Abstract

A novel coupling process using an aerobic bacterial reactor with nitrification and sulfur–oxidization functions followed by a microalgal reactor was proposed for simultaneous biogas desulfurization and anaerobic digestion effluent (ADE) treatment. ADE nitrified by bacteria has a potential to be directly used as a culture medium for microalgae because ammonium nitrogen, including inhibitory free ammonia (NH3), has been converted to harmless NO3. To demonstrate this hypothesis, Chlorella sorokiniana NIES-2173, which has ordinary NH3 tolerance; that is, 1.6 mM of EC50 compared with other species, was cultivated using untreated/treated ADE. Compared with the use of a synthetic medium, when using ADE with 1–10-fold dilutions, the specific growth rate and growth yield maximally decreased by 44% and 88%, respectively. In contrast, the algal growth using undiluted ADE treated by nitrification–desulfurization was almost the same as with using synthetic medium. It was also revealed that 50% of PO43− and most metal concentrations of ADE decreased following nitrification–desulfurization treatment. Moreover, upon NaOH addition for pH adjustment, the salinity increased to 0.66%. The decrease in metals mitigates the bioconcentration of toxic heavy metals from wastewater in microalgal biomass. Meanwhile, salt stress in microalgae and limiting nutrient supplementation, particularly for continuous cultivation, should be of concern.

INTRODUCTION

Anaerobic digestion (AD), which generates biogas by anaerobic microbes, is an economical and environmentally friendly method for the treatment of organic waste/wastewater. However, generated biogas contains hydrogen sulfide (H2S), which is both highly toxic and corrosive, and biogas desulfurization treatment by physicochemical or biological methods is necessary. AD effluent (ADE), which contains a high concentration of nutrients, is generally treated using the nitrification–denitrification process, which involves mechanical aeration and a supply of organic carbon (Fux & Siegrist 2004). The cost and environmental loads of these post-treatments have been a bottleneck for the further spread of AD technology. Mass cultivation of microalgae using ADE has been intensively studied as a possible alternative to conventional ADE treatments (de Godos et al. 2009; Kumar et al. 2010; Wang et al. 2010; Lee et al. 2015; González-Camejo et al. 2018). This is because the commercialization of microalgal products for purposes such as animal feeds, dietary supplements, and cosmetics is increasing, and microalgae production has the potential to be highly profitable. Both pilot- and full-scale microalgal cultivation facilities using ADE have already been applied for various ADE treatments. For example, Arthrospira sp. was cultivated with pig-waste ADE in a 23.6-m2 raceway pond with 84%–91% NH4+-N and 84%–87% PO43−-P removal (Olguín et al. 2003). ADE from sago starch factory wastewater was treated in a 0.71-m2 high-rate algal pond (HRAP), and Arthrospira platensis was produced with 99% NH4+-N removal (Phang et al. 2000). Park & Craggs (2011) treated ADE derived from a wastewater sludge treatment facility with a microalgal–bacterial consortium using HRAP with a supply of CO2. They achieved 24.7 g VSS m−2 d−1 of high biomass productivity and a maximum removal of 84.5% NH4+, and then it was scaled up to a 5-ha full-scale plant in New Zealand (Craggs et al. 2014). However, with ADE treatment using microalgae, the need to dilute the substrate with a large amount of freshwater has been a serious issue. ADE usually contains 1,000–3,000 mg N L−1 of total ammonium nitrogen (TAN) (Xia & Murphy 2016), which can take two forms, free ammonia (NH3) and ammonium ions (NH4+), depending on the pH, as follows:  
formula
(1)

Since NH3 severely inhibits microalgae growth, most high efficiency treatments of ADE, including the ones mentioned above, have been achieved by using a 2- to 50-fold dilution of the substrate (Olguín et al. 2003; de Godos et al. 2009; Kumar et al. 2010; Wang et al. 2010; Park & Craggs 2011; Lee et al. 2015; Xia & Murphy 2016), requiring large amounts of freshwater to be consumed. As an alternative, although we can use other water sources, such as seawater (Sepúlveda et al. 2015) and other wastewater with low TAN concentration (e.g. domestic wastewater (Dickinson et al. 2015) and secondary effluent (Bohutskyi et al. 2016)), the areas near other wastewater treatment facilities were limited and the composition of wastewater must be carefully considered to avoid harmful contamination, particularly for advanced use of microalgae. Using seawater is suitable for seawater species but limits the application of freshwater species.

