2. Photosynthetic Characteristics Results of photosynthetic performance of light-acclimated cultures, obtained after PAM measurements, are reported in Figures 4–7 and in Tables SI 1–8 are those obtained by statistical analysis to evaluate the effect of experimental conditions on photosynthetic parameters.

Microorganisms 2019, 7, 252 7 of 18

3.2. Photosynthetic Characteristics Results of photosynthetic performance of light-acclimated cultures, obtained after PAM measurements, are reported in Figures4–7 and in Tables S1–S8 are those obtained by statistical Microorganismsanalysis to evaluate 2019, 7, x FOR the ePEERffect REVIEW of experimental conditions on photosynthetic parameters. 3 of 19

FigureFigure 4 4.. BoxBox-plots-plots relative relative to to the the comparison comparison of of quantum quantum yield yield (ΔF/F (∆F/Fmm’)’) of of photosystem photosystem II II (PSII) (PSII) measurementsmeasurements determined onon allall light-acclimated light-acclimated biofilms biofilms (Run1–Run4) (Run1–Run4) at initialat initial phase phase (A– (DA)– andD) and last lastsampling sampling day day (E– H(E).–H Run1-LC1in). Run1-LC1in= not = not sampled. sampled.

TheThe effective effective quantumquantum yield yield of of photosynthesis photosynthesis (∆ F(ΔF/F/Fm’)m represents’) represents the the effi ciencyefficiency of the of whole-chain the whole- chainphotosynthetic photosynthetic electron electron transport transport of steady-state of steady photosynthesis-state photosynthesis in the light in [57 the]. Values light measured[57]. Values on measuredthe surface on of the intact surface biofilms, of intact were biofilms, included were between included 0.282 between0.015 (LC4in0.282 ± Run1)0.015 (LC4in and 0.742 Run1)0.001, and ± 2 1 1± 0.742with the± 0.001, latter with occurring the latter in 30-daysoccurring biofilms in 30-days at 60 biofilmsµmol photons at 60 μmol m− photonss− , 20 ◦ mC− and2 s−1, 2520 L°C h −and(LC2ls 25 L hRun1).−1 (LC2l Mosts Run1). yield Most values yield were values included were betweenincluded 0.5 between and 0.7, 0.5 falling and 0.7, in the falling ranges in the reported ranges for reported natural forbiofilms natural from biofilms different from environments, different environments, thus exposed thus at various exposed ambient at various light ambient intensities light [31 intensities,58–60]. [31,58Analyzing–60]. separately the effect of experimental parameters on biofilm photosynthetic performance, we observedAnalyzing that, separately at initial stages, the effect biofilm of ∆ experimentalF/Fm’ was not para affectedmeters by variations on biofilm in light photosynthetic conditions performance,(Figure4A–D we Table observed S1). Conversely, that, at initial in the stages, last experimentalbiofilm ΔF/Fm day’ was in not Run1 affected∆F/F bym’ increasedvariations fromin light 15 2 1 conditionsto 60 µmol (Figurephoton m 4 − A–sD− Table(LC3ls-LC4ls SI 1). Conversely,p = 0, LC2ls-LC3ls in the p last= 0) experimental followed by aday significant in Run1 decrease ΔF/Fm’ 2 1 increasedfrom 60 to from 120 µ 15mol to photon 60 μmol m −photons− (p m= −0.00001254).2 s−1 (LC3ls-LC4ls In Run2 p = a 0, significant LC2ls-LC3ls increase p = 0) occurred followed from by 15a 2 1 significantto 120 µmol decrease photon from m− s60− to(Run2 120 μmol LC3ls-LC4ls photon mp −=2 s0.0001251,−1 (p = 0.00001254). LC2ls-LC3ls In Run2p = a0, significant LC1ls-LC2ls increasep = 0) occurred(Table S2). from 15 to 120 μmol photon m−2 s−1 (Run2 LC3ls-LC4ls p = 0.0001251, LC2ls-LC3ls p = 0, 2 1 LC1lsMoreover,-LC2ls p = 0) in (Table all Runs SI 2). but Run2, cultures at 60 µmol photon m− s− showed significantly higherMoreover, yield than in biofilmsall Runs grownbut Run2, at the cultures highest at irradiance60 μmol photon (p < 0.05 m−2 see s−1 Tableshowed S2) significantly indicating a higher proper yieldfunctioning than biofilms of the phototrophic grown at the community highest irradianceat this irradiance (p < 0.05 and see prospecting Table SI a 2) suitable indicating light conditiona proper functioningfor cultivating of phototrophic the phototrophic biofilms community in photobioreactors at this irradiance for WWT. and prospecting a suitable light conditionThe eforffect cultivating of temperature phototrophic was clearer biofilms in biofilms in photobioreactors at the highest lightfor WWT. intensities were significantly higherThe values effect wereof temperature obtained atwas 20 clearer◦C than in 30 biofilms◦C at both at the flow highest velocities light intensities (p < 0.05 see were Table significantly S2), likely higherbecause values rates ofwere enzymatic obtained reactions at 20 °C involved than 30 °C in electronat both flow transport velocities are temperature-dependent (p < 0.05 see Table SI 2), [61 likely–64]. becauseHigh temperature rates of enzymatic represents reactions an excess involved energy in andelectron may transport lead to an are imbalance temperature between-dependent the energy [61– 64supply]. High and temperature energy consumption represents an by excess the redox energy reactions and may in thelead electron to an imbalance transport between chain resulting the energy in a supplyprogressive and energy photoinactivation consumption of PSIIby the reaction redox centersreactions [63 in]. the electron transport chain resulting in a progressive photoinactivation of PSII reaction centers [63]. As for the effect of flow rate on biofilm photosynthesis, initial stages yield showed a scattered distribution. The most relevant outcome was obtained at 60 μmol photon m−2 s−1 where LC2 cultures attained significantly higher yields at 25 L h−1 than 100 L h−1 at 20 °C (Run1 LC2in-Run2 LC2in p = 0). This was also recorded at the runs’ end at 20 °C (Run1 LC2ls-Run2 LC2ls p = 0) while no significant difference was found between yields at different flow rates at 30 °C (p > 0.05 Table SI 2). The combined effect of light, temperature, and flow was also evaluated on biofilms photosynthesis. In initial stage biofilms ΔF/Fm’ was significantly higher at 30 μmol photon m−2 s−1, 20

