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Phototrophic bacteria for nutrient recovery from domestic wastewater
w a t e r r e s e a r c h 5 0 ( 2 0 1 4 ) 1 8 e2 6
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/watres
Phototrophic bacteria for nutrient recovery from domestic wastewater Tim Hu¨lsen a,b, Damien J. Batstone a,b, Ju¨rg Keller a,b,* a
Advanced Water Management Centre, Gehrmann Building, The University of Queensland, Brisbane, Queensland 4072, Australia b CRC for Water Sensitive Cities, PO Box 8000, Clayton, Victoria 3800, Australia
article info
abstract
Article history:
The organics and nutrients in industrial and domestic wastewater are increasingly being
Received 24 May 2013
regarded as a valuable resource for energy and nutrient recovery. Emerging concepts to
Received in revised form
redesign wastewater treatment as resource recovery systems include the use of different
2 October 2013
bacteria and algae to partition carbon and nutrients to the particulate phase through
Accepted 22 October 2013
assimilation or bio-accumulation. This study evaluates the use of purple phototrophic bac-
Available online 31 October 2013
teria (PPB) (also known as purple non-sulphur bacteria or PNSB) for such a biological concentration process through a series of batch tests. The key objectives are to (a) demonstrate
Keywords:
consistent selection and enrichment of PPB using infrared light in a non-sterile medium, and
Purple non-sulphur bacteria
(b) achieve effective partitioning of soluble organics, ammonium and phosphate into the PPB
Domestic wastewater
culture. PPB were successfully enriched from pre-settled domestic wastewater within 2e3
COD
days and identified as members of the order Rhodobacterales. Under anaerobic conditions
Nutrient recovery
with infrared irradiation the enrichment culture was able to simultaneously remove COD
Assimilation
(63 5%), NH4eN (99.6%e0.12 0.03 mgN L1) and PO4eP (88%e0.8 0.6 mgP L1) from primary settled domestic wastewater in 24 h. In this experiment, acetate was added as an additional carbon source to demonstrate the maximal nitrogen and phosphorous elimination potential. Almost all the COD removed was assimilated into biomass rather than oxidised to CO2, with the total COD actually increasing during the batch experiments due to phototrophic synthesis. NH4eN and PO4eP were also assimilated by the biomass rather than removed through destructive oxidation or accumulation. The process offers the opportunity to concentrate organics and macronutrients from wastewater in one solids stream that can be anaerobically digested to generate energy and recover nutrients from the concentrated digestate. Technical challenges include the design of a continuous reactor system, as well as efficient delivery of electrons, either through light or chemical sources. ª 2013 Elsevier Ltd. All rights reserved.
1.
Introduction
Increases in environmental and financial costs of energy, as well as the availability and cost of mineral and synthetic
fertilizers have shifted the focus of wastewater management from treatment towards nutrient and energy recovery (Verstraete et al., 2009). Physical separation, e.g. using membranes, has been proposed as a method to accumulate carbon
* Corresponding author. Advanced Water Management Centre, Gehrmann Building, The University of Queensland, St. Lucia, Brisbane, Queensland 4072, Australia. Tel.: þ61 (0)7 3365 4727; fax: þ61 (0)733654726. E-mail address: [email protected] (J. Keller). 0043-1354/$ e see front matter ª 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.watres.2013.10.051
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and nutrients and subsequently recover them (Verstraete et al., 2009). More recently, the use of biological concentration has been proposed as a low energy alternative to membrane separation. Phosphorous and carbon can be readily accumulated through polyphosphate (poly-P) and polyhydroxyalkanoates respectively, but biological concentration of nitrogen is far more challenging, as the key route is assimilation through growth rather than accumulation (Suhaimi et al., 1987). Due to the high N:COD ratio commonly found in wastewater (Tchobanoglous et al., 2003), typical aerobic bacterial growth does not achieve sufficient N removal with the available COD and some COD is lost to oxidation. Thus, fast growing organisms that can also be readily supplied with electron equivalents are required. Algae and plants have been proposed for this purpose (Zimmo et al., 2004), but are relatively slow growing and can form macroscopic structures that interfere with high-rate reactor operation. In this paper, we propose the use of infra-red selected phototrophic bacteria, generally known as purple phototrophic bacteria (PPB) (largely consisting of purple non-sulphur bacteria e PNSB), as an alternative mediator for combined biological carbon, nitrogen and phosphorous removal. They are generally referred to as “purple” bacteria due to the presence of carotenoids, and can range from orange to purple in colour. PPB are widely found in soil, freshwater, marine, and wastewaters and can be readily isolated from these sources (Zhang et al., 2003). They are characterized by versatile metabolic modes including photoautotrophic, with light as energy source, H2 as the electron donor and CO2 as the electron acceptor and carbon source (Koku et al., 2002), chemoheterotrophic growth without light, using organics as electron donor and carbon source and O2 as electron acceptor (Dubbs et al., 2000) and photoheterotrophic, using some sugars and a variety of organic acids as electron donors and carbon source, with light as energy source (Kim et al., 2004). There has been limited application of pure cultures of PPB to treat synthetic and sterilized wastewaters. These studies include the removal of chemical oxygen demand (COD) and orthophosphate (PO4eP) from swine wastewater by Rhodopseudomonas palustris (Rps. palustris) grown in modified Lascelles basal medium (Kim et al., 2004) and COD removal from tuna condensate and a mix of tuna condensate and shrimpblanching water by Rhodocyclus gelatinosus grown in G5 medium (Prasertsan et al., 1993). Rubrivivax gelatinosus was used to remove COD from latex processing effluent grown in G5 media (Choorit et al., 2002) and wastewater from a poultry slaughterhouse grown in Pfennig’s medium (Ponsano et al., 2008). de Lima et al. (2011) applied the latter organism with same medium to treat effluent from tilapia fish processing. The COD removal from non-sterilized sardine processing wastewater by Rhodovulum sulfidophilum was reported by Azad et al. (2001). COD removal efficiencies have generally ranged from 43% (de Lima et al., 2011) to 90% (Ponsano et al., 2008; Kantachote et al., 2005). Reports of domestic wastewater treatment by PPB are limited and deal predominantly with the isolation and identification of phototrophs from wastewater sources (Zhang et al., 2003), (Hiraishi and Ueda, 1994). Siefert et al. (1978) found Rhodopseudomonas in all biological stages of a wastewater treatment plant (WWTP). Nagadomi et al. (2000)
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reported COD, PO4eP, NO3eN and H2S removal from synthetic sewage by immobilized Rhodobacter sphaeroides (R. sphaeroides) S, R. sphaeroides NR-3 and Rps. palustris. PPB have significant potential for wastewater treatment due to the assimilation of COD e.g. as polyhydroxybutyrate (PHB) (Khatipov et al., 1998) but also due to nutrient removal by inorganic polyP formation (Hiraishi et al., 1991), N removal by denitrification (Kim et al., 1999) and assimilation (Takabatake et al., 2004). Compared to other phototrophic organisms, their key advantages include high growth rates, versatile metabolic modes (Tabita, 1988) and the utilisation of IR light, which lowers the power required per photon emission and facilitates the specific selection and enrichment over algae (Bertling et al., 2006) that are unable to utilise light at wavelengths above 750 nm (Bidigare et al., 1990). One of the key considerations in any wastewater treatment technology development is the fact that for such applications wastewaters cannot be sterilized and hence mixed culture biotechnology must be used. Therefore, appropriate phototroph cultures need to be consistently enriched using suitable selective pressures. PPB grow specifically on infrared light (IR) (Bertling et al., 2006), which is a suitable selection mechanism. This study aims to evaluate the utility of selectively enriched phototrophs through batch tests, with the two key objectives of (a) demonstrating consistent selection of suitable phototrophs through irradiation with a suitable wavelength, and (b) demonstration that the IR enriched culture can effectively partition soluble organics, NH4eN, and PO4eP to the biomass phase, which would allow this treatment to be used as a single-stage, combined carbon and nutrient removal method.
2.
