This work investigated the effects of eight metal ions on Rhodopseudomonas palustris growth and 5-aminolevulinic acid (ALA) yield in wastewater treatment. Results show that metal ions (Mg2+ of 15 mmol/L, Fe2+ of 400 μmol/L, Co2+ of 4 μmol/L, Ni2+ of 8 μmol/L and Zn2+ of 4 μmol/L) could effectively improve the chemical oxygen demand (COD) removal, Rp. palustris biomass and ALA yield. The highest ALA yield of 13.1 mg/g-biomass was achieved with Fe2+ of 400 μmol/L. ALA yields were differentially increased under different metal ions in the following order: Fe2+ group > Mg2+ group > Co2+ group = Ni2+ group > Zn2+ group = Mo2+ group > control. Cu2+ and Mn2+ inhibited Rp. palustris growth and ALA production. Mechanism analysis revealed that metal ions changed ALA yields by influencing the activities of ALA synthetase and ALA dehydratase.

INTRODUCTION

Purple non-sulfur bacteria (PNSB), used to treat various wastewaters, have received increasing attention since the 1960s. They could effectively remove the pollutants from different wastewaters including dairy wastewater, soybean wastewater, olive mill wastewater, and domestic wastewater (Kaewsuk et al. 2010; Eroglu et al. 2011; Wu et al. 2012; Tim et al. 2014). Simultaneously, the accumulated PNSB biomass contains a variety of valuable materials, including single cell proteins, bacteriochlorophylls, biopolymers, 5-aminolevulinic acid (ALA), carotenoids, and CoQ10 (Kang et al. 2012; Kuo et al. 2012). Among those materials, ALA attracted special attention because it is an important photo-dynamic chemical involved in the biosynthesis of tetrapyrrole compounds, and it is widely applied in both medical and agricultural fields (Sasaki et al. 2002, 2005).

ALA production largely depends on different influencing factors, including light-oxygen conditions, pH, carbon and nitrogen sources, temperature, and metal ions (Choi et al. 2004; Chung et al. 2005; Liu et al. 2014). Trace metal ions, such as iron (Fe), magnesium (Mg), manganese (Mn), molybdenum (Mo), cobalt (Co), copper (Cu), zinc (Zn) and nickel (Ni), play essential roles in regulating osmotic pressure and redox processes and stabilizing macromolecules through electrostatic interactions. Most of them are transition metals with strong abilities to form complex compounds (Hunter et al. 2008). In previous studies, Fe2+, Mg2+, Ca2+ and Ni2+ have been proved to improve PNSB growth of Rhodobacter sphaeroides and Rhodopseudomonas faecalis (Liu et al. 2009; Hakobyan et al. 2012; Wu et al. 2012). In addition, some metal ions were activators to various enzymes. Previous work revealed that metal ions (Fe2+, Cu2+ and Ca2+) had important effects on the activity of ALA synthetase (ALAS) (Sasaki et al. 1989; Tangprasittipap et al. 2007). Fe2+ and Co2+ were important elements for regulating tetrapyrrole biosynthesis of R. sphaeroides (Sasikala et al. 1994).

This study was aimed at investigating the effects of eight different metal ions on Rp. palustris growth and ALA yield in wastewater treatment; optimizing the metal ion concentration to increase the ALA yield; investigating the potential mechanism on influencing ALA yield by changing ALAS and ALA dehydratase (ALAD) activities.

MATERIALS AND METHODS

Materials

An Rp. palustris strain (ACCC10649) used in this study was obtained from Agricultural Culture Collection of China. It was cultivated with 0317 medium (detailed information can be found at www.accc.org.cn/show.asp) under light-anaerobic conditions in a serum bottle (120 rpm, 30 °C). The medium consisted of 3 g/L yeast extract, 0.5 g/L magnesium sulfate heptahydrate, 3 g/L peptone, 0.3 g/L calcium chloride. pH was adjusted to 7.0. The inoculated bacteria in logarithmic growth phase were used for experiments, and the density was 7.5 × 108 colony forming units (CFU)/mL.