Microorganisms 2019, 7, 252 8 of 18

As for the effect of flow rate on biofilm photosynthesis, initial stages yield showed a scattered 2 1 distribution. The most relevant outcome was obtained at 60 µmol photon m− s− where LC2 cultures 1 1 attained significantly higher yields at 25 L h− than 100 L h− at 20 ◦C (Run1 LC2in-Run2 LC2in p = 0). This was also recorded at the runs’ end at 20 ◦C (Run1 LC2ls-Run2 LC2ls p = 0) while no significant diffMicroorganismserence was found2019, 7, betweenx FOR PEER yields REVIEW at different flow rates at 30 ◦C(p > 0.05 Table S2). 3 of 19 The combined effect of light, temperature, and flow was also evaluated on biofilms photosynthesis. 2 1 1 In initial°C, 25 stage L h−1 biofilms (Run1-LC3)∆F/F m than’ was at significantly all other conditions higher at ( 30p <µ mol0.05 photon Table SI m − 1) s suggesting− , 20 ◦C, 25 that L h these− (Run1-LC3)irradiance, than temperature at all other, conditions and flow ( p values< 0.05 Table were S1) the suggesting most favorable that these for irradiance, light utili temperature,zation in the andphotosynthetic flow values were process. the most Noteworthy, favorable for ΔF/F lightm’ utilizationwas higher in at the 60 photosynthetic μmol photon process.m−2 s−1, Noteworthy,20 °C, 25 L h−1 2 1 1 ∆F/(Run1Fm’ was-LC2) higher than at at 60 almostµmol photon all the mother− s− conditions, 20 ◦C, 25 L(Table h− (Run1-LC2) SI 1). The only than atexception almost all was the the other non- conditionssignificant (Table difference S1). The between only exception Run1-LC2 was and the Run1 non-significant-LC3 (p = 1). diInff theerence last phase, between 60 Run1-LC2μmol photon and m−2 2 1 1 Run1-LC3s−1, 20 °C (p and= 1). 25 In L the h−1 last(Run1 phase, LC2) 60 wereµmol the photon conditions m− s −providing, 20 ◦C and higher 25 L hyield− (Run1 values LC2) (p < were 0.05 theTable conditionsSI 2). This providing suggested higher that yield to maintain values (p biofilm< 0.05Table photosynthesis S2). This suggested in optimal that conditions, to maintain and biofilm all the photosynthesisprocesses related in optimal to it, conditions,which are essential and all the for processes WWT, a relatedfull-scale to it,photobioreactor which are essential might for be WWT, operated a 1 full-scaleat 20 °C, photobioreactor 25 L h−1 and at might an irradiance be operated of 30 at or 20 60◦C, μmol 25 L photon h− and m at−2 ans−1, irradianceinitially, and of 30then or 60at 60µmol μmol 2 1 2 1 photonphoton m− ms−−2 s,−1 initially,. and then at 60 µmol photon m− s− .