Material and methods
The approach for this experimental study used a series of batch tests with various inocula and domestic wastewater containing carbon, nitrogen, and phosphorous compounds.
2.1.
Raw wastewater
Domestic wastewater was collected at a local pump station (38B Heroes Avenue, Brisbane QLD, Australia) and stored immediately in a cold room at 4 C. Wastewater was settled in 200 L tanks for 1 day. The supernatant was used as primary settled domestic wastewater without any pre-treatment or chemical addition, expect where specified. The primary settled wastewater contained on average: 526 99 mg L1 total chemical oxygen demand (TCOD); 395 30 mg L1 soluble COD (SCOD); 46 3 mg L1 NH4eN; and 6.2 0.7 mg L1 PO4eP. The pH was 6.85.
2.2.
Enrichment
Four 150 ml serum flasks with septa were inoculated with 130 ml of wastewater and flushed with N2 for 1 min. The pH after flushing was 7.46. The flasks were incubated under anaerobic conditions and exposed to light at 30 1 C for 11 days. The flasks were continuously shaken at 100 rpm (Orbital shaker, Edwards Instrument Company) and illuminated with 2 150 W fluorescence lamps (Nelson Clamp Flood Light) from
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both sites, each at a distance of 80 cm from the shaker. The flasks were covered with UVeVIS absorbing foil (ND 1.2 299, Transformation Tubes). The foil absorbed about 90% of the wavelength below 790 nm. On the lamp side of the foil, illuminance was 2700 (100) lux. On the enrichment side, illuminance of 420 (40) lux was measured. Daily samples (5 mL) were taken from each flask with a syringe (flushed with N2) to analyse nutrients, COD, volatile fatty acids (VFA), pH, temperature, optical density at 660 nm (OD660), wavelength scan from 300 to 1100 nm and total suspended solids (TSS)/volatile suspended solids (VSS). Following enrichment, for analytical and inoculum purposes, the culture was continuously stirred at 500 rpm (RCT basic, Kika Labortechnik) in a 10 L Schott bottle and fed weekly, with 90% fresh wastewater under the above described conditions. The biomass from this bottle was characterized by Raman spectroscopy and fluorescence in-situ hybridisation (FISH) before 1.5 L of the biomass were centrifuged for 20 min at a centrifugal force of 3270 g at 20 C (Allegra X-12 centrifuge) in two 750 mL beakers. The supernatant was discarded and the pellet was resuspended in 50 mL 0.85% NaCl solution in 50 mL falcon tubes. This washing and re-centrifuging was repeated twice in the falcon tubes, and the pelletised biomass resuspended in 0.85% NaCl solution was immediately used as inoculum for the accumulation experiments.
2.3.
Accumulation/assimilation experiments
Six glass flasks with septa (150 mL) were filled with 90 mL of wastewater and 10 mL of the phototroph enriched solution. Three flasks were inoculated with 100 mL wastewater as control. The headspace was flushed with N2 for 1 min. The rest of the PPB enriched solution was used to determine the initial TSS/VSS concentration of the experiment. The flasks were continuously shaken at 100 rpm (Orbital shaker, Edwards Instrument Company) and illuminated with 2 150 W fluorescence lamps (Nelson Clamp Flood Light) in the same configuration as described above. Samples (5 mL) were taken at 5 points in the first hour, at 5 points in the first day, and then daily for the total batch of 6 days (144 h). Six bottles were sampled and triplicates sampled across the range of the analysis. One set of triplicates was sampled in the first hour, the other set of triplicates over the remainder of the first 24 h, and all six were sampled to the end of the batch test. A separate set of experiments were done to identify the limits of N and P assimilation and to determine the achievable minimum concentration of NH4eN and PO4eP in the treated wastewater. Serum flasks were inoculated with 5 mL PPB solution, 110 mL of wastewater as well as 200 mg VFA-COD L1 at t ¼ 0 h, 96 mg VFA-COD L1 at t ¼ 4 h and 91 mg VFA-COD L1 at t ¼ 8 h as glacial acetic acid. Samples were taken in 4 h interval for 24 h.
2.4.
were measured using an Oakton pH 11 Series. The TSS and VSS were determined after glass fibre filtration (Whatman, GF/C). The residue was dried at 105 C for 24 h to determine the TSS. The VSS was determined after 2 h in a furnace at 550 C. The optical density at 660 nm (OD660) and the absorption from 200 nm to 1100 nm were measured using a spectrophotometer (Cary 50 conc, Varian). Illuminance was measured with a Lux/ FC light metre (Sper Scientific, Model 840020). RAMAN spectroscopy (Alpha 300 Raman/AFM,WITek GmbH, Ulm, Germany) equipped with a 100X objective (Nikon) was used to determine the polyP and polyhydroxyalkanoates (PHA) presence. VFA samples were analysed by gas chromatography (Agilent Technologies 7890A GC System) equipped with a flame ionisation detector (GC/FID) and a polar capillary column (DB-FFAP). Sudan black staining with safranin counter-staining was used to qualitatively determine PHA as described elsewhere (Chen et al., 2012). Methylene blue staining was applied to determine polyP presence (Bond et al., 1999). The stained samples were analysed with an epifluorescence microscope (Zeiss, Axiophot 2, Diagnostic Instruments Inc with Spot camera).
2.5.
Microbial identification
Raman spectroscopy and confocal microscopy combined with fluorescent in-situ hybridisation (FISH) were used to identify the bacteria as described below. Raman measurements were performed at room temperature in air using an Alpha 300 Raman/AFM (WITek GmbH, Ulm, Germany) equipped with a 100X objective (Nikon). A frequency-doubled continuous-wave Nd:YAG laser stabilized at 532 nm was used for excitation. Raman signals were collected with a 50 mm optical fibre with a resolution of 4 cm1. For all the measurements the laser power at the sample was less than 5 mW. Typically, spectral acquisition was done at integration times of 0.08 s, which proved to be sufficient to obtain high-contrast resonance spectra for the spheroidene chromophore. All images were constructed by collecting spectra on 50 points/line with a scan line width of 15 mm. Raman scans were performed on a biomass sample dried overnight in a desiccator. All data have been processed using WITec Project 2.1 software. For identification by FISH, the methodology according to Batstone et al. (2004) was used. Samples were fixed in paraformaldehyde-PBS as described previously (Oda et al., 2000). The probes used were ARCH 915 (Stahl and Amann, 1991) with Cy3, PARA 739 (Thayanukul et al., 2010) with Cy5 and EUB mix (EUB 338 (Amann et al., 1990) þ EUB338II þ EUB338III (Daims et al., 1999)) with FITC. Hybridisation at 40% formamide concentration was performed based on recommended stringency ranges. An overview of the probes is shown in Table 1.
3.
