Potential of Rhodobacter Capsulatus Grown in Anaerobic-Light Or Aerobic-Dark Conditions As Bioremediation Agent for Biological Wastewater Treatments

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Article Potential of Rhodobacter capsulatus Grown in Anaerobic-Light or Aerobic-Dark Conditions as Bioremediation Agent for Biological Wastewater Treatments

Stefania Costa 1, Saverio Ganzerli 2, Irene Rugiero 1, Simone Pellizzari 2, Paola Pedrini 1 and Elena Tamburini 1,* 1 Department of Life Science and Biotechnology, University of Ferrara, Via L. Borsari, 46 | 44121 Ferrara, Italy; [email protected] (S.C.); [email protected] (I.R.); [email protected] (P.P.) 2 NCR-Biochemical SpA, Via dei Carpentieri, 8 | 40050 Castello d’Argile (BO), Italy; [email protected] (S.G.); [email protected] (S.P.) * Correspondence: [email protected]; Tel.: +39-053-245-5329

Academic Editors: Wayne O’Connor and Andreas N. Angelakis Received: 4 October 2016; Accepted: 2 February 2017; Published: 10 February 2017

Abstract: The use of microorganisms to clean up wastewater provides a cheaper alternative to the conventional treatment plant. The efficiency of this method can be improved by the choice of microorganism with the potential of removing contaminants. One such group is photosynthetic bacteria. Rhodobacter capsulatus is a purple non-sulfur bacterium (PNSB) found to be capable of different metabolic activities depending on the environmental conditions. Cell growth in different media and conditions was tested, obtaining a concentration of about 108 CFU/mL under aerobic-dark and 109 CFU/mL under anaerobic-light conditions. The biomass was then used as a bioremediation agent for denitrification and nitrification of municipal wastewater to evaluate the potential to be employed as an additive in biological wastewater treatment. Inoculating a sample of mixed liquor withdrawn from the municipal wastewater treatment plant with R. capsulatus grown in aerobic-dark and anaerobic-light conditions caused a significant decrease of N-NO3 (>95%), N-NH3 (70%) and SCOD (soluble chemical oxygen demand) (>69%), independent of the growth conditions. A preliminary evaluation of costs indicated that R. capsulatus grown in aerobic-dark conditions could be more convenient for industrial application.

Keywords: bioremediation; Rhodobacter capsulatus; wastewater treatment; NUR; OUR

1. Introduction Wastewater treatment and reuse is not new, and knowledge on this topic has evolved and advanced in the past years. As the human population continues to grow and urbanize, the challenges for securing water resources and disposing of wastewater will increasingly demand new and sustainable technologies [1]. The general purpose of wastewater treatments is to remove pollutants that can harm the aquatic environment and natural ecosystems in case they are discharged. Because of the deleterious effects of the low concentration of dissolved oxygen in aquatic life, wastewater treatment has been historically focused on the removal of pollutants that would deplete dissolved oxygen and favor eutrophication in receiving waters [2]. Traditionally, both chemical and biological treatments or a combination of them have been employed [3]. However, biological treatments have many advantages because they are cheaper, more reliable, can be done on site and their efﬁciency can be improved by the choice of a variety of microorganisms [4]. Bioremediation processes explore the use of biological mechanisms to destroy, transform or immobilize environmental contaminants

Water 2017, 9, 108; doi:10.3390/w9020108 www.mdpi.com/journal/water Water 2017, 9, 108 2 of 11 to protect the environment. Living organisms are emerging as one of the most useful alternative technologies for restoring contaminated sites and removing contaminants from soils and waters [5]. Both aerobic and anaerobic microorganisms are used for treating wastewaters. One such group of potential microorganisms is the purple non-sulfur bacteria (PNSB), belonging to the broader family of purple phototrophic bacteria (PPB) [6]. They are Gram-negative bacteria, widely distributed in various habitats such as soil, freshwater and ocean, and can be readily isolated from these sources [7]. They are characterized by versatile metabolic activities including photoautotrophic activity, with light as the energy source, H2 as the electron donor and CO2 as the electron acceptor/carbon source; chemoheterotrophic growth in darkness, using organics as the electron donor/carbon source and O2 as the electron acceptor; and photoheterotrophic activity, using some sugars and organic acids as the electron donors/carbon source, with light as the energy source [8]. Since the 1960s, the application of PSNB in wastewater bioremediation has been extensively studied, namely in treating olive mill wastewater, dairy wastewater, poultry slaughterhouse wastewater, seafood wastewater, palm oil mill effluent and domestic wastewater [9]. This method has provided high efficiency in removing COD (Chemical Oxygen Demand) and nitrogen compounds, as well as hydrogen sulphide [10]. The most studied species is Rhodobacter spheroides, but applications of Rhodopseudomonas palustris, Rubrivirax gelatinosus, Rhodocyclus gelatinosus and Rhodovulum sulfidophilum have been also reported [11–13]. Notwithstanding the promising evidence, PNSB-based technology has not been used in the large-scale industrial application of wastewater treatments, and even the most recent studies regard almost exclusively their potential as bio-hydrogen producers [14], or as generators of value-added products from wastewater fermentation. In fact, it is known that PNSB can convert organic carbon into biomass that can be used as a source of single-cell proteins, carotenoids, macromolecules and pantothenic acid for fertilizer or feed [15]. Rhodobacter capsulatus is a selected PNSB bacterium that has been used in the past for the investigation of the bacterial photosynthetic metabolism and related characteristics [16]. It stands out as a particularly interesting individual species as it can rapidly grow in several photosynthetic and dark modes [17]. Due to its capability of using nitrate as an electron acceptor and ammonium as a substrate [18], the role of R. capsulatus could be tested in both wastewater denitrification and nitrification processes, and not only for its denitrifying potential as is usually cited in the literature [19–21]. It is also hardy and stable for long-term storage under appropriate conditions [19], making it very attractive for industrial use. However, no application of R. capsulatus in wastewater treatment on a large scale has been so far reported. Taking into account its proven versatile metabolic behavior depending on environmental conditions [22], the aim of this study was to compare R. capsulatus cell growth in anaerobic-light and aerobic-dark conditions, using appropriate culture media. Then, the two biomasses obtained were added as bioremediation agents in samples of mixed liquor withdrawn from a municipal wastewater treatment plant, both in aerobic (nitrification) and anoxic (denitrification) conditions. The capacity of nitrogen and COD removal was tested at a 1 L batch scale, and our results are reported below.