TAN can be oxidized via nitrification by nitrifying bacteria, producing NO3, which is harmless to microalgae. It is expected that the conversion of TAN to NO3 via biological nitrification would enable the direct use of ADE in microalgal cultivation without requiring dilution. Furthermore, the optimal growth conditions for nitrifying bacteria; that is, a temperature of less than 30 °C, neutral pH, and aerobic conditions, are similar to those for sulfur-oxidizing bacteria, which can desulfurize biogas (Antoniou et al. 1990; Muñoz et al. 2015). Therefore, there is the possibility that ADE nitrification could be simultaneously combined with biogas desulfurization in a single reactor (Figure 1). In this aerobic bacterial reactor, H2S in biogas and TAN in ADE are oxidized to harmless SO42− and NO3 by sulfur-oxidizing bacteria and nitrifying bacteria, respectively, using O2 produced in the algal reactor. Thus, in this novel coupling process, microalgae are cultivated using undiluted ADE that has been treated by the nitrification–desulfurization process.

Figure 1

A proposed coupling process of biogas desulfurization and effluent nutrients removal after anaerobic digestion using aerobic bacterial reactor and microalgal reactor.

Figure 1

A proposed coupling process of biogas desulfurization and effluent nutrients removal after anaerobic digestion using aerobic bacterial reactor and microalgal reactor.

In our previous study, we confirmed in a laboratory-scale experiment that nitrification and desulfurization can be stably performed in a single reactor (Sekine et al. 2020). However, microalgae productivity when using ADE treated by nitrification–desulfurization remains unknown. Praveen et al. (2018) attempted to cultivate microalgae using nitrified ADE and obtained 97% nitrogen removal efficiency. However, they treated ADE after diluting it 3–20-fold with municipal wastewater; therefore, the suitability of nitrified, undiluted ADE for microalgae cultivation remains to be demonstrated. High concentrations of digestate components other than NH3 can inhibit microalgal growth under conditions without dilution. Moreover, there is the possibility that concentrations of some components associated with microalgal growth change through nitrification–desulfurization, causing inhibition or improvement of microalgal growth. Therefore, to evaluate the suitability of the proposed coupling process, the present study was conducted with a focus on microalgae cultivation using ADE treated by nitrification–desulfurization. Chlorella sorokiniana NIES-2173 was used as a target species in this research because the genus Chlorella is a typical green microalgae that breeds in lakes, rivers, and ponds and has been widely studied and used. First, the NH3 tolerance of the microalgae Chlorella sorokiniana NIES-2173 was evaluated as a preparatory experiment to understand the nitrogen utilization characteristics of this species. Second, a comprehensive characterization of ADE treated by nitrification–desulfurization was conducted. Third, the productivity of C. sorokiniana using ADE treated by nitrification–desulfurization was investigated compared with using untreated ADE with/without dilution.

METHODS

Microorganism and preculture condition

The green algae C. sorokiniana NIES-2173 was obtained from the National Institute for Environmental Studies, Tsukuba, Japan, and pre-cultivated in C-medium with 0.6-g C L−1 of NaHCO3 at 25 °C under 150 μmol photons m−2 s−1 continuous light illumination. The composition of C-medium (per liter) was as follows (Ichimura 1971): 500-mg Tris (hydroxymethyl) aminomethane, 150-mg Ca(NO3)2·4H2O, 100-mg KNO3, 50-mg β-Na2 glycerophosphate·5H2O, 40-mg MgSO4·7H2O, 100-μg vitamin B12, 100-μg biotin, 10-mg thiamine HCl, and 3-mL PIV trace metals solution (Provasoli & Pintner 1959; per liter): 1-g Na2 EDTA·2H2O, 200-mg FeCl3·6H2O, 36-mg MnCl2·4H2O, 10.4-mg ZnCl2, 4-mg CoCl2·6H2O, 2.5-mg NaMoO4·2H2O. The concentrations of NO3, PO43−, and SO42− calculated based on the composition of this medium were 32 mg N L−1, 7 mg P L−1, and 5 mg S L−1, respectively, and the N/P molar ratio was 10. Before and after the addition of NaHCO3, the salinity was 0.02% and 0.22%, respectively.

NH3 tolerance of Chlorella sorokiniana

C. sorokiniana was cultivated under different NH3 concentrations (0–3.6 mM). Cells in the exponential growth stage were used as the inoculum. C-medium was used after the addition of 0.6-g C L−1 NaHCO3 and 941-mg N L−1 (67 mM) nitrogen with different ratios of KNO3 and NH4Cl (NO3-N/NH4+-N: 67/0, 50/17, 34/34, 17/50, and 0/67 mM). The pH was adjusted to 7.99 ± 0.09 by adding 4.8 g L−1 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and NaOH solution. These TAN concentrations and pH values resulted in NH3 concentrations of 0, 0.9, 1.8, 2.7, and 3.6 mM (a total of five conditions, with three to four replicates). After the pH was adjusted, each medium was sterilized by filtration using a 0.22-μm pore size syringe-driven filter unit (Millex GV; Merck Millipore, USA). Twelve-well microplates were used as the cultivation vessels, with 2 mL medium. The initial optical density at 750 nm (OD750) was adjusted to 0.021 ± 0.001. The temperature was maintained at 25 °C ± 1 °C and the illumination was set to 150-μmol photons m−2 s−1. During the experimental period, the OD750 was periodically measured after mixing the culture by pipetting. The pH value and the concentrations of TAN, NO3, and NO2 were measured before and after cultivation.