Synthetic wastewater was selected in this study. It comprised peptone (2 g/L), sodium acetate (2 g/L), yeast extract (1.5 g/L) and potassium dihydrogen phosphate (0.5 g/L). The characteristics were as follows: chemical oxygen demand (COD), total nitrogen (TN) and total phosphorus (TP) were 3,425, 232 and 85 mg/L, respectively. pH was adjusted to around 7.0. The wastewater was used for the experiments after filtration and autoclaving to avoid environmental contamination (121 °C, 30 min).

Methods

Experimental

The bioreactors for the experiments were glass conical flasks of 1 L volume, and each reaction volume was 500 mL, as shown in Figure 1. They were sterilized at 121 °C for 30 min before use. Synthetic wastewater (460 mL) and Rp. palustris (40 mL) were added to each bioreactor. The initial concentration of Rp. palustris was 550 mg/L.
Figure 1

Schematic drawing of the experimental bioreactor.

Figure 1

Schematic drawing of the experimental bioreactor.

Light and oxygen conditions were as follows: light intensity was 3,000–6,000 lux; anaerobic conditions were maintained by continuous nitrogen inflow from nitrogen cylinder to bioreactor. The cultivating temperature was 30 °C.

Metal ions addition

Different metal ions were individually added to bioreactors according to the trace elements formula of No. 0259 medium in China General Microbiological Culture Collection Center. The formula contained the following ingredients (mg/L): 1.8 FeCl2·4H2O, 0.5 MgCl2, 0.25 CoCl2·6H2O, 0.01 NiCl2·6H2O, 0.01 CuCl2·2H2O, 0.7 MnCl2·4H2O, 0.1 ZnCl2, 0.5 H3BO3, 0.03 Na2MoO4·2H2O, and 0.01 Na2SeO3·5H2O. In this study, 100, 200, 400 and 500 μmol/L for FeCl2, 5, 10, 15 and 20 mmol/L for MgCl2, 2, 4, 8 and 12 μmol/L for CoCl2, NiCl2, ZnCl2, Na2MoO4, CuCl2 and MnCl2 were added into the reaction systems, respectively. No metal ion was added in the blank group.

Analysis methods

Each sample collected from a bioreactor was centrifuged at 9,000 rpm for 10 min. The supernatant was used to test COD, TN and TP. The collected cells were used to measure the biomass and intracellular ALA. pH and dissolved oxygen were detected by a pH tester and dissolved oxygen meter, respectively. COD and biomass were tested according to American Public Health Association Standard Methods (Clesscerl et al. 1998). The COD detection method was potassium dichromate oxidation spectrophotometric method. Biomass was measured by dry weight (103 °C, overnight). The intracellular ALA concentration was examined according to the description (Liu et al. 2010). The ALA yield (mg/g-biomass) was calculated by Equation (1): 
formula
1
where Y denotes the ALA yield, C (mg/L) denotes the ALA concentration at 96 h, W (g/L) denotes the dry biomass at 96 h.

The assay for ALAS and ALAD activities were measured by the method described (Lin et al. 2009). The reaction containing ALAS extract was performed with acetylacetone addition for 15 min at 100 °C. After the reaction, the mixture was cooled down to room temperature and mixed with modified Ehrlich's reagent for 30 min, then the absorbance at 554 nm was measured. One unit of ALAS activity was defined as the amount of the enzyme needed to produce 1 nmol of ALA in 1 min. The assay for ALAD activity involved a total volume of 500 μL, consisting of 50 mmol/L of potassium phosphate buffer (pH = 7.5), 50 mmol/L of ZnCl2, 1 mmol/L of MgCl2, and 5 mmol/L of ALA. The reaction (37 °C, 10 min) was stopped by 20% trichloroacetic acid. The next procedures were the same as the assay for ALAS activity (15 m reaction with acetylacetone at 100 °C, cooled down, 30 min reaction with modified Ehrlich's reagent, and then measured at 554 nm absorbance). One unit of ALAD activity was defined as the amount of the enzyme needed to produce 1 nmol of porphobilinogen (PBG) in 1 h.