2. Materials and Methods

2.1. Bacterial Strain and Media The strain of PSNB Rhodobacter capsulatus DSM 1710 (ATCC 11166) was purchased as lyophilized tab from the culture collection of Leibniz Institute DSMZ—German Collection of Microrganisms and Cell Cultures (Braunschweig, Germany). It is formerly known as Rhodopseudomonas capsulata [23]. For anaerobic-light (AN-LT) growth, a modiﬁed Rhodospirillaceae medium (MR medium) was used: yeast extract, 0.30 g/L (Difco); Na2-succinate 1.00 g/L; (NH4)-acetate 0.50 g/L; Fe(III)-citrate solution (0.1% in H2O), 5.00 mL; KH2PO4, 0.50 g/L; MgSO4·7H2O, 0.40 g/L; NaCl, 0.40 g/L; NH4Cl, 0.40 g/L; CaCl2·2H2O, 0.05 g/L; vitamin B12 solution (10 mg in 100 mL H2O), 0.40 mL; trace element solution SL-6, 1.00 mL/L (see below); L-cysteinium chloride, 0.30 g/L; resazurin, (0.1%) 0.50 mL/L. Water 2017, 9, 108 3 of 11

Trace elements solution SL-6: ZnSO4·7H2O, 0.10 g/L; MnCl2·4H2O, 0.03 g/L; H3BO3, 0.30 g/L; CoCl2·6H2O, 0.20 g/L; CuCl2·2H2O, 0.01 g/L; NiCl2·6H2O, 0.02 g/L; Na2MoO4·2H2O, 0.03 g/L. Complex culture medium for aerobic-dark (AE-DK) growth was prepared mixing bacteriological peptone, 5 g/L and meat extract, 3 g/L, as suggested by Weaver [16]. The master cell bank was maintained at −20 ◦C in cryovials on 1 mL of MR medium, mixed with 0.5 mL of glycerol as crioprotectant agent. The working cell bank was conserved at 4 ◦C in MR medium-agar tubes for six months and used as seed cultures. All complex media were purchased from Difco (BD, Franklin Lakes,Water 2017 NJ,, 9, 108 USA), whereas all chemicals were purchased from Fluka Analytical3 of (Steinheim,11 Germany),culture unless medium otherwise for aerobic stated.‐dark (AE‐DK) growth was prepared mixing bacteriological peptone, 5 g/L and meat extract, 3 g/L, as suggested by Weaver [16]. The master cell bank was maintained at −20 2.2. Anaerobic-Light°C in cryovials (AN-LT) on 1 mL Growth of MR medium, mixed with 0.5 mL of glycerol as crioprotectant agent. The working cell bank was conserved at 4 °C in MR medium‐agar tubes for six months and used as seed First,cultures. 1 mL from All complex a cryovial media of wereRhodobacter purchased from capsulatus Difco (BD,cells Franklin was inoculated Lakes, NJ, USA), in sterile whereas MR all medium (9 mL; thechemicals medium were was purchased boiled for from 15 Fluka min Analytical and bubbled (Steinheim, with sterilizedGermany), unless nitrogen otherwise gas). stated. After 48 h the cell suspension was used to inoculate completely filled screw-cap Erlenmeyer flask containing 90 mL of 2.2. Anaerobic‐Light (AN‐LT) Growth sterile MR medium, then incubated in a light cabinet and exposed to a LED light, at 30 ◦C for 72 h. A slow agitationFirst, rate 1 mL was from maintaineda cryovial of Rhodobacter to assure capsulatus homogenous cells was illumination, inoculated in sterile and MR pH medium adjusted to 6.8. (9 mL; the medium was boiled for 15 min and bubbled with sterilized nitrogen gas). After 48 h the Samples were withdrawn every 12 h up to 72 h of fermentations, and analyzed for biomass content. cell suspension was used to inoculate completely filled screw‐cap Erlenmeyer flask containing 90 mL of sterile MR medium, then incubated in a light cabinet and exposed to a LED light, at 30 °C for 72 h. 2.3. Aerobic-DarkA slow agitation (AE-DK) rate Growth was maintained to assure homogenous illumination, and pH adjusted to 6.8. First,Samples 1 mL were from withdrawn a cryovial everyRhodobacter 12 h up to 72 capsulatus h of fermentations, cells was and analyzed inoculated for biomass in the content. sterile medium containing2.3. bacteriological Aerobic‐Dark (AE‐ peptoneDK) Growth and meat extract (9 mL). After 48 h the cell suspension was used to inoculate aFirst, sterile 1 mL complex from a cryovial medium Rhodobacter (90 mL) capsulatus in 250 mL cells Erlenmeyer was inoculated flask in the closing sterile withmedium cotton plug, ◦ and thencontaining incubated bacteriological in dark at peptone 30 C for and 72 meat h. extract The pH (9 mL). was After adjusted 48 h the to cell 7.0. suspension The aerobic was used conditions were givento byinoculate agitation a sterile at complex 120 rpm. medium Samples (90 mL) were in 250 withdrawn mL Erlenmeyer every flask 12 closing h up to with 72 cotton h of fermentations, plug, and then analysedand then incubated for biomass in dark content. at 30 °C for 72 h. The pH was adjusted to 7.0. The aerobic conditions were given by agitation at 120 rpm. Samples were withdrawn every 12 h up to 72 h of fermentations, and 2.4. Mixedthen Liquor analysed Samples for biomass content. Samples2.4. Mixed of mixed Liquor Samples liquor was provided by the wastewater treatment plant of Bologna, a city in the North ofSamples Italy of with mixed about liquor 500,000 was provided inhabitants. by the wastewater The plant treatment has an plant overall of Bologna, potential a city capacityin of 900,000 inhabitantsthe North of equivalentsItaly with about and 500,000 consists inhabitants. of a series The plant of unit has processesan overall potential that receive capacity polluted of raw 900,000 inhabitants equivalents and consists of a series of unit processes that receive polluted raw sewage directlysewage directly from thefrom sewer the sewer system system and and progressively progressively clean clean it to it a topoint a pointthat it thatcan be it safely can be safely dischargeddischarged to a receiving to a receiving water. water. It It includes includes a abiological biological treatment treatment with activated with‐ activated-sludgesludge process and process and a downstreama downstream sewage sewage treatment treatment plant. Figure Figure 1 1shows shows a simplified a simplified flow diagram flow diagram for biological for biological processesprocesses used: the used: influent the influent wastewater wastewater goes goes through through stages stages of of denitrification denitrification and and nitrification nitrification in in which which different compound (namely, nitrogen and biodegradable COD) are removed out of the different compound (namely, nitrogen and biodegradable COD) are removed out of the wastewater. wastewater.