Nitrification–desulfurization treatment of ADE

The centrifuged liquid fraction of ADE and nitrifying sludge were obtained from a mesophilic AD reactor treating sewage sludge and an anaerobic–anoxic–aerobic (A2O) bioreactor treating ADE, respectively, in the Hokubu Sludge Treatment Center, Yokohama, Japan. A sequencing batch reactor with an effective volume of 2.1 L was used as the nitrification–desulfurization reactor for the nitrifying sludge. The reactor was operated with a 24-h cycle consisting of a filling and reaction phase (23.5 h), a sedimentation phase (20 min), and a decanting phase (10 min). The hydraulic retention time was adjusted to 3 days. ADE was supplied with NaHS solution to imitate biogas desulfurization. The TAN concentration of the influent was 870 mg N L−1. S2− concentration was adjusted to 384 mg S L−1, after increasing in a stepwise manner from 0 to 96, and 192 mg S L−1 to allow the microbes to acclimatize to S2− based on the assumption of 1:70 of ADE and biogas production volume ratio in AD and 3,000 ppm of H2S concentration in biogas. The pH was adjusted to 7.5 by the addition of NaOH using a process controller (EYALA EPC-2000; Tokyo Rika, Japan). The temperature and agitation rate were maintained at 30 °C ± 1 °C and 200 rpm, respectively, using a water bath and a magnetic stirrer. The dissolved oxygen concentration was maintained at 1.5–4.0 mg O2 L−1 by aeration through two air stones. As a result of sequential batch operation, 170 ± 26 days of long sludge retention time were maintained throughout the operation period. Further details of the reactor operation were previously described in Sekine et al. (2020). In the 384 mg S L−1 of S2− concentration phase, the influent and effluent of the reactor were filtered through a 0.45-μm pore size glass fiber filter (GC-50; Advantec, Japan) and stored in a freezer at −30 °C until use for the composition analysis and the next experiment (microalgal cultivation). For the composition analysis of the influent and effluent, the concentrations of phosphate and dissolved metals and the salinity were determined. Additionally, previously reported values for nitrogen, sulfur, and dissolved organic compound (DOC) concentrations in the influent and effluent (Sekine et al. 2020) were used for analysis.

Microalgae cultivation using ADE treated by nitrification–desulfurization

C. sorokiniana was cultivated using different media: C-medium, untreated ADE with a 1-, 3-, 6-, or 10-fold dilution, and ADE treated by nitrification–desulfurization (a total of six conditions performed in triplicate). Cells in the exponential growth stage were used as the inoculum. The influent and the effluent of the nitrification–desulfurization reactor were used as the medium for the conditions of untreated ADE and ADE treated by nitrification–desulfurization, respectively, after addition of MgSO4·7H2O and PIV metals solution adjusted to the same concentration with C-medium. Each medium was supplemented with 0.6-g C L−1 NaHCO3 and the pH was adjusted to 7.5 by adding HCl solution; the solutions were then filtered using a 0.22-μm pore size syringe-driven filter unit (Millex GV; Merck Millipore, USA). For microalgae cultivation, 10-mL glass vials with silicone stoppers were used with 5 mL medium. Temperature and light intensity were maintained at 25 °C ± 1 °C and 150 μmol photons m−2 s−1, respectively. The cultures were mixed thoroughly by inversion every 12 h, and the OD750 was measured each time. The silicone stopper was removed every 24 h to allow air exchange. The pH value and nutrient (TAN, NO3, NO2, PO43−) concentrations were determined before and after cultivation.

Measurements and calculations

The OD750 was measured using a microplate reader (EPOCH 2; BioTek, USA) and a spectrophotometer (DR2800; HACH, USA) for the NH3 tolerance test and the other experiments, respectively. The pH was measured using a compact pH meter (LAQUAtwin-pH-22B; Horiba, Japan). Nutrient (TAN, NO3, NO2, and PO43−) concentrations were measured using a high-performance liquid chromatography system with a conductometric detector (CDD-10AVP; Shimadzu, Japan) and two types of columns (an IC YS-50 column for cation analysis and an IC I-524A column for anion analysis; Showa Denko, Japan). Dissolved metals and salinity were determined by inductively coupled plasma spectroscopy (ICPS-7000ver2.1; Shimadzu, Japan) and a digital salt meter (Conductivity Method, ES-421; Aatago, Japan), respectively.