Statistical analysis

In order to ensure data accuracy, parallel experiments were carried out and all results were represented as mean ± standard deviation. Significant differences between the treatments were evaluated by one-way analysis of variance with the Statistical Package for Social Sciences for Windows (version 19.0; SPSS Inc., Chicago, IL, USA). Duncan's multiple range tests (DMRT) were used for pairwise or individual (one-to-one) comparisons. Significant difference was considered at P < 0.05.

RESULTS AND DISCUSSION

Optimization of metal ions on COD removal, biomass production and ALA yield in Rp. palustris wastewater treatment

The first step was to determine the optimal dosages of various metal ions. Metal ions at different concentrations were added to the wastewater, and the COD removal, biomass production and ALA yield in Rp. palustris wastewater treatment were examined. The results are summarized in Table 1. Table 1 shows some metal ions (Fe2+, Mg2+, Co2+, Ni2+, Zn2+ and Mo2+) improved the COD removal, biomass production and ALA yield at different levels. The optimal dosage of each metal ion was 400 μmol/L for Fe2+, 15 mmol/L for Mg2+, 4 μmol/L for Co2+, 8 μmol/L for Ni2+, 4 μmol/L for Zn2+, and 4 μmol/L for Mo2+, respectively. Metal ions of Cu2+ and Mn2+ decreased the COD removal, biomass production and ALA yield. The less Cu2+ or Mn2+ was added, the weaker the inhibition on ALA yield.

Table 1

Effects of metal ions on COD removal, biomass production and ALA yield in Rp. palustris wastewater treatment Means sharing no common letter (± s.e.m.) are significantly different (P < 0.05)