Figure 1. Simplified flow diagram of biological processes of the wastewater plant treatment of the city Figure 1. Simpliﬁedof Bologna, where ﬂow samples diagram used of in biological this study were processes withdrawn. of the wastewater plant treatment of the city of Bologna, where samples used in this study were withdrawn. Mixed liquor samples characteristics at the moment of withdrawal were as follows: MLSS (Mixed Liquor Suspended Solids), 3000 mg/L; soluble COD (Chemical Oxygen Demand), 74.10 mg/L; Mixed liquor samples characteristics at the moment of withdrawal were as follows: MLSS (Mixed N‐NO3, 1.25 mg/L; N‐NO2, 0.08 mg/L; N‐NH3, 1.42 mg/L. All samples were stored and maintained at Liquor Suspended4 °C for 24 Solids),h until analyzed 3000 mg/L; and used soluble for experiments. COD (Chemical Before the Oxygen tests, samples Demand), were 74.10 sanitized mg/L; in N-NO3, ◦ 1.25 mg/L;autoclave N-NO (1202, 0.08 °C for mg/L; 15 min). N-NH 3, 1.42 mg/L. All samples were stored and maintained at 4 C for 24 h until analyzed and used for experiments. Before the tests, samples were sanitized in autoclave (120 ◦C for 15 min).

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2.5. Nitrate Uptake Rate (NUR) and Oxygen Uptake Rate (OUR) Tests In the NUR determination, two samples of mixed liquor from denitrification tank (250 mL) of municipal wastewater were diluted to 50% with distilled water and mixed with 0.75 g/L of CH3COONa and 0.3 g/L KNO3 in denitrification conditions (anoxia). One sample was inoculated with 100 ppm of R. capsulatus AN-LT grown, the other with 100 ppm of AE-DK grown. The decrease in N-NO3 content was followed for 4 h and plotted against sampling time. The NUR (mg/gVSS·h) of the samples was obtained from the slope of the graph divided by the volatile suspended solid (VSS, mg/L). In the OUR measurement, two aliquots of 250 mL of mixed liquor from oxidation tank (nitrification) of municipal wastewater was taken, added with 0.75 g of CH3COONa and aerated until saturation of Dissolved Oxygen (DO) (nitrification); then, the DO fall was continuously monitored by using an automatic probe (Mettler Toledo, Garvens, Germany). The OUR (mg/gVSS·h) of each sample equaled the slope of the DO depletion versus time divided by the volatile suspended solid (VSS, mg/L). All tests were carried out in triplicate against blank.

2.6. Batch Tests All bioreactors were 1 L glass tanks. They were sanitized under steam stripping conditions (100 ◦C for 30 min), filled with 700 gr of sterile (120 ◦C for 15 min in autoclave) mixed liquor and inoculated with 100 mg/L of biomass suspension diluted to 107 CFU/mL. For inoculum, biomass was withdrawn in exponential phase from the cultivation tank, properly diluted and stored at 4 ◦C until inoculating bioreactors. A magnetic stirrer was used to mix biomass and mixed liquor homogeneously. Denitrification bioreactors were unaerated and oxygen levels kept at 0.4 mg/L. Nitrification bioreactors were aerated with an air flow of 90 L/h as to guarantee oxygen saturation. Simulating denitrification of municipal wastewater, two different batches were set up as follows:

- Batch DM1: mixed liquor inoculated with 100 mg/L (107 CFU/mL) aerobic-dark (AE-DK) grown R. capsulatus - Batch DM2: mixed liquor inoculated with 100 mg/L (107 CFU/mL) of anaerobic-light (AN-LT) grown R. capsulatus

Simulating nitriﬁcation of municipal wastewater, other two different batches were set up as follows:

- Batch NM1: mixed liquor inoculated with 100 mg/L (107 CFU/mL) of aerobic-dark (AE-DK) grown R. capsulatus. - Batch NM2: mixed liquor inoculated with 100 mg/L (107 CFU/mL) of anaerobic-light (AN-LT) grown R. capsulatus.