The specific growth rate of C. sorokiniana was calculated using Equation (2).  
formula
(2)
where μ is specific growth rate (d−1) and x1 and x2 are the OD750 at time t1 and t2 (d), respectively. The NH3 ratio in TAN (%) was calculated using Equation (3) (Anthonisen et al. 1976):  
formula
(3)
where T is the temperature (K). The half-maximal effective concentration (EC50) of NH3 on microalgae was determined by fitting microalgal specific growth rates at the different NH3 concentrations to the following four-parameter logistic curve-regression using SigmaPlot 11.0 software (Systat Software, UK):  
formula
(4)
where min is the bottom of curve, max is the top of curve, and Hillslope is the slope of the curve at its midpoint.

RESULTS AND DISCUSSION

NH3 tolerance of Chlorella sorokiniana

After 56-h cultivation, the OD750 of the conditions with 0-, 0.9-, 1.8-, 2.7-, and 3.6-mM initial NH3 concentration conditions tended to be lower with higher initial NH3 concentration conditions, from 0.097 ± 0.018 to 0.030 ± 0.021 (Figure 2, Table 1). The pH increased to a maximum of 9.47 based on the level of microalgal growth with inorganic carbon consumption. The TAN concentrations at the end of the cultivation were decreased by 21%–78% in the 0.9-, 1.8-, 2.7-, and 3.6-mM initial NH3 concentration conditions. The main reason for TAN reduction was considered to be NH3 volatilization because the NO3 concentration was almost unchanged in all conditions, including the condition using only NO3 where we obtained the highest microalgal growth, indicating that no nitrification had occurred and the added nitrogen was almost not assimilated into microalgal biomass. The specific growth rates calculated from the OD750 between 8 and 24 h of the logarithmic growth period were 1.85 ± 0.22, 1.32 ± 0.53, 0.77 ± 0.37, −0.14 ± 0.37, and −0.16 ± 0.28 d−1 with the initial NH3 concentrations of 0, 0.9, 1.8, 2.7, and 3.6 mM, respectively (Table 1); that is, there was a decreasing trend with increasing initial NH3 concentration. NH3 strongly inhibits the growth of microalgae via two main mechanisms that lead to the breakdown of photosystem II (PSII) function: (1) by diminishing the proton gradient across the thylakoid membrane and suppressing adenosine triphosphate (ATP) production, which is necessary for repairing photodamaged PSII (McCarty 1969; Gutierrez et al. 2016); and (2) by ligation to the organo-metallic reactor core in the D1 subunit of PSII, which is essential for its correct functioning (Britt et al. 2004; Gutierrez et al. 2016). These inhibitory mechanisms associated with NH3 might have led to the decreased growth of C. sorokiniana NIES-2173 we observed.

Table 1

Specific growth rate and final optical density (OD750) of Chlorella sorokiniana NIES-2173 and cultivation conditions using different NH3 concentrations

Initial NH3 [TAN] concentration (mM)Specific growth ratea (d−1)Final OD750Final pHTAN reduction (%)Final TAN concentration (mM)
0 [0] 1.85 ± 0.22 0.10 ± 0.02 9.47 – – 
0.9 [17] 1.32 ± 0.53 0.13 ± 0.02 9.21 78 3.7 ± 1.2 
1.8 [34] 0.77 ± 0.37 0.08 ± 0.02 8.63 69 10.5 ± 3.6 
2.7 [50] −0.14 ± 0.37 0.03 ± 0.01 8.01 44 28.2 ± 3.5 
3.6 [67] −0.16 ± 0.28 0.03 ± 0.02 7.93 21 53.2 ± 1.2 
Initial NH3 [TAN] concentration (mM)Specific growth ratea (d−1)Final OD750Final pHTAN reduction (%)Final TAN concentration (mM)
0 [0] 1.85 ± 0.22 0.10 ± 0.02 9.47 – – 
0.9 [17] 1.32 ± 0.53 0.13 ± 0.02 9.21 78 3.7 ± 1.2 
1.8 [34] 0.77 ± 0.37 0.08 ± 0.02 8.63 69 10.5 ± 3.6 
2.7 [50] −0.14 ± 0.37 0.03 ± 0.01 8.01 44 28.2 ± 3.5 
3.6 [67] −0.16 ± 0.28 0.03 ± 0.02 7.93 21 53.2 ± 1.2 

aCalculated from the OD750 between 8 and 24 hours of the logarithmic growth period.