Metal irons COD removal (%) Biomass (g/L) ALA yield (mg/g-biomass) 
Blank for Fe2+ 88.46 ± 0.69a 2.44 ± 0.07a 5.42 ± 0.66ba 
Fe2+ (100 μmol/L) 88.83 ± 0.41a 2.58 ± 0.08b 7.91 ± 0.54bc 
Fe2+ (200 μmol/L) 89.03 ± 0.62a 2.66 ± 0.08b 8.99 ± 0.42c 
Fe2+ (400 μmol/L) 91.17 ± 1.09b 2.85 ± 0.05c 13.1 ± 0.68d 
Fe2+ (500 μmol/L) 88.60 ± 0.73a 2.80 ± 0.03c 6.81 ± 0.27b 
Blank for Mg2+ 88.46 ± 0.69a 2.44 ± 0.07a 5.42 ± 0.66a 
Mg2+ (5 mmol/L) 89.37 ± 0.41ab 2.85 ± 0.07b 7.92 ± 0.11b 
Mg2+ (10 mmol/L) 90.93 ± 1.02b 2.91 ± 0.06b 8.24 ± 0.46b 
Mg2+ (15 mmol/L) 93.93 ± 0.53c 3.16 ± 0.06c 10.56 ± 0.46c 
Mg2+ (20 mmol/L) 90.43 ± 0.78b 2.94 ± 0.06b 7.61 ± 0.52b 
Blank for Co2+ 88.46 ± 0.69a 2.44 ± 0.07a 5.42 ± 0.66a 
Co2+ (2 μmol/L) 88.87 ± 0.76a 2.67 ± 0.11b 7.68 ± 0.11a 
Co2+ (4 μmol/L) 89.73 ± 0.45a 2.72 ± 0.10b 8.35 ± 0.23b 
Co2+ (8 μmol/L) 89.40 ± 0.51a 1.64 ± 0.10ab 8.08 ± 0.51b 
Co2+ (12 μmol/L) 88.93 ± 0.46a 2.71 ± 0.09b 7.78 ± 0.17b 
Blank for Ni2+ 88.46 ± 0.69a 2.44 ± 0.07a 5.42 ± 0.66a 
Ni2+ (2 μmol/L) 90.00 ± 0.43b 2.66 ± 0.12b 7.00 ± 0.49b 
Ni2+ (4 μmol/L) 90.26 ± 0.82b 2.76 ± 0.07b 7.76 ± 0.07b 
Ni2+ (8 μmol/L) 90.67 ± 0.56b 2.71 ± 0.11b 9.12 ± 0.56c 
Ni2+ (12 μmol/L) 90.20 ± 0.73b 2.67 ± 0.06b 7.34 ± 0.88b 
Blank for Zn2+ 88.46 ± 0.69a 2.44 ± 0.07a 5.42 ± 0.66a 
Zn2+ (2 μmol/L) 89.37 ± 0.45ab 2.63 ± 0.05b 5.43 ± 0.36a 
Zn2+ (4 μmol/L) 90.07 ± 0.37b 2.68 ± 0.09b 5.97 ± 0.42a 
Zn2+ (8 μmol/L) 90.00 ± 0.22b 2.75 ± 0.06b 5.87 ± 0.58a 
Zn2+ (12 μmol/L) 89.17 ± 0.58ab 2.72 ± 0.10b 5.89 ± 0.28a 
Blank for Mo2+ 88.46 ± 0.69a 2.44 ± 0.07a 5.42 ± 0.66a 
Mo2+ (2 μmol/L) 88.43 ± 0.74a 2.62 ± 0.06b 5.62 ± 0.87a 
Mo2+ (4 μmol/L) 88.77 ± 0.25a 2.72 ± 0.05b 6.11 ± 0.11a 
Mo2+ (8 μmol/L) 88.43 ± 0.68a 2.64 ± 0.05b 5.97 ± 0.48a 
Mo2+ (12 μmol/L) 88.33 ± 0.56a 2.59 ± 0.06b 5.92 ± 0.42a 
Blank for Cu2+ 88.46 ± 0.69c 2.44 ± 0.07b 5.42 ± 0.66b 
Cu2+ (2 μmol/L) 83.83 ± 1.44b 2.21 ± 0.09a 3.77 ± 0.38a 
Cu2+ (4 μmol/L) 83.67 ± 0.79ab 2.32 ± 0.04ab 3.68 ± 0.43a 
Cu2+ (8 μmol/L) 82.37 ± 0.66ab 2.24 ± 0.06a 3.67 ± 0.31a 
Cu2+ (12 μmol/L) 81.63 ± 0.57a 2.21 ± 0.07a 2.96 ± 0.59a 
Blank for Mn2+ 88.46 ± 0.69c 2.44 ± 0.07b 5.42 ± 0.66c 
Mn2+ (2 μmol/L) 83.80 ± 0.33b 2.12 ± 0.07a 3.81 ± 0.17b 
Mn2+ (4 μmol/L) 82.67 ± 0.61ab 2.10 ± 0.06a 3.10 ± 0.06ab 
Mn2+ (8 μmol/L) 82.13 ± 0.62ab 2.11 ± 0.06a 2.77 ± 0.53ab 
Mn2+ (12 μmol/L) 81.63 ± 1.17a 2.06 ± 0.07a 2.72 ± 0.50a 