Results and discussion
3.1. light
PPB can be consistently enriched under infrared
Analytical methods
TCOD and SCOD were determined by COD cell test (Merck, 1.14541.0001). Dissolved NH4eN, NO2eN and PO4eP were determined by flow injection analysis (FIA). All parameters except TCOD were determined after filtering with a 0.45 mm membrane filter (Millipore, Millex-HP). Temperature and pH
Enrichment experiments using the method described above with domestic wastewater only, consistently resulted in a colour change in the serum flasks from yellowish to red within
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Table 1 e FISH probes used in this study and information relevant to FISH oligonucleotides. Probe EUB338þ EUB338IIþ EUB338IIIGCT PARA739 40 ARCH 915
Sequence (50 e30 )
rRNA target site
Specificity
% Formamide
GCTGCCTCCCGTAGGAGT GCA GCC ACC CGT AGG TGT GCC ACC CGT AGG TGT GCG TCA GTA TCG AGC CAG GTG CTC CCC CGC CAA TTC CT
16S, 338e355 16S, 338e355 16S, 338e355 16S, 739e756 16S rRNA, 915e934
Bacteria Planctomycetales Verrucomicrobiales Order Rhodobacterales Archaea
40 40 40 40 40
100 h (Fig. 1A). Samples taken at this point had developed light adsorption peaks at 805 and 865 nm, consistent with BChl-a (Jones, 1982), as well as a peak at 590 nm which likely was related to the carotenoid content causing the colour change (Do et al., 2003). Absorption spectra are provided in supplementary information (Figure S1). As shown in Fig. 1C, these samples also had Raman spectra matching the emission profile of Rhodobacter sphaeroides (Kniggendorf and MeinhardtWollweber, 2011). Analysis using FISH identified that approximately 50% of the total bacterial community (identified by EUB-338) bound to the PARA 739 probe (Fig. 1B). The combination of the matching Raman emission spectra and the FISH binding to the PARA-739 probe specific for the order Rhodobacterales indicates that the sample is heavily populated by these organisms. According to the ARCH 915 probe, between 2% and 10% of the total population (data not shown) of the enrichment serum flask was Archaea (measured at t ¼ 76, t ¼ 146 and t ¼ 242 h). The role of the Archaea and their potential detrimental impact in terms of acetate consumption need to be further evaluated. However, given the chemical results and relatively low numbers, it is unlikely they consumed a substantial amount of VFAs. The maximum
specific growth rates of acetoclastic methanogens at mesophilic conditions are 0.33 d1 for the fast growing Methanosarcina (Demirel and Scherer, 2008) and 0.12 d1 for Methanosaeta (Conklin et al., 2006) and decrease considerably with decreasing temperature. Therefore, the growth can be controlled with a suitable sludge retention time (SRT). Chemical and OD analysis of the enrichments (Fig. 2) showed that after the initial lag of approximately 3 days (72 h), the culture entered a logarithmic growth phase, and was stationary after about 150 h. During the growth phase, 60% of the soluble organics measured as SCOD were removed. NH4eN and PO4eP concentrations followed the same trend. During the lag phase, 15.5% NH4eN and 15.2% PO4eP were released and this was then re-assimilated, with a further removal of a cumulative 30.5% and 26.8% by the end of the batch. Total COD increased slightly during the growth phase. Phototrophic bacteria can generate sufficient adenosine triphosphate (ATP) and reducing power during the anoxygenic photosynthesis and can assimilate the majority of the substrate directly into biomass, with little CO2 production (Van Niel, 1944). That is, catabolic metabolic energy is derived from light, with anabolic activity being mainly assimilative.
Fig. 1 e Enrichment colour change over time from t [ 27 h to t [ 190 h (A) and confocal laser scanning micrographs of an IR enriched PPB sample dual hybridized with EUB mix (FITC labelled) and a PARA 739 (Cy3 labelled) (B), RAMAN emission signal of PPB inoculum (red) and Rhodobacter sphaeroides signal (blue line) measured by Kniggendorf and MeinhardtWollweber (2011) (C). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 2 e SCOD (:), NH4eN (,), PO4eP (B) and Absorbance at 660 nm (OD660 (A)) trends over the enrichment period (n [ 4 ± standard deviation).
Fixing of COD is mainly due to reduction of substrates such as organic acids to match the carbon oxidation state of the biomass. SCODremoved/Nremoved/Premoved (SCOD/N/P) ratios of 100/22.6/2.9 (from 76 to 148 h) and 100/3.9/0.29 (from 148 to 170 h) were determined. The ratio in the period 76e148 h was determined during the log phase, which results in higher values compared to the ratio of the residual growth during the stationary phase (Fig. 2), meaning that more NH4eN and PO4eP are consumed (relative to SCOD uptake) in the log phase. The SCOD/N/P ratio from 0 to 76 h was negative i.e. NH4eN and PO4eP concentrations increased, most likely due to the mobilisation of organic N and P compounds in the wastewater. However, after the colour change (due to PPB growth) the NH4eN and PO4eP is consumed concurrently with the SCOD. The trends in Fig. 2 are consistent with COD, N, and P assimilation into biomass rather than accumulation of storage products. The pH after 266 h was on average 8.2, the increase being likely due to the removal of organic acids and phototrophic uptake of CO2.
3.2.
Batch experiments on settled wastewater
Fig. 3 shows the batch test results of COD (total and soluble), NH4eN, PO4eP using an inoculum from the enrichment culture described in the previous section. The maximum removal efficiencies for SCOD, NH4eN and PO4eP were on average (n ¼ 3); 64 14% after 120 h, 39 2.5% after 120 h and 21 2.8% after 24 h, respectively. The TCOD increased significantly (27 6% after 120 h) with increasing SCOD consumption. Nitrate remained below 1 mgN L1 during the test (data not shown). The control culture removed after 266 h on average (n ¼ 4) 50 6.5% SCOD, 0.1 1.8% TCOD, 30.5 2.7% NH4eN and 26.8 4.7% PO4eP. As described for the enrichment experiment, all of the control experiments turned reddish/ purple after 72 h indicating PPB growth (confirmed by FISH) with inoculation from native bacteria present in the wastewater. The SCOD removal of the wastewater was largely related to VFA removal, as the initial concentration of 184 mg COD L1 (as VFAs) was completely consumed within 15 h, representing
Fig. 3 e Average SCOD (:), TCOD (A), NH4eN (,), PO4eP (B) data (n [ 3 ± standard deviation) in batch tests with IR enriched inoculum.
approximately 60% of the soluble COD removed (data not shown). This is consistent with previous reports of rapid uptake of VFAs by PPB (Kim et al., 2004). The TCOD increased significantly over time, indicating that not only was SCOD assimilated (mainly VFA), but also a substantial amount (around 200 mg COD L1) was generated from phototrophic activity (Fig. 3). Some of the soluble COD was accumulated as PHA, and PHA could be detected 15 min after inoculation through Sudan black staining and counter staining with safranin (details in Supporting material). PPB are known to accumulate PHA (Ali Hassan et al., 1997). This rapid SCOD uptake and PHA accumulation may have caused the short term fluctuations in soluble COD seen in the first 4 h. However, longer term reduction of soluble COD, nitrogen and phosphorous due to assimilation rather than accumulation was observed. Raman spectroscopy could not be used to identify poly-P and PHA peaks due to a high background signal of the sample. Longer term NH4eN removal is directly linked to the SCOD assimilation. PPB contain around 60% of proteins (Kobayashi and Kobayashi, 1995). Phosphate can be accumulated as polyP (Hiraishi et al., 1991) but a staining with methylene blue did not show intracellular polyP storage, and PO4eP uptake appears to be mainly due to assimilation. The COD:N:P uptake ratios after 24 and 144 h assimilation were 100:5.6:0.6 and 100:6.2:0.3 (n ¼ 5) at an average sludge loading rate (SLR) of 0.78 and 0.17 gSCOD/gVSS/d and 0.27 gVSS L1, respectively.
3.3.