Figure 2

Time course of the optical density at 750 nm (OD750) under different initial NH3 concentration: 0 mM (♦), 0.9 mM (▪), 1.8 mM (▴), 2.7 mM (□), and 3.6 mM (×).

Figure 2

Time course of the optical density at 750 nm (OD750) under different initial NH3 concentration: 0 mM (♦), 0.9 mM (▪), 1.8 mM (▴), 2.7 mM (□), and 3.6 mM (×).

The correct functioning of PSII tends to be maintained until the NH3 concentration exceeds a certain concentration and the above-mentioned mechanisms take effect, therefore the NH3 inhibition level can be easily fitted to a sigmoid regression curve to obtain the EC50 (Azov & Goldman 1982). Based on this fitting regression, the EC50 of NH3 for the C. sorokiniana NIES-2173 used in the present study was 1.6 mM (p < 0.01, Figure S1, Supplementary Material). This value falls within the range of EC50 (from 0.003 to 3.3 mM) for other species, both reported by Collos & Harrison (2014) and calculated from other studies (Figure S1; Azov & Goldman 1982; Belkin & Boussiba 1991; Tan et al. 2016). Therefore, it is reasonable to use C. sorokiniana NIES-2173 as a representative species for the validation of nitrification–desulfurization treatments to avoid NH3 inhibition.

Composition of ADE treated by nitrification–desulfurization

Table 2 shows the composition of the influent and effluent of the nitrification–desulfurization reactor under a supply of 384 mg S L−1 of S2−. TAN was completely converted to NO3; S2− was removed at 100% efficiency, 40% of which was converted to SO42−, leading to an increase in SO42− concentration from 25 mg S L−1 to 183 mg S L−1. H2S was not detected from the exhaust gas, and white precipitates were observed attached to the reactor; therefore, a part of the removed S2− was probably converted to elemental sulfur (Sekine et al. 2020). The concentration of SO42− tends to be between 0.3 and 1.6 mg S L−1 in the soil/freshwater environments where microalgae grow (Bochenek et al. 2013), and between 3 and 160 mg S L−1 in microalgal synthetic medium (Mera et al. 2016), therefore the SO42− concentrations of both influent and effluent were probably sufficient for microalgal cultivation. SO42− does not inhibit the freshwater microalgae Chlamydomonas moewusii, even at concentrations of more than 600 mg S L−1 (Mera et al. 2016). The phosphate concentration of the effluent fluctuated mainly in the range of 15 to 101 mg P L−1, with an average of 58 mg L−1, and tended to be lower than the average influent concentration of 119 ± 11 mg P L−1. Microbes need to assimilate phosphorus into their cells to grow. Moreover, phosphate can easily precipitate in the presence of some components that exist in ADE, such as TAN, Mg, Al, Ca, and Fe, particularly under alkaline pH conditions. Al, Ca, and Fe were also removed with more than 50% efficiency in this experiment, as described below. The precipitation with these components possibly contributed to the reduction in the phosphate content of the effluent. In particular, Ca, which does not precipitate with sulfide, probably affected PO43− precipitation. The molar ratio of N/P in the treated ADE was 53 ± 42, which was higher than the ratio in the untreated ADE (15 ± 0.7). Microalgae can live in freshwater environments with a wide range of N/P molar ratios, from 8 to 45, by changing their cellular composition according to the surrounding nutrients in their environment, but a high enough level of phosphorus (N/P < 22) is required to maintain a high growth rate (Hecky et al. 1993; Beuckels et al. 2015). There is a possibility that the treatment under pH lower than 7.5 can suppress PO43− precipitation; however, lower pH can promote sulfide inhibition in nitrification because the portion of H2S inhibiting microbes more than HS or S2− increased under lower pH. Therefore, especially with continuous algal cultivation, it may be necessary to add phosphate to ADE that has been treated with nitrification–desulfurization to adjust the N/P molar ratio to <22.