In all cases, temperature was set at 30 ◦C and pH not controlled, mimicking as closest as possible what happens in industrial plant treatment. SCOD, N-nitric, N-nitrous, N-ammonia and biomass concentration were monitored and measured every 12 h.

2.7. Analytical Assays Biomass concentration was determined as optical density at 600 nm (Shimadzu, Kyoto, Japan) and as colony forming units (UFC)/mL in Petri dishes, after 72 h of incubation on agar-MR medium or agar-complex medium at 30 ◦C, respectively. Supernatant, collected by centrifuging samples at 4500 rpm for 15 min, was used for SCOD, N-NO3, N-NO2 and N-NH3 measurements, and following the ASTM standard methods [24]. All the experiments were carried out in triplicate to ensure the accuracy of data and the reported results were the average values. Water 2017, 9, 108 5 of 11

3. Results

3.1. Comparison between R. capsulatus Cell Growth in Aerobic-Dark and Anaerobic-Light Conditions Growth curves were obtained by plotting the cell concentration (CFU/mL) versus the time for bothWater aerobic-dark 2017, 9, 108 and anaerobic-light conditions. Figure2 shows the comparative proﬁles of5 of the 11 two growths,growths, and and it shows it shows that that when whenR. R. capsulatus capsulatus waswas grown anaerobically anaerobically in inlight, light, the themaximum maximum cell cell concentrationconcentration reached reached an order an order of magnitude of magnitude higher higher than thatthan obtainedthat obtained in aerobic in aerobic and dark and conditionsdark 9 8 (3 × 10conditionsversus (3 3 × 10109 versusCFU/mL, 3 × 108 respectively).CFU/mL, respectively).

Figure 2. Experimental profile of growth curves for R. capsulatus grown in aerobic‐dark and Figure 2. Experimental profile of growth curves for R. capsulatus grown in aerobic-dark and anaerobic‐light conditions. anaerobic-light conditions. In both case, lag phases were conveniently short, and replication was terminated after 30 h, Inprobably both case, due to lag some phases substrate were convenientlydepletion. In the short, absence and of replication external nutrient was terminated supplies, in after the 30 h, probablyremaining due to80 h, some the anaerobically substrate depletion. grown biomass In the lost absence an order of of externalmagnitude nutrient of CFU/mL. supplies, In these in the remainingconditions, 80 h, recovering the anaerobically the cell suspension grown biomass by 28–30 lost h from an the order beginning of magnitude of the process of CFU/mL. could assure In these conditions,the maximum recovering viable thecell cellconcentration. suspension Previous by 28–30 studies h revealed from the that beginning the standardized of the processcount that could 5 6 assurecan the be maximumused to inoculate viable the cell media concentration. of biodegrading Previous systems studiesis within revealed the range thatof 10 the–10 standardizedCFU/mL [25]. For anaerobic‐light growth, the stationary phase was very short and in few hours kinetic death count that can be used to inoculate the media of biodegrading systems is within the range of 5 prevailed.6 In the exponential phase, R. capsulatus showed a doubling time of 19 h. For aerobic‐dark 10 –10growthCFU/mL the doubling [25]. For time anaerobic-light was 36 h, and up growth, to 60 h, the the stationary cell concentration phase wasseemed very not short to decrease and in few hourssignificantly. kinetic death The prevailed. optimal light In the intensity exponential for biomass phase, growthR. capsulatus varies showedfrom microorganism a doubling timeto of 19 h.microorganism, For aerobic-dark and light growth also influenced the doubling the presence time was and 36 the h, composition and up to of 60 the h, bacteriochlorophylls the cell concentration seemedand not carotenoids to decrease produced significantly. by the cells, The which optimal in turn light has an intensity effect on the for cell biomass suspension’s growth color varies [26]. from microorganismIn this case, tothe microorganism, biomass grown in and chemotrophic light also influencedconditions gave the presencea dark pink and to thered compositioncolor, probably of the bacteriochlorophyllsdue to the biosynthesis and carotenoids of lycopene, produced rhodopin, by and the cells,spirilloxanthin, which in turnwhereas has anin phototrophic effect on the cell suspension’sconditions color the color [26]. turned In this to case, brownish the biomass‐red or greenish grown‐ inbrown, chemotrophic probably due conditions to the presence gave a of dark the pink to redspheroidenone color, probably series due [27]. to theIt is biosynthesis worth noting ofthat, lycopene, independent rhodopin, of the environment and spirilloxanthin, of growth, whereas after in several weeks of storage at room temperature in closed bottles, the cells’ viability was 106–105 phototrophic conditions the color turned to brownish-red or greenish-brown, probably due to the CFU/mL (data not shown). presence of the spheroidenone series [27]. It is worth noting that, independent of the environment of growth,3.2. afterNUR and several OUR weeks of storage at room temperature in closed bottles, the cells’ viability was 106–105 CFU/mL (data not shown). Most activated sludge processes used for biological nitrogen removal have a mixed, non‐aerated 3.2. NURanoxic and zone OUR before the oxygenation tank, where no oxygen is present and nitrate reduction occurs [28]. Usually, the rate of substrate utilization has been observed to be lower when nitrate instead of Mostoxygen activated is used as sludge the electron processes acceptor, used principally for biological because nitrogen not all removal of the endogenous have a mixed, heterotrophic non-aerated anoxicbacteria zone before present the in the oxygenation activated sludge tank, system where noare oxygenfacultative is presentorganisms and capable nitrate of reduction nitrate reduction occurs [28]. Usually,[29]. the Inoculating rate of substrate R. capsulatus utilization could enhance has been the overall observed nitrogen to be uptake lower rate, when while nitrate reducing instead the of time oxygen is usedof astreatment the electron and improving acceptor, the principally efficiency because of the process. not all ofWe the used endogenous the NUR test heterotrophic to evaluate bacteriathe potential of the nitrogen removal rate with the external inoculum of R. capsulatus grown in different present in the activated sludge system are facultative organisms capable of nitrate reduction [29]. conditions. Mixed liquor from a municipal wastewater biological treatment plant was used as the soluble substrate for this experiment, as well as for OUR tests. In the NUR tests, nitrate and an