Use of IR selected PPB for wastewater BNR
In order to achieve effective wastewater treatment, the PPBbased process needs to attain nutrient concentrations comparable to Biological Nutrient Removal (BNR) plant effluent primarily through assimilation. To test these assimilation limits, batch experiments with organic acids added were used in preference to phototrophic assimilation, since (a) it is relatively simple to make more organic acids available from complex organics through pre-acidification, and (b) providing assimilation through light delivery is energetically inefficient unless light delivery efficiency is dramatically improved. Fig. 4 demonstrates the achieved assimilation limits with acetic
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acid as chemical electron source added (178 mg VFA-COD at t ¼ 0 h, 91 mg at t ¼ 4 h and 96 mg at t ¼ 8 h). The SCOD removal after 24 h reached 62.5 5.3%, while PO4eP was removed by 88.2% to 0.8 0.55 mgP L1 and NH4eN removal achieved 99.6% to 0.12 0.03 mgN L1. The average SCOD/N/P ratio from 8 to 24 h was 100/8.6/1.4. This ratio was consistent over these 24 h as well as in previous batch tests, which again indicates that the likely uptake mechanism is through assimilation rather than accumulation. The average SLR, from 4 to 24 h was 0.23 gSCOD/gVSS/d with a maximum of 0.47 gSCOD/gVSS/d after 12 h and 0.015 gNH4eN/gVSS/ d with a maximum of 0.033 gNH4eN/gVSS/d after 8 h. This compares with the conventional activated sludge process which usually achieves SLRs of 0.2e1.0 gCOD/gVSS/d and of 0.05e0.1 kgN/kgVSS/d (Tchobanoglous et al., 2003). Volumetric removal rates are shown in Table 2. The average SCOD removal rate was higher than typically found in extended aeration reactors, oxidation ditches or anaerobic ponds (all 0.2e0.6 kgCOD/m3/d (Tchobanoglous et al., 2003). It is comparable with conventional activated sludge and membrane bioreactor processes (1.2e3.2 kgCOD/m3/d) (Tchobanoglous et al., 2003). The growth rate based on OD660 measurements from 4 to 24 h was 0.013 h1 with a maximum rate of 0.063 h1 from 0 to 4 h. These growth rates are comparable with literature values for mixed phototrophic populations (Kaewsuk et al., 2010) and microalgae such as Chlorella. Higher growth rates have been reported for pure cultures of PPB; e.g., 0.217 h1 (Eroglu et al., 1999) and 0.296 h1 (Ponsano et al., 2008) for R. sphaeroides. These results demonstrate the ability of phototrophs to achieve very low effluent nitrogen concentrations, which is of key importance for wastewater treatment since phosphorous removal can be enhanced by accumulation or chemical precipitation. However, all batch tests showed that the assimilation of N and P depends on the amount of degradable COD, and particularly, VFA-COD present in the wastewater. When acetate was added, the COD/N/P ratio required to remove almost all NH4eN and PO4eP was found to be 100/8.6/1.5. The relatively linear slope for both ammonia and phosphate to <3 mg/L indicates that the substrate half-saturation constant KS values for these compounds are low, and performance is
Fig. 4 e Average SCOD (:), VFA-COD (A),NH4eN (,), PO4eP (B) concentrations treating wastewater over time and calculated HAc-VFA added (–A–) (n [ 4 ± standard deviation) in IR enriched cultures.
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not limited by affinity to these compounds (Fig. 4). This additional COD can be delivered either through wastewater fermentation, external carbon addition, or increased light intensity. The latter is of particular relevance for PPB since they are able to use infrared (IR) light. Compared to the use of incandescent lamps, IR light from light emitting diodes (LEDs) can save up to 70% of the power requirements (Bertling et al., 2006) since the quantum efficiency of IR-LEDs can be close to 100% for emission wavelengths around 650 nm and higher. However this cannot fully compensate for the reported low light conversion efficiency of 6e8% (defined as combustion energy of hydrogen gas produced/incident light energy) for R. sphaeroides (Miyake and Kawamura, 1987). Therefore, the best way to enhance nitrogen removal to very low limits is through pre-acidification of the wastewater, possibly combined with internal carbon recycling to optimise the use of carbon for assimilation. Failing this, however, the use of PPB does provide the option of either delivering electrons through external carbon addition, or through increased light (and therefore power) input. Compared to denitrification, the stoichiometric COD:N ratio for the enriched cultures assessed here is approximately two times higher (COD:N ratio for denitrification including sludge production is 4 (Gustavsson, 2010)). However, denitrification destroys the nitrogen and COD whereas assimilation preserves nitrogen as well as the COD in the produced biomass.
3.4. Significance and application to the wastewater industry The application of IR selected phototrophs (PPB) for the removal of degradable COD, NH4eN and PO4eP from domestic or industrial wastewater achieves both key criteria of reproducible selection and enrichment of the suitable biocatalyst (through infrared light), as well as the ability to remove the major nutrients in a single-step process to achieve low effluent concentrations. A treatment process utilising phototrophic bacteria has a number of advantages over algae. The selection can be done at lower energy consumption, because PPBs can efficiently utilise wavelengths in the IR spectrum. This allows to limit the growth of non-desired organisms (e.g., algae and cyanobacteria) based on light delivery. Both can assimilate N and P as proteins and other organic biomass compounds. It is generally accepted that closed photo-bioreactors achieve higher removal rates but are more expensive to build and operate compared to open systems (Posten, 2009). PPB do not require CO2 addition and are not inhibited by O2 production, which are typically the most limiting factors in phototrophic systems, especially in closed reactors (Mun˜oz and Guieysse, 2006). This represents an opportunity to apply PPB particularly for highrate closed cell reactors in comparison with open and closed cell systems for algae, with delivery of only sufficient light to remove residual nitrogen. Assimilative partitioning of wastewater macronutrients and organics in one solids stream (i.e. biomass) that can be digested in a dedicated process to generate biogas, as well as a concentrated nitrogen and phosphorous stream might be a major advantage for product recovery. As an alternative, many PPB (as well as algae) contain high amounts of protein
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Table 2 e Volumetric removal rates for SCOD, NH4eN and PO4eP. Parameter SCOD NH4eN PO4eP
Unit
MIN
MAX
AVG
kgCOD/m3/d kgN/m3/d kgP/m3/d
0.231 (20e24 h) 0.027 (20e24 h) 0.007 (20e24 h)
2.629 (8e12 h) 0.305 (4e8 h) 0.045 (4e8 h)
1.658 (4e20 h) 0.183 (4e20 h) 0.024 (4e20 h)
(>60%) and were reported to be suitable as animal food (Sasaki et al., 1991) and organic fertilizer (Xu, 2001). A key technical challenge is the reactor design. This requires optimal light delivery to compensate for the relatively low photoconversion efficiency of 6e8% reported previously for R. sphaeroides (Miyake and Kawamura, 1987), which is, however, still higher than typically achieved values of <5% photoconversion efficiency from full sunlight spectra for microalgae (Posten, 2009). Interactions between fluid dynamics, biochemical reaction, and light transfer in photobioreactors are crucial (Posten, 2009) and considerable progress has been made in the last decade (Hankamer et al., 2007). A photo-anaerobic membrane-bioreactor (PAnMBR) is a possible process configuration. Compared to a conventional anaerobic membrane bioreactor (AnMBR) this reactor type will consume additional energy for the continuous IR light supply (for PPB selection). This extra consumption can be balanced with the efficient nutrient removal and recovery potential, and hence avoiding aerobic nitrogen removal. Phosphate removal can also be achieved by assimilative growth instead of biological accumulation or chemical precipitation. Continuous operation allows for optimisation options that were not available in the batch experiments, and is likely to further increase the applicability of this technology for practical wastewater treatment. The impact of pre-acidification, use of recycle streams, application of illuminated sections for selection and polishing, and recovery of organic carbon to remove residual nutrients will need to be determined in continuous processes. A major advantage in this context is the fact that the basic process platform has a high degree of flexibility due to the provision of additional control handles in the form of light and organic carbon dosing.
4.
Conclusions
The use of IR selected phototrophic purple bacteria offers potential as an alternative microbial agent for partitioning of nutrients and organic carbon to the solid (biomass) phase for subsequent recovery, with a number of key elements shown in this paper. These include: - Reliable enrichment of the target microbes from wastewater directly through application of infra-red light, showing that culture selection can be directed by this method with short start-up times. - Good removal of carbon and nutrients (N & P) present in wastewater. - Proof of achievable nutrient removal to meet discharge limits (N, P < 1.0 mg/L).
- Ability to supply supplementary electrons either by light or chemicals (e.g., acetate). The outcomes from the current work provide a proof-ofconcept demonstration of the suitability of PPB for wastewater treatment. However there are a number of technical challenges remaining, including verifying that nutrients can be effectively released, and determining optimal configurations for reactor design.
Acknowledgements We gratefully acknowledge Dr Beatrice Keller, Ms Jianguang Li and Nathan Clayton for assistance with FIA and GC/FID measurements. We also thank Dr Bogdan Donose for assisting with the Raman spectroscopy. This work was jointly funded by the Smart Water Fund (project 10OS-023) and the CRC for Water Sensitive Cities (project C2.1).
Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.watres.2013.10.051.
references
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Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/watres
Phototrophic bacteria for nutrient recovery from domestic wastewater Tim Hu¨lsen a,b, Damien J. Batstone a,b, Ju¨rg Keller a,b,* a
Advanced Water Management Centre, Gehrmann Building, The University of Queensland, Brisbane, Queensland 4072, Australia b CRC for Water Sensitive Cities, PO Box 8000, Clayton, Victoria 3800, Australia
article info
abstract
Article history:
The organics and nutrients in industrial and domestic wastewater are increasingly being
Received 24 May 2013
regarded as a valuable resource for energy and nutrient recovery. Emerging concepts to
Received in revised form
redesign wastewater treatment as resource recovery systems include the use of different
2 October 2013
bacteria and algae to partition carbon and nutrients to the particulate phase through
Accepted 22 October 2013
assimilation or bio-accumulation. This study evaluates the use of purple phototrophic bac-
Available online 31 October 2013
teria (PPB) (also known as purple non-sulphur bacteria or PNSB) for such a biological concentration process through a series of batch tests. The key objectives are to (a) demonstrate
Keywords:
consistent selection and enrichment of PPB using infrared light in a non-sterile medium, and
Purple non-sulphur bacteria
(b) achieve effective partitioning of soluble organics, ammonium and phosphate into the PPB
Domestic wastewater
culture. PPB were successfully enriched from pre-settled domestic wastewater within 2e3
COD
days and identified as members of the order Rhodobacterales. Under anaerobic conditions
Nutrient recovery
with infrared irradiation the enrichment culture was able to simultaneously remove COD
Assimilation
(63 5%), NH4eN (99.6%e0.12 0.03 mgN L1) and PO4eP (88%e0.8 0.6 mgP L1) from primary settled domestic wastewater in 24 h. In this experiment, acetate was added as an additional carbon source to demonstrate the maximal nitrogen and phosphorous elimination potential. Almost all the COD removed was assimilated into biomass rather than oxidised to CO2, with the total COD actually increasing during the batch experiments due to phototrophic synthesis. NH4eN and PO4eP were also assimilated by the biomass rather than removed through destructive oxidation or accumulation. The process offers the opportunity to concentrate organics and macronutrients from wastewater in one solids stream that can be anaerobically digested to generate energy and recover nutrients from the concentrated digestate. Technical challenges include the design of a continuous reactor system, as well as efficient delivery of electrons, either through light or chemical sources. ª 2013 Elsevier Ltd. All rights reserved.
1.
Introduction
Increases in environmental and financial costs of energy, as well as the availability and cost of mineral and synthetic
fertilizers have shifted the focus of wastewater management from treatment towards nutrient and energy recovery (Verstraete et al., 2009). Physical separation, e.g. using membranes, has been proposed as a method to accumulate carbon
* Corresponding author. Advanced Water Management Centre, Gehrmann Building, The University of Queensland, St. Lucia, Brisbane, Queensland 4072, Australia. Tel.: þ61 (0)7 3365 4727; fax: þ61 (0)733654726. E-mail address: [email protected] (J. Keller). 0043-1354/$ e see front matter ª 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.watres.2013.10.051
w a t e r r e s e a r c h 5 0 ( 2 0 1 4 ) 1 8 e2 6
and nutrients and subsequently recover them (Verstraete et al., 2009). More recently, the use of biological concentration has been proposed as a low energy alternative to membrane separation. Phosphorous and carbon can be readily accumulated through polyphosphate (poly-P) and polyhydroxyalkanoates respectively, but biological concentration of nitrogen is far more challenging, as the key route is assimilation through growth rather than accumulation (Suhaimi et al., 1987). Due to the high N:COD ratio commonly found in wastewater (Tchobanoglous et al., 2003), typical aerobic bacterial growth does not achieve sufficient N removal with the available COD and some COD is lost to oxidation. Thus, fast growing organisms that can also be readily supplied with electron equivalents are required. Algae and plants have been proposed for this purpose (Zimmo et al., 2004), but are relatively slow growing and can form macroscopic structures that interfere with high-rate reactor operation. In this paper, we propose the use of infra-red selected phototrophic bacteria, generally known as purple phototrophic bacteria (PPB) (largely consisting of purple non-sulphur bacteria e PNSB), as an alternative mediator for combined biological carbon, nitrogen and phosphorous removal. They are generally referred to as “purple” bacteria due to the presence of carotenoids, and can range from orange to purple in colour. PPB are widely found in soil, freshwater, marine, and wastewaters and can be readily isolated from these sources (Zhang et al., 2003). They are characterized by versatile metabolic modes including photoautotrophic, with light as energy source, H2 as the electron donor and CO2 as the electron acceptor and carbon source (Koku et al., 2002), chemoheterotrophic growth without light, using organics as electron donor and carbon source and O2 as electron acceptor (Dubbs et al., 2000) and photoheterotrophic, using some sugars and a variety of organic acids as electron donors and carbon source, with light as energy source (Kim et al., 2004). There has been limited application of pure cultures of PPB to treat synthetic and sterilized wastewaters. These studies include the removal of chemical oxygen demand (COD) and orthophosphate (PO4eP) from swine wastewater by Rhodopseudomonas palustris (Rps. palustris) grown in modified Lascelles basal medium (Kim et al., 2004) and COD removal from tuna condensate and a mix of tuna condensate and shrimpblanching water by Rhodocyclus gelatinosus grown in G5 medium (Prasertsan et al., 1993). Rubrivivax gelatinosus was used to remove COD from latex processing effluent grown in G5 media (Choorit et al., 2002) and wastewater from a poultry slaughterhouse grown in Pfennig’s medium (Ponsano et al., 2008). de Lima et al. (2011) applied the latter organism with same medium to treat effluent from tilapia fish processing. The COD removal from non-sterilized sardine processing wastewater by Rhodovulum sulfidophilum was reported by Azad et al. (2001). COD removal efficiencies have generally ranged from 43% (de Lima et al., 2011) to 90% (Ponsano et al., 2008; Kantachote et al., 2005). Reports of domestic wastewater treatment by PPB are limited and deal predominantly with the isolation and identification of phototrophs from wastewater sources (Zhang et al., 2003), (Hiraishi and Ueda, 1994). Siefert et al. (1978) found Rhodopseudomonas in all biological stages of a wastewater treatment plant (WWTP). Nagadomi et al. (2000)
19
reported COD, PO4eP, NO3eN and H2S removal from synthetic sewage by immobilized Rhodobacter sphaeroides (R. sphaeroides) S, R. sphaeroides NR-3 and Rps. palustris. PPB have significant potential for wastewater treatment due to the assimilation of COD e.g. as polyhydroxybutyrate (PHB) (Khatipov et al., 1998) but also due to nutrient removal by inorganic polyP formation (Hiraishi et al., 1991), N removal by denitrification (Kim et al., 1999) and assimilation (Takabatake et al., 2004). Compared to other phototrophic organisms, their key advantages include high growth rates, versatile metabolic modes (Tabita, 1988) and the utilisation of IR light, which lowers the power required per photon emission and facilitates the specific selection and enrichment over algae (Bertling et al., 2006) that are unable to utilise light at wavelengths above 750 nm (Bidigare et al., 1990). One of the key considerations in any wastewater treatment technology development is the fact that for such applications wastewaters cannot be sterilized and hence mixed culture biotechnology must be used. Therefore, appropriate phototroph cultures need to be consistently enriched using suitable selective pressures. PPB grow specifically on infrared light (IR) (Bertling et al., 2006), which is a suitable selection mechanism. This study aims to evaluate the utility of selectively enriched phototrophs through batch tests, with the two key objectives of (a) demonstrating consistent selection of suitable phototrophs through irradiation with a suitable wavelength, and (b) demonstration that the IR enriched culture can effectively partition soluble organics, NH4eN, and PO4eP to the biomass phase, which would allow this treatment to be used as a single-stage, combined carbon and nutrient removal method.
2.
Material and methods
The approach for this experimental study used a series of batch tests with various inocula and domestic wastewater containing carbon, nitrogen, and phosphorous compounds.
2.1.