Table 2

Nitrogen, sulfur, and other compounds' concentrations of anaerobic digestate before and after desulfurization-nitrification treatment

ElementsInfluentEffluent
TAN-N (mg L−1856 ± 12 n.d. 
NO2-N (mg L−1n.d. n.d. 
NO3-N (mg L−1n.d. 847 ± 54 
S2−-S (mg L−1384a n.d. 
S2O32−-S (mg L−1n.d. n.d. 
SO42−-S (mg L−125 ± 1 183 ± 34b 
PO43−-P (mg L−1119 ± 11 58 ± 38b 
DOC (mg L−163 ± 1 81 ± 1d 
Salinity (%) 0.35 ± 0.06 0.66 ± 0.04c 
ElementsInfluentEffluent
TAN-N (mg L−1856 ± 12 n.d. 
NO2-N (mg L−1n.d. n.d. 
NO3-N (mg L−1n.d. 847 ± 54 
S2−-S (mg L−1384a n.d. 
S2O32−-S (mg L−1n.d. n.d. 
SO42−-S (mg L−125 ± 1 183 ± 34b 
PO43−-P (mg L−1119 ± 11 58 ± 38b 
DOC (mg L−163 ± 1 81 ± 1d 
Salinity (%) 0.35 ± 0.06 0.66 ± 0.04c 

TAN, Total ammonium nitrogen; DOC, Dissolved organic carbon; n.d., not detected.

aCalculated from supplied solution concentration. Student's t-test was conducted on SO42−, PO43− and DOC concentrations and salinity of influent and effluent (n ≥ 3).

bp < 0.05.

cp < 0.005.

dp < 0.001.

The concentration of most metals decreased in the nitrification–desulfurization reactor (Table 3). Aerobic sludge, such as activated sludge and nitrifying sludge, has the potential to remove metals both through its assimilation into cells and by sorption to flocs (Üstün 2009). Additionally, several metals (Al, Mn, Zn, Fe, Ni, Cd, Sn, Pb, Cu, Hg, Ag, Pt, and Au) can easily precipitate in the presence of S2− (Lewis 2010). Precipitation with phosphorous was also considered because PO43− concentration decreased at the same time. Therefore, these metals were probably taken into bacteria and precipitated with S2− and PO43−. A nitrification–desulfurization reactor can decrease the effects of inhibitory metals (Al, Co, Ni, Cu, Cd, and Pb) in microalgal cultivation, although the addition of some essential metals (Mg, K, Ca, Fe, Mn, and Zn) may be necessary if they are present in insufficient quantities. Furthermore, from a commercial perspective, the accumulation of heavy metals (which are toxic to humans) in microalgae cultivated using ADE should be avoided to realize the utilization of microalgae to high-added-value products, such as feed and food supplements (Chamorro-Cevallos & Barrón 2007). There is a report of toxic heavy metals being detected in Arthrospira platensis, at a higher concentration than the upper limit of the acceptable range for human intake, following the contamination of a cultivation pond with environmental pollutants (Boudene et al. 1975). Removal of these toxic metals by a nitrification–desulfurization reactor may mitigate the accumulation of these metals in microalgae.

Table 3

Metals concentrations of anaerobic digestate before and after desulfurization-nitrification treatment

Essential metals for microalgae
Inhibitory metals for microalgae
MetalConcentration (mg L−1)
Change (%)MetalConcentration (mg L−1)
Change (%)
InfluentEffluentInfluentEffluent
Mg 3.2 2.9 −10.5 Al 0.35 0.18 −50.1 
31.2 39.5 26.6 Co 0.16 0.04 −73.8 
Ca 19.4 5.3 −72.5 Ni 0.29 0.19 −34.4 
Fe 0.34 0.10 −70.6 Cu 0.44 0.13 −70.7 
Mna 0.08 0.02 −71.4 Cd 0.19 0.07 −66.3 
Zna 0.19 0.06 −65.4 Pb 0.75 0.15 −80.5 
Essential metals for microalgae
Inhibitory metals for microalgae
MetalConcentration (mg L−1)
Change (%)MetalConcentration (mg L−1)
Change (%)
InfluentEffluentInfluentEffluent
Mg 3.2 2.9 −10.5 Al 0.35 0.18 −50.1 
31.2 39.5 26.6 Co 0.16 0.04 −73.8 
Ca 19.4 5.3 −72.5 Ni 0.29 0.19 −34.4 
Fe 0.34 0.10 −70.6 Cu 0.44 0.13 −70.7 
Mna 0.08 0.02 −71.4 Cd 0.19 0.07 −66.3 
Zna 0.19 0.06 −65.4 Pb 0.75 0.15 −80.5 

aMetals which inhibit microalgae under high concentration.

The salinity of ADE increased from 0.35% to 0.66% following nitrification–desulfurization, because NaOH was added to increase the reactor pH as it was decreased by nitrification and desulfurization. It has been reported that the final biomass concentration of Chlorella vulgaris decreased by approximately 30%, even under 0.3% salinity, in batch cultivation for 24 days (Kebeish et al. 2014). This increase in salinity may negatively affect microalgal growth depending on the salinity tolerance of the algal strains used.