Figure 5. Box-plots relative to the comparison of rel.ETR values determined on all light-acclimated Figure 5. Box-plots relative to the comparison of rel.ETRmax max values determined on all light-acclimated biofilmsbiofilms (Run1–Run4) (Run1–Run4) at initial at initial phase phase (A– D(A)– andD) and last samplinglast sampling day day (E–H (E).– Run1-LC1inH). Run1-LC1in=not= not sampled. sampled.

Photoacclimation of cultured biofilms to different experimental irradiances was indicated also by Photoacclimation of cultured biofilms to different experimental irradiances was indicated also variations in ETR/I curve parameters such as rel.ETRmax,Ik, and α (Figures5–7). Rel.ETR max (Figure5) by variations in ETR/I curve parameters such as rel.ETRmax, Ik , and α (Figures 5–7). Rel.ETRmax (Figure varied between 5.5 0.9 and 43.4 1.3 µmol e m 2 s 1 (LC4in Run1 and LC1ac Run2, respectively). 5) varied between± 5.5 ± 0.9 and ±43.4 ± 1.3 μmol− e−− m−−2 s−1 (LC4in Run1 and LC1ac Run2, respectively). Plotting rel.ETRmax variations with irradiance did not show coherent effects both in initial and Plotting rel.ETRmax variations with irradiance did not show coherent effects both in initial and last day biofilms (Figure5). In both cases, the most relevant result was the significant increase from 60 last day biofilms (Figure 5). In both cases, the most relevant result was the significant increase from to 120 µmol photon m 2 s 1 in Run2 (p = 0.0005674), reflecting the photoacclimation of the benthic 60 to 120 μmol photon− −m−2 s−1 in Run2 (p= 0.0005674), reflecting the photoacclimation of the benthic phototrophic communities at increasing irradiance [32,60,65]. At higher temperature initial and last phototrophic communities at increasing irradiance [32,60,65]. At higher temperature initial and last day biofilms did not show significant rel.ETRmax variations at both flow conditions (Tables S3 and S4); day biofilms did not show significant rel.ETRmax variations at both flow conditions (Table SI 3,4); 1 except for the higher rel.ETRmax of LC1ls cultures at 25 L h− at 20 ◦C(p = 0.0004123). As for the effect except for the higher rel.ETRmax of LC1ls cultures at 25 L h−1 at 20 °C (p = 0.0004123). As for the effect of flow rate, only few variations were significant both in samples at the initial phase and at the last run of flow rate, only few variations were significant both in samples at the initial phase and at the last day (Tables S3 and S4). The interaction of light, temperature and flow rate variation on rel.ETRmax run day (Tables SI 3,4). The interaction of light, temperature and flow rate variation on rel.ETRmax yielded different results between initial biofilms and cultures sampled at the last day. In the former yielded different results between initial biofilms and cultures sampled at the last day. In the former 2 1 1 cases, rel.ETRmax was significantly higher at 120 µmol photon m− s− and 100 L h− , both at 20 ◦C cases, rel.ETRmax was significantly higher at 120 μmol photon m−2 s−1 and 100 L h−1, both at 20 °C and 30 °C, than at most of the parameters combinations tested (Table SI 3); while in the latter cases, rel.ETRmax, significantly increased at 60 and 120 μmol photon m−2 s−1, at 20 °C and 25 L h−1 (Table SI 4).

Microorganisms 2019, 7, 252 9 of 18

and 30 ◦C, than at most of the parameters combinations tested (Table S3); while in the latter cases, 2 1 1 Microorganismsrel.ETRmax, significantly 2019, 7, x FOR PEER increased REVIEW at 60 and 120 µmol photon m− s− , at 20 ◦C and 25 L h− (Table3 of S4). 19

FigureFigure 6. 6. BoxBox-plots-plots relative relative to to the the comparison comparison of of αα valuesvalues determined determined on on all all light light-acclimated-acclimated biofilms biofilms (Run1(Run1–Run4)–Run4) at at initial phase (A––D)) and and last last sampling day ( E–H). Run1 Run1-LC1in-LC1in = notnot sampled. sampled.