Metal irons COD removal (%) Biomass (g/L) ALA yield (mg/g-biomass) 
Blank for Fe2+ 88.46 ± 0.69a 2.44 ± 0.07a 5.42 ± 0.66ba 
Fe2+ (100 μmol/L) 88.83 ± 0.41a 2.58 ± 0.08b 7.91 ± 0.54bc 
Fe2+ (200 μmol/L) 89.03 ± 0.62a 2.66 ± 0.08b 8.99 ± 0.42c 
Fe2+ (400 μmol/L) 91.17 ± 1.09b 2.85 ± 0.05c 13.1 ± 0.68d 
Fe2+ (500 μmol/L) 88.60 ± 0.73a 2.80 ± 0.03c 6.81 ± 0.27b 
Blank for Mg2+ 88.46 ± 0.69a 2.44 ± 0.07a 5.42 ± 0.66a 
Mg2+ (5 mmol/L) 89.37 ± 0.41ab 2.85 ± 0.07b 7.92 ± 0.11b 
Mg2+ (10 mmol/L) 90.93 ± 1.02b 2.91 ± 0.06b 8.24 ± 0.46b 
Mg2+ (15 mmol/L) 93.93 ± 0.53c 3.16 ± 0.06c 10.56 ± 0.46c 
Mg2+ (20 mmol/L) 90.43 ± 0.78b 2.94 ± 0.06b 7.61 ± 0.52b 
Blank for Co2+ 88.46 ± 0.69a 2.44 ± 0.07a 5.42 ± 0.66a 
Co2+ (2 μmol/L) 88.87 ± 0.76a 2.67 ± 0.11b 7.68 ± 0.11a 
Co2+ (4 μmol/L) 89.73 ± 0.45a 2.72 ± 0.10b 8.35 ± 0.23b 
Co2+ (8 μmol/L) 89.40 ± 0.51a 1.64 ± 0.10ab 8.08 ± 0.51b 
Co2+ (12 μmol/L) 88.93 ± 0.46a 2.71 ± 0.09b 7.78 ± 0.17b 
Blank for Ni2+ 88.46 ± 0.69a 2.44 ± 0.07a 5.42 ± 0.66a 
Ni2+ (2 μmol/L) 90.00 ± 0.43b 2.66 ± 0.12b 7.00 ± 0.49b 
Ni2+ (4 μmol/L) 90.26 ± 0.82b 2.76 ± 0.07b 7.76 ± 0.07b 
Ni2+ (8 μmol/L) 90.67 ± 0.56b 2.71 ± 0.11b 9.12 ± 0.56c 
Ni2+ (12 μmol/L) 90.20 ± 0.73b 2.67 ± 0.06b 7.34 ± 0.88b 
Blank for Zn2+ 88.46 ± 0.69a 2.44 ± 0.07a 5.42 ± 0.66a 
Zn2+ (2 μmol/L) 89.37 ± 0.45ab 2.63 ± 0.05b 5.43 ± 0.36a 
Zn2+ (4 μmol/L) 90.07 ± 0.37b 2.68 ± 0.09b 5.97 ± 0.42a 
Zn2+ (8 μmol/L) 90.00 ± 0.22b 2.75 ± 0.06b 5.87 ± 0.58a 
Zn2+ (12 μmol/L) 89.17 ± 0.58ab 2.72 ± 0.10b 5.89 ± 0.28a 
Blank for Mo2+ 88.46 ± 0.69a 2.44 ± 0.07a 5.42 ± 0.66a 
Mo2+ (2 μmol/L) 88.43 ± 0.74a 2.62 ± 0.06b 5.62 ± 0.87a 
Mo2+ (4 μmol/L) 88.77 ± 0.25a 2.72 ± 0.05b 6.11 ± 0.11a 
Mo2+ (8 μmol/L) 88.43 ± 0.68a 2.64 ± 0.05b 5.97 ± 0.48a 
Mo2+ (12 μmol/L) 88.33 ± 0.56a 2.59 ± 0.06b 5.92 ± 0.42a 
Blank for Cu2+ 88.46 ± 0.69c 2.44 ± 0.07b 5.42 ± 0.66b 
Cu2+ (2 μmol/L) 83.83 ± 1.44b 2.21 ± 0.09a 3.77 ± 0.38a 
Cu2+ (4 μmol/L) 83.67 ± 0.79ab 2.32 ± 0.04ab 3.68 ± 0.43a 
Cu2+ (8 μmol/L) 82.37 ± 0.66ab 2.24 ± 0.06a 3.67 ± 0.31a 
Cu2+ (12 μmol/L) 81.63 ± 0.57a 2.21 ± 0.07a 2.96 ± 0.59a 
Blank for Mn2+ 88.46 ± 0.69c 2.44 ± 0.07b 5.42 ± 0.66c 
Mn2+ (2 μmol/L) 83.80 ± 0.33b 2.12 ± 0.07a 3.81 ± 0.17b 
Mn2+ (4 μmol/L) 82.67 ± 0.61ab 2.10 ± 0.06a 3.10 ± 0.06ab 
Mn2+ (8 μmol/L) 82.13 ± 0.62ab 2.11 ± 0.06a 2.77 ± 0.53ab 
Mn2+ (12 μmol/L) 81.63 ± 1.17a 2.06 ± 0.07a 2.72 ± 0.50a 