Microalgae cultivation using ADE treated by nitrification–desulfurization

Batch cultivation of C. sorokiniana NIES-2173 was conducted using C-medium, untreated ADE with 1-, 3-, 6- and 10-fold dilutions, and ADE treated by nitrification–desulfurization (Figure 3). The specific growth rates within the first two days were very similar (0.43–0.57 d−1) for all conditions except untreated ADE without dilution (0.32 d−1) (Table 4). The highest initial NH3 concentration (0.91 mM) of untreated ADE without dilution decreased the specific growth rate by 44% compared with the C-medium. Therefore, ADE treated using nitrification–desulfurization is effective for use in microalgal cultivation without dilution. In this connection, although TAN generally improves microalgal growth rate more than NO3 because energy requirement for TAN assimilation is lower than for NO3 assimilation (Sanz-Luque et al. 2015), it appeared that the specific growth rate was not affected by differences in nitrogen sources (TAN of untreated and diluted ADE and NO3 of C-medium and treated ADE). There is a possibility that increasing contaminant concentrations (e.g. DOC, SO42−) or decreasing toxicity of metals via nitrification–desulfurization treatment improved microalgal growth, apart from the suppression of NH3 inhibition. However, the observed negligible effect of salinity might differ if other microalgal species were to be used. The reported salinity tolerance of Chlorella spp. and Chlorococcum spp. that are commonly found in terrestrial habitats (EC50: 0.9%–5%) is higher than that of other freshwater microalgae (EC50: 0.3%–1.8%) (Kim et al. 2016; von Alvensleben et al. 2016; Figler et al. 2019). Therefore, for microalgal cultivation using ADE treated by nitrification–desulfurization without dilution, it may be necessary to select species which exhibit a high salinity tolerance.

Table 4

Specific growth rate and final optical density (OD750) of Chlorella sorokiniana NIES-2173 and cultivation conditions using different mediums

MediumSpecific growth rate (d−1)Final OD750Final pHNH3 concentration (mM)
InitialFinal
C-medium 0.57 ± 0.16 0.71 ± 0.01 10.5 
Untreated anaerobic digestion effluent (ADE) Without dilution 0.32 ± 0.04a 0.08 ± 0.01b 8.9 0.85 13.2 
3-fold dilution 0.43 ± 0.05 0.23 ± 0.02b 9.3 0.35 7.15 
6-fold dilution 0.45 ± 0.11 0.26 ± 0.03b 9.6 0.16 3.40 
10-fold dilution 0.47 ± 0.04 0.43 ± 0.03b 10.1 0.08 2.12 
Treated ADE 0.54 ± 0.09 0.71 ± 0.02 11.2 
MediumSpecific growth rate (d−1)Final OD750Final pHNH3 concentration (mM)
InitialFinal
C-medium 0.57 ± 0.16 0.71 ± 0.01 10.5 
Untreated anaerobic digestion effluent (ADE) Without dilution 0.32 ± 0.04a 0.08 ± 0.01b 8.9 0.85 13.2 
3-fold dilution 0.43 ± 0.05 0.23 ± 0.02b 9.3 0.35 7.15 
6-fold dilution 0.45 ± 0.11 0.26 ± 0.03b 9.6 0.16 3.40 
10-fold dilution 0.47 ± 0.04 0.43 ± 0.03b 10.1 0.08 2.12 
Treated ADE 0.54 ± 0.09 0.71 ± 0.02 11.2 

Dunnett's test was conducted on specific growth rate and final OD750 values (n = 3) assuming C-medium condition was the control.

aSignificantly different at the 5% level.

bSignificantly different at the 1% level.

Figure 3

Time course of the optical density at 750 nm (OD750) under using C-medium (▪), 1- (without), 3-, 6-, or 10-fold diluted untreated anaerobic digestion effluent (ADE) (▪, •, ▴, ×), and ADE treated by desulfurization-nitrification (□).

Figure 3

Time course of the optical density at 750 nm (OD750) under using C-medium (▪), 1- (without), 3-, 6-, or 10-fold diluted untreated anaerobic digestion effluent (ADE) (▪, •, ▴, ×), and ADE treated by desulfurization-nitrification (□).

In all conditions using untreated ADE, the microalgal growth rate, which was initially almost the same as with the other medium conditions, decreased gradually throughout the cultivation period, with a final OD750 that was 40% to 88% lower than that in conditions that used C-medium (Figure 3). During the cultivation period, the pH increased to 8.9–11.2 in all conditions (Table 4). This increase in pH increased the NH3 concentration of untreated ADE from 0.08–0.85 mM to 2.12–13.2 mM (Table 4) and inhibited microalgal growth. However, in the condition using ADE treated using nitrification–desulfurization, the same final OD750 of that in C-medium condition was achieved.