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Inoculating R. capsulatus could enhance the overall nitrogen uptake rate, while reducing the time of treatment and improving the efficiency of the process. We used the NUR test to evaluate the potential Waterof the 2017 nitrogen, 9, 108 removal rate with the external inoculum of R. capsulatus grown in different conditions.6 of 11 Mixed liquor from a municipal wastewater biological treatment plant was used as the soluble substrate externalfor this experiment, carbon source as wellwere as added for OUR in excess tests. Into theavoid NUR nutrient tests, nitratedepletion and disguising an external the carbon results. source We foundwere added a significantly in excess different to avoid denitrification nutrient depletion rate disguisingdepending theon results.the type We of foundbiomass a significantlyused as the inoculumdifferent denitrification (Figure 3). rate depending on the type of biomass used as the inoculum (Figure3).

Figure 3. Nitrate depletiondepletion with with time time during during NUR NUR test test for forR. R. capsulatus capsulatusgrown grown in aerobic-darkin aerobic‐dark (AE-DK) (AE‐ DK)and anaerobic-lightand anaerobic‐light (AN-LT) (AN conditions.‐LT) conditions.

The inoculum of anaerobic‐light (AN‐LT) biomass almost permitted us to obtain a slope of The inoculum of anaerobic-light (AN-LT) biomass almost permitted us to obtain a slope of depletion. When aerobic‐dark (AE‐DK) biomass was added, we observed a short lag phase at the depletion. When aerobic-dark (AE-DK) biomass was added, we observed a short lag phase at the beginning which could be due to the need for a period of adaptation to the anoxic environment by beginning which could be due to the need for a period of adaptation to the anoxic environment by the the cells. When excluding the first hour of the experiment from the calculation, the NO3 consumption cells. When excluding the ﬁrst hour of the experiment from the calculation, the NO consumption rate rate was similar to the one obtained with the AN‐LT biomass. NUR values in 3the two cases are was similar to the one obtained with the AN-LT biomass. NUR values in the two cases are reported in reported in Table 1. It is clear that in the presence of AN‐LT biomass, NUR is more efficient, probably Table1. It is clear that in the presence of AN-LT biomass, NUR is more efﬁcient, probably because in because in this case the biomass re‐started growing in a similar environment of cell growth. this case the biomass re-started growing in a similar environment of cell growth.

Table 1. NUR and OUR tests results. Table 1. NUR and OUR tests results. Test NUR (mg/gVSS∙h) OUR (mg/gVSS∙h) AETest‐DK NUR10.08 (mg/gVSS ± 2.75 ·h) OUR6.20 (mg/gVSS± 1.23 ·h) AE-DKAN‐LT 13.90 10.08 ±± 2.672.75 4.73 6.20 ± 0.98± 1.23 AN-LT 13.90 ± 2.67 4.73 ± 0.98 In the biotechnological field, microbial respiration is typically expressed as the oxygen uptake rate (OUR).In the biotechnological Although OUR is field, a general microbial term respiration which does is not typically presuppose expressed any asspecific the oxygen conditions uptake in itsrate determination, (OUR). Although in the OUR wastewater is a general field term it is whichusually does reserved not presuppose for on‐line anymeasurements specific conditions of oxygen in consumptionits determination, through in the the wastewater measurement field of itthe is residual usually reserved dissolved for oxygen on-line (DO). measurements Indeed, OUR of oxygenis often referredconsumption to as the through dynamic the measurementrespirometric index, of the as residual a measure dissolved of the oxygenaerobic degradation (DO). Indeed, of OURorganic is materialoften referred by aerobic to as the microorganisms dynamic respirometric [30]. Furthermore, index, as a OUR measure can of provide the aerobic much degradation information of concerningorganic material the treatment by aerobic plant’s microorganisms performance, [30 wastewater]. Furthermore, characteristics, OUR can the provide degradability much information of special concentratedconcerning the streams treatment as plant’swell as performance, the parameters wastewater needed characteristics, to predict possible the degradability optimizations of special of a treatmentconcentrated plant. streams The asOUR well measurements as the parameters proved needed that to the predict oxygen possible‐depletion optimizations bioprocesses of a treatmentin mixed liquorplant. Theare OURa necessary measurements but not proved sufficient that thecondition oxygen-depletion for nitrification bioprocesses [31]. Figure in mixed 4 shows liquor the are respirogramsa necessary but of not mixed sufficient liquor condition with the foraddition nitrification of R. capsulatus [31]. Figure as4 the shows inoculum, the respirograms both in AE of‐DK mixed and ANliquor‐LT with growth the addition conditions. of R. The capsulatus OUR measurementsas the inoculum, can both be performed in AE-DK and in various AN-LT growthways which conditions. have beenThe OUR described measurements in detail [32]. can be In performedthis case, tests in various were carried ways which out by have adding been a described readily biodegradable in detail [32]. substrateIn this case, such tests as wereacetate carried to avoid out byfalse adding‐negative a readily results. biodegradable They could be substrate caused such by the as acetateexcess of to nitrogen due to the presence of human waste in the sludge, which could lead to an imbalance in the optimal COD:N ratio of 100:5 [33], or to the presence of inhibitory streams, or to an occurrence of endogenous, slowly biodegradable organic carbon. In fact, as it is well known while the carbon sources and oxygen have been consumed, DO progressively decreases due to the cells’ respiration. It is worthwhile to note that the oxygen was completely depleted in 45 min. In this case, as expected,