As for photosynthetic efficiency (α), it was included between 0.14 0.02 (LC4 Run2) and 0.43 0.01 As for photosynthetic efficiency (α), it was included between 0.14± ± 0.02 (LC4 Run2) and 0.43± ± 0.01(LC1ac (LC1 Run1).ac Run1). The visualThe visual analysis analysis of α values of α invalues relation in rel to irradianceation to irradiance showed thatshowed in initial that biofilms in initial of 2 1 biofilmsRun1–3, ofα Run1increased–3, α fromincreased 15 to from 30 µ mol15 to photon 30 μmol m −photons− and m−2 decreased s−1 and decreased from 30 from to 60 30µmol to 60 photon μmol 2 1 1 photonm− s− m(Figure−2 s−1 (Figure6). Nevertheless, 6). Nevertheless, only the only variation the variation between between LC3 and LC3 LC4 and at 20 LC4◦C at and 20 100°C and L h− 100was L hsignificant−1 was significant (p = 0.004918) (p = 0.004918 (Table) S5).(Table In SI biofilms 5). In biofilms collected collected at runs’ at end, runs’α increasedend, α increased at 20 ◦C, atboth 20 °C, at 2 1 bothlow andat low high and flow high rate, flow from rate, 15 from to 120 15µ tomol 120 photon μmol photon m− s− m. Some−2 s−1. Some of the of variations the variations observed observed were weresignificant, significant, i.e., Run2LC1-Run2LC2 i.e., Run2LC1-Run2L (pC2= 0.01241), (p = 0.01241), Run2LC2-Run2LC3 Run2LC2-Run2 (LC3p = 0.03837) (p = 0.03837 (Table) (Table S6).At SI the6). Atinitial the initial stage ofstage development, of development, temperature temperature variations variations did not did have not ahave significant a significant effect oneffectα while on α while at the atlast the sampling last sampling day some day significant some significant variations variations occurred. occurred. In detail, In at detail, low flow, at lowα was flow, higher α was at 20higher◦C than at 20at 30°C◦ thanC(p

Microorganisms 2019, 7, 252 10 of 18 Microorganisms 2019, 7, x FOR PEER REVIEW 3 of 19

FigureFigure 7. 7. BoxBox-plots-plots relative relative to to the the comparison comparison of of I Ikk valuesvalues determined determined on on all all light light-acclimated-acclimated biofilms biofilms (Run1(Run1–Run4)–Run4) at at initial initial phase phase ( (AA––DD)) and and last last sampling sampling day day ( (E–H).). Run1 Run1-LC1in-LC1in = notnot sampled. sampled.

2 1 I ranged (Figure7) between 34.8 3.2 (LC3in Run2) and 128.1 5.2 µmol photon m −2 s−1 (LC1ac Ikk ranged (Figure 7) between 34.8 ±± 3.2 (LC3in Run2) and 128.1± ± 5.2 μmol photon m− s− (LC1ac Run2).Run2). Most Most values values of of rel.ETR maxmax andand I Ik kobtainedobtained matched matched those those found found for for sub sub-tidal-tidal phototrophic biofilmsbiofilms from from di differentfferent geographical geographical areas areas [32,60 [32,60]. In biofilms]. In biofilms at initial at stage initial and stage at the lastand experimental at the last experimentalday, light variations day, light influenced variations Ik only influence in fewdcases Ik only (Tables in few S7 cases and S8)(Table whiles SI temperature 7,8) while t hademperatur no effecte 2 1 hadon cultures no effect at o bothn cultures phases at ofboth development, phases of development except for initial, except biofilms for initial at 15 biofilmsµmol photon at 15 μmol m− photons− and 1 m100−2 s L−1h and− with 100 higherL h−1 with values higher at 20 values◦C than at at20 30 °C◦ C(thanp = at0.005464). 30 °C (p = As 0.005464 for the). flow As rate,for the Ik didflow not rate have, Ik dida significant not have variationa significant in initial variation cultures in initial while beingcultures significantly while being higher significantly at 20 ◦C andhigher lower at 20 at 30°C◦ andC at 1 lower100 L hat− 30at °C almost at 100 all L lighth−1 at conditionsalmost all light (Table conditions S8). For the (Table combined SI 8). For effect the of combined light, temperature, effect of light, and temperatureflow rate results, and were flowdi ratefferent results between were initialdifferent biofilms between and initial cultures biofilms at the lastand sampling cultures day.at the In last the samplingformer cases, day. I k Inwas the significantly former case highers, Ik was at the significantly lowest light higher intensity, at the low lowest temperature light intensity, and high flowlow 2 1 temperature(Table S7); in and the high latter flow cases, (Table Ik was SI significantly7); in the latter higher cases at, I 60k was and si 120gnificantly◦C µmol higher photon at m60− ands− 120, 20 °C◦C, 1 μmol25 L hphoton− (Table m− S8).2 s−1, 20 °C, 25 L h−1 (Table SI 8). Photoinhibition,Photoinhibition, mea measuredsured as as the the reduction reduction of of rel.ETR rel.ETR during during ETR/I ETR/I curve curve registration, registration, occurred occurred 2 1 inin almost almost all all LC1, LC1, LC2 LC2,, and LC3 samples at 470 µμmolmol photonphoton mm−−2 ss−−1, irrespective, irrespective of of growth growth irradiance irradiance andand biofilm biofilm developmental developmental stage stage ( (FiguresFigures 88,9 and). 9).