At the end of the experiment, both nitrogen and phosphate remained, in all conditions, more than 20.0-mg N L−1 and 3.5-mg P L−1, respectively (Figure 4). The growth of C. sorokiniana in conditions using C-medium and treated ADE was probably suppressed by light limitation, increased pH, and/or lack of dissolved inorganic carbon, trace metals, and other micronutrients, rather than by nitrogen or phosphate limitation. In this batch cultivation, TAN volatilization can be reduced by using a glass tube with a silicone stopper as a cultivation vessel compared with the NH3 tolerance assay using microplates, which resulted in a loss of 21%–78% TAN. However, in the case of conventional microalgal cultivation using untreated ADE, TAN would easily volatilize by culture mixing, gas exchange, and/or increases in pH. The prevention of nitrogen loss in a large-scale reactor should be an advantage of ADE nitrification treatment because of the conversion of TAN to NO3.

Figure 4

Nitrogen compounds and phosphate concentrations of medium before (filled bar) and after (hollow bar) Chlorella sorokiniana NIES-2173 cultivation.

Figure 4

Nitrogen compounds and phosphate concentrations of medium before (filled bar) and after (hollow bar) Chlorella sorokiniana NIES-2173 cultivation.

The proposed post-AD process and further prospects

In microalgal cultivation using ADE, in addition to the dilution of ADE, sustaining the pH at less than 7.5 or controlling the concentration of TAN inside the reactor are also ways to avoid inhibition of growth by NH3. However, to maintain uniform pH control requires complete agitation, which is difficult to achieve in a large full-scale reactor (Eroglu et al. 2014), and the TAN removal rate in a reactor fluctuates widely depending on the algal growth rate, particularly with outdoor cultivation (González-Camejo et al. 2018). In the present study, it was confirmed that microalgae grow well in ADE that has been treated by nitrification–desulfurization, as well as in a synthetic medium even without dilution. It is considered that the proposed process can be an effective option to recover nutrients from the ADE as a variable microalgal biomass that will contribute to the further spread of the AD process. Moreover, the present study also highlighted some problems; for example, a higher N/P molar ratio and salinity of treated ADE than is appropriate for microalgal cultivation. The need to add PO43− is probably unavoidable, especially for continuous microalgal cultivation. The high salinity can limit various algal species that can be cultured in this process.

For the real application, first, we note that all ADE composition analysis and microalgal cultivation in this research was conducted using ADE after freezing and thawing to unify the analysis and cultivation conditions. Therefore, this freezing and thawing step might have changed the treated/untreated ADE composition; that is, by precipitation of dissolved contaminates and decomposition of organic compounds, and affected the microalgal growth. The effect of this experimental step required further confirmation. Furthermore, the handling of gas phases (biogas, O2) in each bacterial/algal reactor will be crucial to achieve effective and stable operation of the proposed system. For instance, (1) an appropriate method for O2 recovery from a microalgal reactor and (2) for further biogas utilization, effective supplementation of biogas and O2 in a nitrification–desulfurization reactor to mitigate O2 contamination into biogas should be established. Biological reactor operation and monitoring technologies, which are developing nowadays, should be appropriately applied.

CONCLUSION

It was confirmed that C. sorokiniana NIES-2173 has an average NH3 tolerance of 1.6 mM EC50. As a result of cultivating this species using different growth media, the final biomass concentration (OD750) decreased by 40% or more in every condition using untreated ADE even after 3-, 6-, and 10-fold dilution compared with the final biomass concentration obtained in the synthetic medium condition. In contrast, high productivity of C. sorokiniana was achieved without dilution by using ADE that had been treated by nitrification–desulfurization, the same as was seen with synthetic medium, indicating that this novel ADE treatment, without dilution, is useful for microalgal cultivation using ADE with suppression of the amount of freshwater consumption.

ACKNOWLEDGEMENTS

We are grateful to the Hokubu Sludge Treatment Center in Yokohama, Japan, for providing the nitrifying sludge and anaerobic digestion effluent. This research was supported by (1) the Japan Science and Technology Agency (JST)/Japan International Cooperation Agency (JICA), Science and Technology Research Partnership for Sustainable Development (SATREPS) and (2) Japan Society for the Promotion of Science (JSPS) KAKENHI, Japan, Grant Number JP19J15095.

CONFLICTS OF INTEREST

M. Sekine, S. Akizuki, T. Toda and M. Kishi are inventors on Japanese Patent Application No. 2019-35294 (Treatment method of anaerobic digestion products).

SUPPLEMENTARY MATERIAL

The Supplementary Material for this paper is available online at https://dx.doi.org/10.2166/wst.2020.153.

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Supplementary data