Water 2017, 9, 108 7 of 11 avoid false-negative results. They could be caused by the excess of nitrogen due to the presence of human waste in the sludge, which could lead to an imbalance in the optimal COD:N ratio of 100:5 [33], or to the presence of inhibitory streams, or to an occurrence of endogenous, slowly biodegradable organic carbon. In fact, as it is well known while the carbon sources and oxygen have been consumed, DO progressively decreases due to the cells’ respiration. It is worthwhile to note that the oxygen was completelyWater 2017 depleted, 9, 108 in 45 min. In this case, as expected, the AE-DK-grown biomass seemed7 of 11 to be betterthe adapted AE‐DK‐ togrown the oxygen-richbiomass seemed environment, to be better adapted contrary to to the the oxygen AN-LT‐rich biomass environment, which contrary showed to a lag phasethe before AN‐LT starting biomass aerobic which respirationshowed a lag (Table phase1 before). starting aerobic respiration (Table 1).

Figure 4. Dissolved oxygen with time during OUR test for R. capsulatus grown in aerobic‐dark (AE‐ Figure 4. Dissolved oxygen with time during OUR test for R. capsulatus grown in aerobic-dark (AE-DK) DK) and anaerobic‐light (AN‐LT) conditions. and anaerobic-light (AN-LT) conditions. Since the kinetic parameters of nitrification by respirometric measurements (stoichiometry of Sinceoxygen the depletion kinetic due parameters to ammonium of nitrification oxidation) byare respirometricwell established measurements [34], an increase (stoichiometry of the OUR of oxygencould depletion be related due to tonitrification ammonium due oxidation) to R. capsulatus are well. established [34], an increase of the OUR could be relatedR. to capsulatus nitrification has confirmed due to R. to capsulatus be very versatile. and able to rapidly adapt its metabolism and its R.enzymatic capsulatus legacyhas to confirmed changes in to environmental be very versatile conditions. and able Moreover, to rapidly it was adapt also clear its metabolism that adding R. and its capsulatus to wastewater as an external inoculum could lay the ground for process optimization in enzymatic legacy to changes in environmental conditions. Moreover, it was also clear that adding both aerobic and anoxic environments. R. capsulatus to wastewater as an external inoculum could lay the ground for process optimization in both3.3. aerobic Batch and Experiments anoxic environments.on Mixed Liquor from Municipal Wastewater Biological Treatment Plant

3.3. BatchWastewater Experiments treatment on Mixed for Liquor the removal from Municipal of organic Wastewater carbon (COD) Biological and nitrogen Treatment [35,36] Plant is usually achieved through several steps in the treatment process such as primary settling, bioadsorption, and Wastewaterbiodegradation, treatment followed for by thea secondary removal settling of organic step to carbon remove (COD) the sludge and flocs nitrogen [37]. [In35 particular,,36] is usually achievednitrogen through can be several transformed steps and in theremoved treatment by biologically process suchmediated as primary nitrification settling, (aerobic bioadsorption, process) and biodegradation,and denitrification followed (anoxic process). by a secondary Nitrification settling is a process step to removewhich converts the sludge ammonia flocs to [37 nitrites]. In particular, and nitrogenthen can to nitrates, be transformed while denitrification and removed results by in biologically the transformation mediated of nitrates nitrification and nitrites (aerobic into nitrogen process) and denitrificationgas [38]. Figure (anoxic 5 shows process). the results Nitrification of the batches is of a processdenitrification which processes converts on ammonia mixed liquor to (batches nitrites and DM1 and DM2) by following N‐NO3, and the SCOD concentration over time. It is worth noting that then to nitrates, while denitrification results in the transformation of nitrates and nitrites into nitrogen in both cases of inoculum of R. capsulatus, the efficiency of the nitrate (Figure 5a) and SCOD removal gas [38]. Figure5 shows the results of the batches of denitrification processes on mixed liquor (batches (Figure 5b) did not show significant differences. When R. capsulatus was added, in 25–30 h the nitrate DM1was and almost DM2) completely by following removed N-NO (>95%)3, and from the SCOD the batches. concentration The corresponding over time. SCOD It is worth moved noting up to that in both69% cases of the of removal. inoculum No of significantR. capsulatus difference, the efficiency could be ofnoted the when nitrate inoculating (Figure5a) mixed and SCODliquor with removal (FigureAE5‐b)DK did or AN not‐ showLT biomass. significant R. capsulatus differences. was confirmed When R. to capsulatus be a versatilewas strain, added, capable in 25–30 of adapting h the nitrate was almostits metabolism completely to variable removed environmental (>95%) from conditions. the batches. The corresponding SCOD moved up to 69% of the removal. No significant difference could be noted when inoculating mixed liquor with AE-DK or AN-LT biomass. R. capsulatus was confirmed to be a versatile strain, capable of adapting its metabolism to variable environmental conditions.