Microorganisms 20192019,, 77,, 252x FOR PEER REVIEW 113 of 1819 Microorganisms 2019, 7, x FOR PEER REVIEW 3 of 19

Figure 8. ElectronElectron Transport Transport Rates Rates versusversus IrradianceIrradiance relative relative to to LC1, LC1, LC2, LC2, LC3 LC3,, and and LC4 LC4 biofilms biofilms at Figure 8. Electron Transport Rates versus Irradiance relative to LC1, LC2, LC3, and LC4 biofilms at atdifferent different stage stage of ofdevelopment development in Run1 in Run1 (a) (anda) and Run2 Run2 (b). (Eachb). Each curve curve represents represents the mean the mean of nine of different stage of development in Run1 (a) and Run2 (b). Each curve represents the mean of nine ninereplicates. replicates. replicates.

Figure 9. Electron transport rates versus irradiance relative to LC1, LC2, LC3, and LC4 biofilms Figure 9. Electron transport rates versus irradiance relative to LC1, LC2, LC3, and LC4 biofilms at at different stage of development in Run3 (a) and Run4 (b). Each curve represents the mean of differentFigure 9. stageElectron of development transport rates in versusRun3 ( airradiance) and Run4 relative (b). Each to LC1, curve LC2, represents LC3, and the LC4 mean biofilms of nine at nine replicates. replicates.different stage of development in Run3 (a) and Run4 (b). Each curve represents the mean of nine replicates. LC4 cultures showed photoinhibition at lower but, nevertheless, quite moderate irradiance LC4 cultures showed photoinhibition2 1 at lower but, nevertheless, quite moderate irradiance (210– (210–350 mmol photon m− s− ) considering that they were grown at the lowest light. This result 350 mmolLC4 cu photonltures showedm−2 s−1) consideringphotoinhibition that atthey lower were but, grown nevertheless, at the lowest quite light.moderate This irradiance result suggests (210– suggests that the phototrophic community studied had the potential to acclimate to higher irradiances that350 mmolthe phototrophic photon m−2 communitys−1) considering studied that hadthey the were potential grown toat theacclimate lowest to light. higher This irradiances result suggests than than those tested in the incubator cultures. Nevertheless, occurrence of photoinhibition in benthic thosethat the tested phototrophic in the incubator community cultures studied. Nevertheless,had the potential occurrence to acclimate of photoinhibition to higher irradiances in benthic than communities during laboratory measurements and its relationship with the growth light regime is still communitiesthose tested during in the laboratory incubator measurements cultures. Nevertheless, and its relationship occurrence with of photoinhibitionthe growth light in regime benthic is quite controversial [33]. Photoinhibition may be often hidden by the self-shading effect within the stillcommunities quite controversial during laboratory [33]. Photoinhibition measurements may and be its often relationship hidden by with the theself growth-shading light effect regime within is biofilm matrix [28]. If the matrix is deep enough, decreases in the rate of oxygen production in the thestill biofilm quite controversial matrix [28]. If[33 the]. Photoinhibition matrix is deep enough,may be oftendecreases hidden in theby therate self of -oxygenshading production effect within in uppermost layer of biofilms with high irradiance are compensated for by increased photosynthesis of thethe biofilm uppermost matrix layer [28] . ofIf the biofilms matrix is with deep high enough, irradiance decreases are in compensatedthe rate of oxygen for byproduction increased in subsurface phototrophs, which receive only saturating or subsaturating irradiance [28,32,66]. On the photosynthesisthe uppermost of layersubsurface of biofilms phototrophs, with which high receive irradiance only saturating are compensated or subsaturating for by irradiance increased contrary, fluorescence measurement of photosynthesis is derived mainly from the uppermost layers of [28,32,66photosynthesis]. On the of contrary,subsurface fluorescence phototrophs, measurement which receive of onlyphotosynthesis saturating oris derivedsubsaturating mainly irradiance from the uppermost[28,32,66]. On layer thes contrary,of phototrophs fluorescence due to measurement light attenuation of photosynthesis with depth, particularly is derived mainlyin thick from mature the uppermost layers of phototrophs due to light attenuation with depth, particularly in thick mature