Raw wastewater
Domestic wastewater was collected at a local pump station (38B Heroes Avenue, Brisbane QLD, Australia) and stored immediately in a cold room at 4 C. Wastewater was settled in 200 L tanks for 1 day. The supernatant was used as primary settled domestic wastewater without any pre-treatment or chemical addition, expect where specified. The primary settled wastewater contained on average: 526 99 mg L1 total chemical oxygen demand (TCOD); 395 30 mg L1 soluble COD (SCOD); 46 3 mg L1 NH4eN; and 6.2 0.7 mg L1 PO4eP. The pH was 6.85.
2.2.
Enrichment
Four 150 ml serum flasks with septa were inoculated with 130 ml of wastewater and flushed with N2 for 1 min. The pH after flushing was 7.46. The flasks were incubated under anaerobic conditions and exposed to light at 30 1 C for 11 days. The flasks were continuously shaken at 100 rpm (Orbital shaker, Edwards Instrument Company) and illuminated with 2 150 W fluorescence lamps (Nelson Clamp Flood Light) from
20
w a t e r r e s e a r c h 5 0 ( 2 0 1 4 ) 1 8 e2 6
both sites, each at a distance of 80 cm from the shaker. The flasks were covered with UVeVIS absorbing foil (ND 1.2 299, Transformation Tubes). The foil absorbed about 90% of the wavelength below 790 nm. On the lamp side of the foil, illuminance was 2700 (100) lux. On the enrichment side, illuminance of 420 (40) lux was measured. Daily samples (5 mL) were taken from each flask with a syringe (flushed with N2) to analyse nutrients, COD, volatile fatty acids (VFA), pH, temperature, optical density at 660 nm (OD660), wavelength scan from 300 to 1100 nm and total suspended solids (TSS)/volatile suspended solids (VSS). Following enrichment, for analytical and inoculum purposes, the culture was continuously stirred at 500 rpm (RCT basic, Kika Labortechnik) in a 10 L Schott bottle and fed weekly, with 90% fresh wastewater under the above described conditions. The biomass from this bottle was characterized by Raman spectroscopy and fluorescence in-situ hybridisation (FISH) before 1.5 L of the biomass were centrifuged for 20 min at a centrifugal force of 3270 g at 20 C (Allegra X-12 centrifuge) in two 750 mL beakers. The supernatant was discarded and the pellet was resuspended in 50 mL 0.85% NaCl solution in 50 mL falcon tubes. This washing and re-centrifuging was repeated twice in the falcon tubes, and the pelletised biomass resuspended in 0.85% NaCl solution was immediately used as inoculum for the accumulation experiments.
2.3.
Accumulation/assimilation experiments
Six glass flasks with septa (150 mL) were filled with 90 mL of wastewater and 10 mL of the phototroph enriched solution. Three flasks were inoculated with 100 mL wastewater as control. The headspace was flushed with N2 for 1 min. The rest of the PPB enriched solution was used to determine the initial TSS/VSS concentration of the experiment. The flasks were continuously shaken at 100 rpm (Orbital shaker, Edwards Instrument Company) and illuminated with 2 150 W fluorescence lamps (Nelson Clamp Flood Light) in the same configuration as described above. Samples (5 mL) were taken at 5 points in the first hour, at 5 points in the first day, and then daily for the total batch of 6 days (144 h). Six bottles were sampled and triplicates sampled across the range of the analysis. One set of triplicates was sampled in the first hour, the other set of triplicates over the remainder of the first 24 h, and all six were sampled to the end of the batch test. A separate set of experiments were done to identify the limits of N and P assimilation and to determine the achievable minimum concentration of NH4eN and PO4eP in the treated wastewater. Serum flasks were inoculated with 5 mL PPB solution, 110 mL of wastewater as well as 200 mg VFA-COD L1 at t ¼ 0 h, 96 mg VFA-COD L1 at t ¼ 4 h and 91 mg VFA-COD L1 at t ¼ 8 h as glacial acetic acid. Samples were taken in 4 h interval for 24 h.
2.4.
were measured using an Oakton pH 11 Series. The TSS and VSS were determined after glass fibre filtration (Whatman, GF/C). The residue was dried at 105 C for 24 h to determine the TSS. The VSS was determined after 2 h in a furnace at 550 C. The optical density at 660 nm (OD660) and the absorption from 200 nm to 1100 nm were measured using a spectrophotometer (Cary 50 conc, Varian). Illuminance was measured with a Lux/ FC light metre (Sper Scientific, Model 840020). RAMAN spectroscopy (Alpha 300 Raman/AFM,WITek GmbH, Ulm, Germany) equipped with a 100X objective (Nikon) was used to determine the polyP and polyhydroxyalkanoates (PHA) presence. VFA samples were analysed by gas chromatography (Agilent Technologies 7890A GC System) equipped with a flame ionisation detector (GC/FID) and a polar capillary column (DB-FFAP). Sudan black staining with safranin counter-staining was used to qualitatively determine PHA as described elsewhere (Chen et al., 2012). Methylene blue staining was applied to determine polyP presence (Bond et al., 1999). The stained samples were analysed with an epifluorescence microscope (Zeiss, Axiophot 2, Diagnostic Instruments Inc with Spot camera).
2.5.
Microbial identification
Raman spectroscopy and confocal microscopy combined with fluorescent in-situ hybridisation (FISH) were used to identify the bacteria as described below. Raman measurements were performed at room temperature in air using an Alpha 300 Raman/AFM (WITek GmbH, Ulm, Germany) equipped with a 100X objective (Nikon). A frequency-doubled continuous-wave Nd:YAG laser stabilized at 532 nm was used for excitation. Raman signals were collected with a 50 mm optical fibre with a resolution of 4 cm1. For all the measurements the laser power at the sample was less than 5 mW. Typically, spectral acquisition was done at integration times of 0.08 s, which proved to be sufficient to obtain high-contrast resonance spectra for the spheroidene chromophore. All images were constructed by collecting spectra on 50 points/line with a scan line width of 15 mm. Raman scans were performed on a biomass sample dried overnight in a desiccator. All data have been processed using WITec Project 2.1 software. For identification by FISH, the methodology according to Batstone et al. (2004) was used. Samples were fixed in paraformaldehyde-PBS as described previously (Oda et al., 2000). The probes used were ARCH 915 (Stahl and Amann, 1991) with Cy3, PARA 739 (Thayanukul et al., 2010) with Cy5 and EUB mix (EUB 338 (Amann et al., 1990) þ EUB338II þ EUB338III (Daims et al., 1999)) with FITC. Hybridisation at 40% formamide concentration was performed based on recommended stringency ranges. An overview of the probes is shown in Table 1.
3.