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(a) (b)

Figure 5. Effect of denitrification of mixed liquor inoculated with R. capsulatus grown in aerobic‐dark Figure 5. Effect of denitrification of mixed liquor inoculated with R. capsulatus grown in aerobic-dark (batch DM1) and anaerobic‐light (batch DM2) on nitrate (a) and SCOD (b) concentration. (batch DM1) and anaerobic-light (batch DM2) on nitrate (a) and SCOD (b) concentration. (a) (b) Under aerobic‐dark conditions, aerobic respiration is the main catabolism pathway for organic Figure 5. Effect of denitrification of mixed liquor inoculated with R. capsulatus grown in aerobic‐dark Undermatter aerobic-darkdegradation through conditions, the glycolysis, aerobic respiration tricarboxylic is theacid main cycle catabolismand electron pathway transport for chain organic (batch DM1) and anaerobic‐light (batch DM2) on nitrate (a) and SCOD (b) concentration. matterprocess, degradation whereas throughunder anaerobic the glycolysis,‐light conditions, tricarboxylic material acidcatabolism cycle pathways and electron are glycolysis transport and chain the following fermentation, or anaerobic respiration [39]. In the first case, oxygen is the final electron process, whereasUnder aerobic under‐dark anaerobic-light conditions, aerobic conditions, respiration material is the catabolismmain catabolism pathways pathway are for glycolysis organic and acceptor and the initial matrixes can be completely mineralized to CO2 (oxidation tank conditions). the followingmatter degradation fermentation, through or anaerobic the glycolysis, respiration tricarboxylic [39]. Inacid the cycle first case,and electron oxygen transport is the final chain electron In the latter, the final electron acceptors could be small products (fermentation) or nitrate (anaerobic acceptorprocess, and whereas the initial under matrixes anaerobic can‐light be completelyconditions, material mineralized catabolism to CO pathways2 (oxidation are glycolysis tank conditions). and respiration). In the nitrification tank (aerobic conditions, batches NM1 and NM2), N‐NH3 was the following fermentation, or anaerobic respiration [39]. In the first case, oxygen is the final electron In thereduced latter, the by 70% final in electron 50 h when acceptors both AE‐ couldDK and be AN small‐LT biomass products were (fermentation) added. or nitrate (anaerobic acceptor and the initial matrixes can be completely mineralized to CO2 (oxidation tank conditions). respiration).Under In the highly nitrification aerobic conditions, tank (aerobic biomass conditions, grown in batches AN‐LT NM1 needed and a long NM2), lag N-NH phase 3towas express reduced In the latter, the final electron acceptors could be small products (fermentation) or nitrate (anaerobic by 70%enzymatic in 50 h whenactivities both for AE-DK supporting and the AN-LT rearrangement biomass of were the added.metabolic pathways. However, after 24 respiration). In the nitrification tank (aerobic conditions, batches NM1 and NM2), N‐NH3 was Underh the rate highly of N‐NH aerobic3 removal conditions, from the biomass mixed liquor grown reached in AN-LT almost neededthe same a amount long lag for phase both inocula, to express reduced by 70% in 50 h when both AE‐DK and AN‐LT biomass were added. corresponding to a SCOD decrease of 70% (Figure 6). The capacity of R. capsulatus to assimilate enzymaticUnder activities highly for aerobic supporting conditions, the rearrangement biomass grown ofin theAN‐ metabolicLT needed pathways.a long lag phase However, to express after 24 h ammonia has been already established [40,41], indicating that it could be heterotrophically consumed the rateenzymatic of N-NH activities3 removal for supporting from the mixedthe rearrangement liquor reached of the almost metabolic the pathways. same amount However, for both after inocula,24 for cell growth. Otherwise, the increase of the nitrate concentration could reflect some kind of correspondingh the rate of toN‐NH a SCOD3 removal decrease from the of mixed 70% liquor (Figure reached6). The almost capacity the same of amountR. capsulatus for bothto inocula, assimilate nitrifying behavior as reported in the literature for other Rhodobacter spp. isolated from the soil [42]. ammoniacorresponding has been to already a SCOD established decrease of [40 70%,41], (Figure indicating 6). The that capacity it could of be R. heterotrophically capsulatus to assimilate consumed Further investigations are necessary to evaluate if heterotrophic nitrification [43] could be one of the for cellammonia growth. has been Otherwise, already established the increase [40,41], of theindicating nitrate that concentration it could be heterotrophically could reflect consumed some kind of well‐known metabolisms of R. capsulatus. for cell growth. Otherwise, the increase of the nitrate concentration could reflect some kind of nitrifyingThese behavior results as indicate reported that in inoculating the literature mixed for liquor other withRhodobacter R. capsulatusspp. promotes isolated the from efficiency the soil of [42]. nitrifying behavior as reported in the literature for other Rhodobacter spp. isolated from the soil [42]. Furtherboth investigations nitrification and are necessarydenitrification to evaluate conditions. if heterotrophic Nevertheless, nitrification it is quite [surprising43] could bethat one the of the Further investigations are necessary to evaluate if heterotrophic nitrification [43] could be one of the well-knownconditions metabolisms in which the of biomassR. capsulatus had grown. seemed not to have an overall great influence on the well‐known metabolisms of R. capsulatus. Theseprocess’s results performances, indicate that except inoculating for the occurrence mixed liquor of a lag with phaseR. capsulatus after the inoculumpromotes due the to efficiency the These results indicate that inoculating mixed liquor with R. capsulatus promotes the efficiency of unavoidable necessity of rearranging the enzymatic legacy in order to adapt to the different of bothboth nitrification nitrification andand denitrificationdenitrification conditions. conditions. Nevertheless, Nevertheless, it is itquite is quite surprising surprising that the that the environmental conditions. conditionsconditions in whichin which the the biomass biomass had grown grown seemed seemed not not to have to have an overall an overall great influence great influence on the on the process’sprocess’s performances, performances, except except for forthe theoccurrence occurrence of a lag of aphase lag phaseafter the after inoculum the inoculum due to the due to the unavoidableunavoidable necessity necessity of of rearranging rearranging the the enzymatic enzymatic legacy legacy in order in order to adapt to adapt to the to different the different environmentalenvironmental conditions. conditions.