Microorganisms 2019, 7, 252 12 of 18 Microorganisms 2019, 7, x FOR PEER REVIEW 3 of 19 phototrophsbiofilms [33,35,67 due to]. lightThus, attenuation the occurrence with of depth, photoinhibition particularly inreflects thick maturemore the biofilms acclimation [33,35 ,of67 ].surface Thus, cellsthe occurrence to incident of light photoinhibition rather than self reflects-shading more effects the acclimation within the of bio surfacefilm [33 cells]. Therefore, to incident ETR/I light curves rather ofthan LC1 self-shading and LC2 biofilms effects withinprobably the reflected biofilm [ 33the]. photoinhibition Therefore, ETR /Iof curves phototrophs of LC1 in and the LC2 uppermost biofilms probablylayers, particularly reflected thein the photoinhibition active and mature of phototrophs phases of development in the uppermost when communities layers, particularly were greatly in the stratified.active and mature phases of development when communities were greatly stratified.

3.3. Biomass Biomass P Productionroduction Biomass accumulation has has previously been been shown shown to to be linearly related to the decrease of subsurface light below the substratum [[40,4840,48].]. The The increase increase of biomass measurements over time indicated that natural mixed inocula were able to grow in culture after biofilmbiofilm structure alteration caused by initial homogenizing treatment. Chl a concentrationconcentration,, used to estimate the phototrophic biomass in the cultured biofilms biofilms,, ranged 2 between 0.16 and 125.10 ± 1.52 mg m−2 (LC4in(LC4in Run1 Run1 an andd LC2ls LC2ls Run1, Run1, respectively respectively,, Figure 10). Mean ± values were in the range reported for 7 7-days-days biofilms biofilms grown in situ on artificial artificial substrata (up to 252.6 2 mg m −2) )[[4646]] and and for for freshwater freshwater biofilms biofilms of of rivers rivers and and streams impacted by nutrient nutrient-rich-rich urban and agriculagriculturaltural waste discharges [[6868–70].].