Results and discussion
3.1. light
PPB can be consistently enriched under infrared
Analytical methods
TCOD and SCOD were determined by COD cell test (Merck, 1.14541.0001). Dissolved NH4eN, NO2eN and PO4eP were determined by flow injection analysis (FIA). All parameters except TCOD were determined after filtering with a 0.45 mm membrane filter (Millipore, Millex-HP). Temperature and pH
Enrichment experiments using the method described above with domestic wastewater only, consistently resulted in a colour change in the serum flasks from yellowish to red within
21
w a t e r r e s e a r c h 5 0 ( 2 0 1 4 ) 1 8 e2 6
Table 1 e FISH probes used in this study and information relevant to FISH oligonucleotides. Probe EUB338þ EUB338IIþ EUB338IIIGCT PARA739 40 ARCH 915
Sequence (50 e30 )
rRNA target site
Specificity
% Formamide
GCTGCCTCCCGTAGGAGT GCA GCC ACC CGT AGG TGT GCC ACC CGT AGG TGT GCG TCA GTA TCG AGC CAG GTG CTC CCC CGC CAA TTC CT
16S, 338e355 16S, 338e355 16S, 338e355 16S, 739e756 16S rRNA, 915e934
Bacteria Planctomycetales Verrucomicrobiales Order Rhodobacterales Archaea
40 40 40 40 40
100 h (Fig. 1A). Samples taken at this point had developed light adsorption peaks at 805 and 865 nm, consistent with BChl-a (Jones, 1982), as well as a peak at 590 nm which likely was related to the carotenoid content causing the colour change (Do et al., 2003). Absorption spectra are provided in supplementary information (Figure S1). As shown in Fig. 1C, these samples also had Raman spectra matching the emission profile of Rhodobacter sphaeroides (Kniggendorf and MeinhardtWollweber, 2011). Analysis using FISH identified that approximately 50% of the total bacterial community (identified by EUB-338) bound to the PARA 739 probe (Fig. 1B). The combination of the matching Raman emission spectra and the FISH binding to the PARA-739 probe specific for the order Rhodobacterales indicates that the sample is heavily populated by these organisms. According to the ARCH 915 probe, between 2% and 10% of the total population (data not shown) of the enrichment serum flask was Archaea (measured at t ¼ 76, t ¼ 146 and t ¼ 242 h). The role of the Archaea and their potential detrimental impact in terms of acetate consumption need to be further evaluated. However, given the chemical results and relatively low numbers, it is unlikely they consumed a substantial amount of VFAs. The maximum
specific growth rates of acetoclastic methanogens at mesophilic conditions are 0.33 d1 for the fast growing Methanosarcina (Demirel and Scherer, 2008) and 0.12 d1 for Methanosaeta (Conklin et al., 2006) and decrease considerably with decreasing temperature. Therefore, the growth can be controlled with a suitable sludge retention time (SRT). Chemical and OD analysis of the enrichments (Fig. 2) showed that after the initial lag of approximately 3 days (72 h), the culture entered a logarithmic growth phase, and was stationary after about 150 h. During the growth phase, 60% of the soluble organics measured as SCOD were removed. NH4eN and PO4eP concentrations followed the same trend. During the lag phase, 15.5% NH4eN and 15.2% PO4eP were released and this was then re-assimilated, with a further removal of a cumulative 30.5% and 26.8% by the end of the batch. Total COD increased slightly during the growth phase. Phototrophic bacteria can generate sufficient adenosine triphosphate (ATP) and reducing power during the anoxygenic photosynthesis and can assimilate the majority of the substrate directly into biomass, with little CO2 production (Van Niel, 1944). That is, catabolic metabolic energy is derived from light, with anabolic activity being mainly assimilative.
Fig. 1 e Enrichment colour change over time from t [ 27 h to t [ 190 h (A) and confocal laser scanning micrographs of an IR enriched PPB sample dual hybridized with EUB mix (FITC labelled) and a PARA 739 (Cy3 labelled) (B), RAMAN emission signal of PPB inoculum (red) and Rhodobacter sphaeroides signal (blue line) measured by Kniggendorf and MeinhardtWollweber (2011) (C). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 2 e SCOD (:), NH4eN (,), PO4eP (B) and Absorbance at 660 nm (OD660 (A)) trends over the enrichment period (n [ 4 ± standard deviation).
Fixing of COD is mainly due to reduction of substrates such as organic acids to match the carbon oxidation state of the biomass. SCODremoved/Nremoved/Premoved (SCOD/N/P) ratios of 100/22.6/2.9 (from 76 to 148 h) and 100/3.9/0.29 (from 148 to 170 h) were determined. The ratio in the period 76e148 h was determined during the log phase, which results in higher values compared to the ratio of the residual growth during the stationary phase (Fig. 2), meaning that more NH4eN and PO4eP are consumed (relative to SCOD uptake) in the log phase. The SCOD/N/P ratio from 0 to 76 h was negative i.e. NH4eN and PO4eP concentrations increased, most likely due to the mobilisation of organic N and P compounds in the wastewater. However, after the colour change (due to PPB growth) the NH4eN and PO4eP is consumed concurrently with the SCOD. The trends in Fig. 2 are consistent with COD, N, and P assimilation into biomass rather than accumulation of storage products. The pH after 266 h was on average 8.2, the increase being likely due to the removal of organic acids and phototrophic uptake of CO2.
3.2.
Batch experiments on settled wastewater
Fig. 3 shows the batch test results of COD (total and soluble), NH4eN, PO4eP using an inoculum from the enrichment culture described in the previous section. The maximum removal efficiencies for SCOD, NH4eN and PO4eP were on average (n ¼ 3); 64 14% after 120 h, 39 2.5% after 120 h and 21 2.8% after 24 h, respectively. The TCOD increased significantly (27 6% after 120 h) with increasing SCOD consumption. Nitrate remained below 1 mgN L1 during the test (data not shown). The control culture removed after 266 h on average (n ¼ 4) 50 6.5% SCOD, 0.1 1.8% TCOD, 30.5 2.7% NH4eN and 26.8 4.7% PO4eP. As described for the enrichment experiment, all of the control experiments turned reddish/ purple after 72 h indicating PPB growth (confirmed by FISH) with inoculation from native bacteria present in the wastewater. The SCOD removal of the wastewater was largely related to VFA removal, as the initial concentration of 184 mg COD L1 (as VFAs) was completely consumed within 15 h, representing
Fig. 3 e Average SCOD (:), TCOD (A), NH4eN (,), PO4eP (B) data (n [ 3 ± standard deviation) in batch tests with IR enriched inoculum.
approximately 60% of the soluble COD removed (data not shown). This is consistent with previous reports of rapid uptake of VFAs by PPB (Kim et al., 2004). The TCOD increased significantly over time, indicating that not only was SCOD assimilated (mainly VFA), but also a substantial amount (around 200 mg COD L1) was generated from phototrophic activity (Fig. 3). Some of the soluble COD was accumulated as PHA, and PHA could be detected 15 min after inoculation through Sudan black staining and counter staining with safranin (details in Supporting material). PPB are known to accumulate PHA (Ali Hassan et al., 1997). This rapid SCOD uptake and PHA accumulation may have caused the short term fluctuations in soluble COD seen in the first 4 h. However, longer term reduction of soluble COD, nitrogen and phosphorous due to assimilation rather than accumulation was observed. Raman spectroscopy could not be used to identify poly-P and PHA peaks due to a high background signal of the sample. Longer term NH4eN removal is directly linked to the SCOD assimilation. PPB contain around 60% of proteins (Kobayashi and Kobayashi, 1995). Phosphate can be accumulated as polyP (Hiraishi et al., 1991) but a staining with methylene blue did not show intracellular polyP storage, and PO4eP uptake appears to be mainly due to assimilation. The COD:N:P uptake ratios after 24 and 144 h assimilation were 100:5.6:0.6 and 100:6.2:0.3 (n ¼ 5) at an average sludge loading rate (SLR) of 0.78 and 0.17 gSCOD/gVSS/d and 0.27 gVSS L1, respectively.
3.3.