(a) (b)

Figure 6. Cont.

Water 2017, 9, 108 9 of 11 Water 2017, 9, 108 9 of 11

Figure 6. Effect of nitrification of municipal mixed liquor inoculated with R. capsulatus grown in Figure 6. Effect of nitrification of municipal mixed liquor inoculated with R. capsulatus grown in aerobic‐dark (batch NM1) and anaerobic‐light (batch NM2) on ammonia (a) and SCOD (b) nitrate (c) aerobic-dark (batch NM1) and anaerobic-light (batch NM2) on ammonia (a) and SCOD (b) nitrate (c) and nitrite (d) concentration. and nitrite (d) concentration. At this point, the discriminant factor that may suggest the use of AE‐DK or AN‐LT conditions is onlyAt this the point, cost of the production, discriminant in terms factor of that substrate may suggest supply theand useequipment. of AE-DK To orgive AN-LT a first conditions and is onlyapproximate the cost ofeconomic production, evaluation in terms between of substratethe two growth supply conditions and equipment. tested, some To preliminary give a first and approximatecalculations economic have been evaluation carried out. between In particular, the the two 19 growth‐component conditions MR medium tested, for anaerobic some preliminary‐light growth had an average cost of 1.6 €/L while the aerobic‐dark medium, which contained only peptone calculations have been carried out. In particular, the 19-component MR medium for anaerobic-light and meat extract, had a cost of about 0.9 €/L. Moreover, both components could be quite easily growthreplaced had an with average low‐cost cost agro of‐ 1.6food€/L byproducts while the as aerobic-dark sources of nutrients, medium, further which reducing contained the onlyaverage peptone andprice. meat For extract, industrial had aapplications, cost of about that 0.9 means€/L. large Moreover, volumes both and componentsbig plants. For could anaerobic be quite‐light– easily replacedgrown with biomass, low-cost higher agro-food costs have byproducts to be unavoidably as sources accounted of nutrients, for due furtherto energy reducing supply for the light average price.and For the industrial equipment applications, for maintaining that an means anaerobic large environment. volumes and Nevertheless, big plants. For it is anaerobic-light–grown worth noting that biomass,in anaerobic higher conditions, costs have the to cell be concentration unavoidably obtained accounted was foran order due toof magnitude energy supply larger forthan light for and the equipmentAE‐DK, which for contributes maintaining to considerable an anaerobic money environment. savings in terms Nevertheless, of the quantity it is worth of biomass noting to be that in anaerobicproduced. conditions, the cell concentration obtained was an order of magnitude larger than for AE-DK, which contributes to considerable money savings in terms of the quantity of biomass to be produced. 4. Conclusions 4. ConclusionsThe use of R. capsulatus as an external inoculum for biological processes in municipal wastewater treatments seems to be promising for both denitrification and nitrification. Moreover, in both cases, R. capsulatus comparingThe use of the cost‐effectivenessas an external and the inoculumperformance, for biologicalbiomass grown processes in aerobic in municipal‐dark conditions wastewater treatmentsseems to seems be more to convenient be promising for large for both‐scale denitrification industrial applications, and nitrification. even though Moreover, the biomass in yield both is cases, comparingof an order the of cost-effectiveness magnitude less than and in anaerobic the performance,‐light conditions. biomass grown in aerobic-dark conditions seems to be more convenient for large-scale industrial applications, even though the biomass yield is of anAuthor order Contributions: of magnitude Stefania less than Costa, in Saverio anaerobic-light Ganzerli and conditions. Irene Rugiero performed all the experiments and carried out all the analytical assays, also giving a great contribution to the discussion. Simone Pellizzari Authorconceived Contributions: and designedStefania the Costa,experiments, Saverio together Ganzerli with and Elena Irene Tamburini, Rugiero performedwho wrote the all themanuscript. experiments As and carriedsupervisor out all the of the analytical research assays, group, alsoPaola giving Pedrini a greatdefined contribution the general research to the discussion. statement. Simone Pellizzari conceived and designedConflicts theof Interest: experiments, The authors together declare with no Elenaconflict Tamburini, of interest. who wrote the manuscript. As supervisor of the research group, Paola Pedrini defined the general research statement. ConflictsReferences of Interest: The authors declare no conflict of interest.

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