Figure 10.10. Temporal development development of of Chl Chla and a and AFDW AFDW in LC1, in LC1, LC2, LC2, LC3, LC3 and,LC4 and biofilms LC4 biofilms (Run1–Run4). (Run1– Run4). 2 1 Looking at Chl a trend, at 120 and 60 µmol photons m− s− , Chl a concentration increased over 2 timeLooking following at biofilm Chl a trend development,, at 120 and up to60 approximatelyμmol photons 120m−2 mg s−1, mChl− ina concentration final cultures. increased This suggested over 2 1 timethat 60followingµmol photons biofilm mdevelopment,− s− was su upffi tocient approximately for biofilm development120 mg m−2 in final and highercultures. intensity This suggest did noted thatappear 60 toμmol exert photons any additional m−2 s−1 was effect. sufficient Nevertheless, for biofilm at this development irradiance a longerand higher lag phase intensity was requireddid not 2 1 appearfor biofilm to exert growth any than additional at 120 µeffect.mol photons Nevertheless, m− s− at. this This irradiance might have a longer implications lag phase in the was selection required of forthe biofilm light condition growth forthan culturing at 120 μmol microbial-algal photons m− consortia2 s−1. This inmight a wastewater have implications photobioreactor. in the selection of the lightWhole condition biofilm for biomass, culturing estimated microbial as-algal AFDW, consortia grew in up a wastewater to 14.30 photobioreactor.3.20 g m 2 (LC1ls Run4) ± − (FigureWhole 10). biofilm This value biomass, fell in estimated the range as reported AFDW, grew for natural up to 14.30 biofilms ± 3.20 from g m streams−2 (LC1ls both Run4) subjected (Figure 2 10or). not This to nutrientvalue fell pollution in the range [71, 72reported]. Lower for values, natural between biofilms ca. from 23 and streams 37 mg both m− subjected, were achieved or not to in nutrientbiofilms grownpollution in artificial[71,72]. Lower streams values, with wastewater between ca. at di23ff anderent 37 sewage mg m− percentage2, were achieved [70]. A in significant biofilms positivegrown in correlation artificial streams was found with between wastewater Chl ata and different AFDW sewage (Table percentage S9), suggesting [70]. A that significant the phototrophic positive correlationcommunity was represented found between the main Chl component a and AFDW of the (Table whole 9 SI) biofilm., suggesting This was that confirmed the phototrophic by the communityautotrophic index represented (AI), which the is main a means component of determining of the the whole metabolism biofilm./ trophicThis was nature confirmed of biofilms by [the53]. autotrophicMost values index were below(AI), which 50 (Table is a1 ),means indicating of determining that biofilms the were metabolism/ autotrophs-dominatedtrophic nature [of73 biofi]. lms [53]. Most values were below 50 (Table 1), indicating that biofilms were autotrophs-dominated [73].

Table 1. Mean values ± standard error of AI at initial (in), active (ac), 85% light absorption, last sampling day of biofilm development in the four runs performed. ns = not sampled.

Microorganisms 2019, 7, 252 13 of 18

Table 1. Mean values standard error of AI at initial (in), active (ac), 85% light absorption, last sampling ± day of biofilm development in the four runs performed. ns = not sampled.

Light Chamber Sample Run1 Run2 Run3 Run4 in ns 9 5 11 2 24 7 ± ± ± ac 38 5 33 6 31 4 LC1 ± ± − ± 85% 44 1 21 2 ± − ± − ma ns 63 18 144 57 141 23 ± ± ± in 62 11 5 45 22 131 73 ± ± ± LC2 ac 19 2 9 3 23 11 23 12 ± ± ± ± ld 21 1 15 8 33 13 87 30 ± ± ± ± in 94 52 55 10 35 15 331 114 ± ± ± ± LC3 ac 57 20 ± − − − ld 68 1 15 7 18 5 48 7 ± ± ± ± in 860 165 454 367 363 742 LC4 ± ± ld 655 284 748 382 ± ± − −

Values of ca. 140 were obtained in LC1 mature cultures at the highest light in Run3 and Run4, indicating a balance between the photo- and heterotrophic components [71,74]. Nevertheless, in LC1 cultures in Run3 and Run4 the heterotrophic component became, in terms of biomass, more important as biofilms grew. The AI values, in fact, increased over time due to the increase of AFDW and the saturation of Chl a in mature communities. The raise of heterotrophs might be explained by the increased amount, in mature biofilms at high light intensity, of algal exudates that represent a high-quality organic substrate for bacteria [71,75,76]. Wagner et al. [77] observed a shift towards bacterial consumption of DOC of autochthonous origin in phototrophic stream biofilms at high light intensity. As explained, cultured mature biofilms were dominated by filamentous cyanobacteria able to produce high amounts of exopolysaccharides [38,40]. Finally, heterotrophs strongly dominated cultures at the lowest irradiances [37] where values were above 200 in all samples. Biomass productivity was included between 0.01 (LC3in Run2, LC4in in Run2 and Run3) and 2.10 0.36 (LC1ma Run4) g DW m 2 d 1 (Table2). ± − − Table 2. Mean values standard error of biomass productivity (DW g m 2 d 1) at initial (in) and active ± − − (ac) biofilms and cultures at last sampling day (ls) in the four runs performed. ns = not sampled.