Use of IR selected PPB for wastewater BNR
In order to achieve effective wastewater treatment, the PPBbased process needs to attain nutrient concentrations comparable to Biological Nutrient Removal (BNR) plant effluent primarily through assimilation. To test these assimilation limits, batch experiments with organic acids added were used in preference to phototrophic assimilation, since (a) it is relatively simple to make more organic acids available from complex organics through pre-acidification, and (b) providing assimilation through light delivery is energetically inefficient unless light delivery efficiency is dramatically improved. Fig. 4 demonstrates the achieved assimilation limits with acetic
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acid as chemical electron source added (178 mg VFA-COD at t ¼ 0 h, 91 mg at t ¼ 4 h and 96 mg at t ¼ 8 h). The SCOD removal after 24 h reached 62.5 5.3%, while PO4eP was removed by 88.2% to 0.8 0.55 mgP L1 and NH4eN removal achieved 99.6% to 0.12 0.03 mgN L1. The average SCOD/N/P ratio from 8 to 24 h was 100/8.6/1.4. This ratio was consistent over these 24 h as well as in previous batch tests, which again indicates that the likely uptake mechanism is through assimilation rather than accumulation. The average SLR, from 4 to 24 h was 0.23 gSCOD/gVSS/d with a maximum of 0.47 gSCOD/gVSS/d after 12 h and 0.015 gNH4eN/gVSS/ d with a maximum of 0.033 gNH4eN/gVSS/d after 8 h. This compares with the conventional activated sludge process which usually achieves SLRs of 0.2e1.0 gCOD/gVSS/d and of 0.05e0.1 kgN/kgVSS/d (Tchobanoglous et al., 2003). Volumetric removal rates are shown in Table 2. The average SCOD removal rate was higher than typically found in extended aeration reactors, oxidation ditches or anaerobic ponds (all 0.2e0.6 kgCOD/m3/d (Tchobanoglous et al., 2003). It is comparable with conventional activated sludge and membrane bioreactor processes (1.2e3.2 kgCOD/m3/d) (Tchobanoglous et al., 2003). The growth rate based on OD660 measurements from 4 to 24 h was 0.013 h1 with a maximum rate of 0.063 h1 from 0 to 4 h. These growth rates are comparable with literature values for mixed phototrophic populations (Kaewsuk et al., 2010) and microalgae such as Chlorella. Higher growth rates have been reported for pure cultures of PPB; e.g., 0.217 h1 (Eroglu et al., 1999) and 0.296 h1 (Ponsano et al., 2008) for R. sphaeroides. These results demonstrate the ability of phototrophs to achieve very low effluent nitrogen concentrations, which is of key importance for wastewater treatment since phosphorous removal can be enhanced by accumulation or chemical precipitation. However, all batch tests showed that the assimilation of N and P depends on the amount of degradable COD, and particularly, VFA-COD present in the wastewater. When acetate was added, the COD/N/P ratio required to remove almost all NH4eN and PO4eP was found to be 100/8.6/1.5. The relatively linear slope for both ammonia and phosphate to <3 mg/L indicates that the substrate half-saturation constant KS values for these compounds are low, and performance is
Fig. 4 e Average SCOD (:), VFA-COD (A),NH4eN (,), PO4eP (B) concentrations treating wastewater over time and calculated HAc-VFA added (–A–) (n [ 4 ± standard deviation) in IR enriched cultures.
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not limited by affinity to these compounds (Fig. 4). This additional COD can be delivered either through wastewater fermentation, external carbon addition, or increased light intensity. The latter is of particular relevance for PPB since they are able to use infrared (IR) light. Compared to the use of incandescent lamps, IR light from light emitting diodes (LEDs) can save up to 70% of the power requirements (Bertling et al., 2006) since the quantum efficiency of IR-LEDs can be close to 100% for emission wavelengths around 650 nm and higher. However this cannot fully compensate for the reported low light conversion efficiency of 6e8% (defined as combustion energy of hydrogen gas produced/incident light energy) for R. sphaeroides (Miyake and Kawamura, 1987). Therefore, the best way to enhance nitrogen removal to very low limits is through pre-acidification of the wastewater, possibly combined with internal carbon recycling to optimise the use of carbon for assimilation. Failing this, however, the use of PPB does provide the option of either delivering electrons through external carbon addition, or through increased light (and therefore power) input. Compared to denitrification, the stoichiometric COD:N ratio for the enriched cultures assessed here is approximately two times higher (COD:N ratio for denitrification including sludge production is 4 (Gustavsson, 2010)). However, denitrification destroys the nitrogen and COD whereas assimilation preserves nitrogen as well as the COD in the produced biomass.
3.4. Significance and application to the wastewater industry The application of IR selected phototrophs (PPB) for the removal of degradable COD, NH4eN and PO4eP from domestic or industrial wastewater achieves both key criteria of reproducible selection and enrichment of the suitable biocatalyst (through infrared light), as well as the ability to remove the major nutrients in a single-step process to achieve low effluent concentrations. A treatment process utilising phototrophic bacteria has a number of advantages over algae. The selection can be done at lower energy consumption, because PPBs can efficiently utilise wavelengths in the IR spectrum. This allows to limit the growth of non-desired organisms (e.g., algae and cyanobacteria) based on light delivery. Both can assimilate N and P as proteins and other organic biomass compounds. It is generally accepted that closed photo-bioreactors achieve higher removal rates but are more expensive to build and operate compared to open systems (Posten, 2009). PPB do not require CO2 addition and are not inhibited by O2 production, which are typically the most limiting factors in phototrophic systems, especially in closed reactors (Mun˜oz and Guieysse, 2006). This represents an opportunity to apply PPB particularly for highrate closed cell reactors in comparison with open and closed cell systems for algae, with delivery of only sufficient light to remove residual nitrogen. Assimilative partitioning of wastewater macronutrients and organics in one solids stream (i.e. biomass) that can be digested in a dedicated process to generate biogas, as well as a concentrated nitrogen and phosphorous stream might be a major advantage for product recovery. As an alternative, many PPB (as well as algae) contain high amounts of protein
24
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Table 2 e Volumetric removal rates for SCOD, NH4eN and PO4eP. Parameter SCOD NH4eN PO4eP
Unit
MIN
MAX
AVG
kgCOD/m3/d kgN/m3/d kgP/m3/d
0.231 (20e24 h) 0.027 (20e24 h) 0.007 (20e24 h)
2.629 (8e12 h) 0.305 (4e8 h) 0.045 (4e8 h)
1.658 (4e20 h) 0.183 (4e20 h) 0.024 (4e20 h)
(>60%) and were reported to be suitable as animal food (Sasaki et al., 1991) and organic fertilizer (Xu, 2001). A key technical challenge is the reactor design. This requires optimal light delivery to compensate for the relatively low photoconversion efficiency of 6e8% reported previously for R. sphaeroides (Miyake and Kawamura, 1987), which is, however, still higher than typically achieved values of <5% photoconversion efficiency from full sunlight spectra for microalgae (Posten, 2009). Interactions between fluid dynamics, biochemical reaction, and light transfer in photobioreactors are crucial (Posten, 2009) and considerable progress has been made in the last decade (Hankamer et al., 2007). A photo-anaerobic membrane-bioreactor (PAnMBR) is a possible process configuration. Compared to a conventional anaerobic membrane bioreactor (AnMBR) this reactor type will consume additional energy for the continuous IR light supply (for PPB selection). This extra consumption can be balanced with the efficient nutrient removal and recovery potential, and hence avoiding aerobic nitrogen removal. Phosphate removal can also be achieved by assimilative growth instead of biological accumulation or chemical precipitation. Continuous operation allows for optimisation options that were not available in the batch experiments, and is likely to further increase the applicability of this technology for practical wastewater treatment. The impact of pre-acidification, use of recycle streams, application of illuminated sections for selection and polishing, and recovery of organic carbon to remove residual nutrients will need to be determined in continuous processes. A major advantage in this context is the fact that the basic process platform has a high degree of flexibility due to the provision of additional control handles in the form of light and organic carbon dosing.
4.
Conclusions
The use of IR selected phototrophic purple bacteria offers potential as an alternative microbial agent for partitioning of nutrients and organic carbon to the solid (biomass) phase for subsequent recovery, with a number of key elements shown in this paper. These include: - Reliable enrichment of the target microbes from wastewater directly through application of infra-red light, showing that culture selection can be directed by this method with short start-up times. - Good removal of carbon and nutrients (N & P) present in wastewater. - Proof of achievable nutrient removal to meet discharge limits (N, P < 1.0 mg/L).
- Ability to supply supplementary electrons either by light or chemicals (e.g., acetate). The outcomes from the current work provide a proof-ofconcept demonstration of the suitability of PPB for wastewater treatment. However there are a number of technical challenges remaining, including verifying that nutrients can be effectively released, and determining optimal configurations for reactor design.
Acknowledgements We gratefully acknowledge Dr Beatrice Keller, Ms Jianguang Li and Nathan Clayton for assistance with FIA and GC/FID measurements. We also thank Dr Bogdan Donose for assisting with the Raman spectroscopy. This work was jointly funded by the Smart Water Fund (project 10OS-023) and the CRC for Water Sensitive Cities (project C2.1).
Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.watres.2013.10.051.
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