Light Chamber Sample Run1 Run2 Run3 Run4 in ns 0.14 0.04 0.26 0.04 0.20 0.02 ± ± ± LC1 ac 0.25 001 0.80 0.08 1.09 0.15 0.91 0.04 ± ± ± ± ls 0.81 0.08 1.08 0.09 1.36 0.40 2.10 0.36 ± ± ± ± in 0.04 0.00 0.32 0.18 0.04 0.00 0.06 0.02 ± ± ± ± LC2 ac 0.13 0.01 0.20 0.04 0.37 0.10 0.24 0.06 ± ± ± ± ls 0.37 0.01 0.33 0.12 0.51 0.09 0.70 0.21 ± ± ± ± in 0.14 0.02 0.01 0.00 0.03 0.01 0.04 0.01 ± ± ± ± LC3 ac 0.29 0.11 ± − − − ls 0.51 0.05 0.09 0.06 0.08 0.01 0.21 0.05 ± ± ± ± in 0.03 0.00 0.01 0.01 0.01 0.00 0.03 0.01 LC4 ± ± ± ± ls 0.02 0.00 0.01 0.01 ± ± − −

Productivity was consistent with those reported [78] for mixed biofilms composed of green algae 2 1 and diatoms in a lab-scale biofilm reactor (0.97–2.08 g DW m− d− ). The highest productivity values obtained here were higher than those attained with three different biofilm-forming cyanobacterial isolates grown in the same system [15] and with the green alga Chlorella vulgaris grown in a rotating algal bioreactor [17]. Higher values were reported for an algal biofilm grown in a photobioreactor [26]. Microorganisms 2019, 7, 252 14 of 18

4. Conclusions This study provided new insights into photosynthetic activity of light-acclimated cultures, contributing to the knowledge of wastewater biofilm ecophysiology, in light of their exploitation in water bioremediation. The difficulty of studying these communities in nature, due to the complex interactions among several environmental factors, was overridden in the flow lane system, which allowed the investigation of key variables that influence photosynthetic performance.

The tested light–dark cycle appeared to be suitable for community development and functioning. • It was possible to identify light intensity, temperature, and flow value favoring photosynthetic • 2 1 1 activity, i.e., 60 µmol photons m− s− , 20 ◦C, 25 l− . This has implications for biomass accumulation and nutrient uptake related to photosynthesis. Therefore, this set of values appears to be appropriate for the optimal operating of a WWT photobioreactor that purifies municipal sewage by means of mixed phototrophic biofilms. Further development would be in the cultivation of these microbial communities in wastewater • effluents to evaluate the treatment efficiency at the set of conditions here identified.

Supplementary Materials: The following are available online at http://www.mdpi.com/2076-2607/7/8/252/s1, Table S1: Results of ANOVA and Tukey’s HSD testing with factors irradiance, temperature and flow on ∆F/Fm’ in initial biofilms. Tukey’s Q below the diagonal, p value above the diagonal. Significant comparisons are red. Table S2: Results of ANOVA and Tukey’s HSD testing with factors irradiance, temperature and flow on ∆F/Fm’ in biofilms at the last sampling day. Tukey’s Q below the diagonal, p value above the diagonal. Significant comparisons are red. Table S3: Results of ANOVA and Tukey’s HSD testing with factors irradiance, temperature and flow on rel.ETRmax in initial biofilms. Tukey’s Q below the diagonal, p value above the diagonal. Significant comparisons are red. Table S4: Results of ANOVA and Mann–Whitney test testing with factors irradiance, temperature and flow on rel.ETRmax in biofilms at the last sampling day. Significant comparisons are red. Table S5: Results of ANOVA and Tukey’s HSD testing with factors irradiance, temperature and flow on α in initial biofilms. Tukey’s Q below the diagonal, p value above the diagonal. Significant comparisons are red. Table S6: Results of ANOVA and Tukey’s HSD testing with factors irradiance, temperature and flow on α in biofilms at the last sampling day. Tukey’s Q below the diagonal, p value above the diagonal. Significant comparisons are red. Table S7: Results of ANOVA and Tukey’s HSD testing with factors irradiance, temperature and flow on Ik in initial biofilms. Tukey’s Q below the diagonal, p value above the diagonal. Significant comparisons are red. Table S8: Results of ANOVA and Tukey’s HSD testing with factors irradiance, temperature and flow on Ik in biofilms at the last sampling day. Tukey’s Q below the diagonal, p value above the diagonal. Significant comparisons are red. Table S9: Results of Spearman’s rho-statistic applied to test the correlation between Chl a and AFDW. Author Contributions: A.G. and F.D.P. collected samples, performed the experimental runs, analyzed the data. A.G., F.D.P. and R.C. wrote and edited the manuscript. Funding: This work was partially conducted within the framework of EU project PHOBIA (QLK3-CT2002-01938). Conflicts of Interest: The authors declare no conflict of interest.

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