a, b, c, dSignificantly different, P < 0.05.

Effects of different metal ions on COD removal, biomass production and ALA yield in Rp. palustris wastewater treatment

Based on the results in Table 1, the optimal dosage of each metal ion was selected in the following experiments.

The biomass production and COD removal

Figure 2(a) shows that, compared with the blank group, metal ions such as Fe2+, Mg2+, Co2+, Ni2+, Zn2+ and Mo2+ increased the COD removals in Rp. palustris wastewater treatment. The COD removals were 91.2, 93.9, 89.7, 90.7, 90.0, 88.4, 88.6, 83.8, and 83.8%, respectively. Metal ion addition was a simple method, and this method could improve the pollutant degradation efficiency in Rp. palustris wastewater treatment from the results above.
Figure 2

Effects of metal ions on (a) COD removal, (b) biomass production, (c) ALA yield. Bar represents the standard error of means for replicates. Means sharing no common letter (above bar ± s.e.m.) are significantly different (P < 0.05).

Figure 2

Effects of metal ions on (a) COD removal, (b) biomass production, (c) ALA yield. Bar represents the standard error of means for replicates. Means sharing no common letter (above bar ± s.e.m.) are significantly different (P < 0.05).

With the same trend, Figure 2(b) shows the biomass production reached the peak of 3.16 g/L with 15 mmol/L for Mg2+. Clearly, Fe2+, Mg2+, Co2+, Ni2+, Zn2+ and Mo2+ stimulated Rp. palustris growth, and the biomass displayed an increase of 16.6, 29.5, 11.5, 10.9, 12.4 and 11.2%, respectively. Analysis results of DMRT show that the biomass increased differentially under different metal ions in the following order: Mg2+ group > Fe2+ group = Co2+ group = Ni2+ group = Zn2+ group = Mo2+ group > control. In addition, Cu2+ and Mn2+ inhibited Rp. palustris growth. The possible reason is that Cu2+ and Mn2+ are toxic to bacteria to some extent. Wu et al. (2012) reported that the optimal Fe2+ content (20 mg/L) could significantly increase the biomass production and COD removal in R. sphaeroides Z08 wastewater treatment. Through further analysis, Wu et al. (2012) found that Fe2+ improved the R. sphaeroides growth and COD removal by enhancing the energy metabolism pathway of R. sphaeroides. Likewise, previous studies showed that Fe2+, Mg2+, Ca2+ and Ni2+ ions promoted the growth of PNSB including R. sphaeroides and Rp. faecalis (Liu et al. 2009; Hakobyan et al. 2012). On the one hand, some metal ions were essential elements for cell growth. On the other hand, it was proved that Mg2+ and Ni2+ could enhance R. sphaeroides growth by synthesizing more pigment complexes. In addition, Hakobyan et al. (2012) reported that Mg2+ also affected R. sphaeroides growth by regulating the pH of the medium. In this study, Fe2+ and Mg2+ both significantly increased Rp. palustris growth and COD removal.

The ALA production

Metal ions improved the biomass and COD removal, and the potential of ALA production in Rp. palustris wastewater treatment under different metal ions was examined. Figure 2(c) shows the ALA yields in the groups with metal ions (Fe2+, Mg2+, Co2+ and Ni2+) were improved to some extent. Compared with the blank group, the ALA yields were increased by 142.5, 95.1, 54.1 and 68.5%, respectively. Metal ions Zn2+ and Mo2+ had no obvious effect on the ALA yield. The ALA yields in the groups with Cu2+ and Mn2+ were lower than that of the blank. Moreover, it was proved that Rp. palustris growth was inhibited by Cu2+ and Mn2+ (Figure 2(b)). So Cu2+ and Mn2+ were possibly toxic to Rp. palustris cells or ALAS activity in the ALA biosynthesis pathway. A previous study reported that Cu2+ had critical effects on ALAS activity (Sasaki et al. 1989). Further, the analysis results of DMRT showed that ALA yields varied differentially under different metal ions in the following order: Fe2+ group > Mg2+ group > Co2+ group = Ni2+ group > Zn2+ group = Mo2+ group > control > Cu2+ group = Mn2+ group. Hence, the addition of Fe2+ could dramatically improve ALA production.

Effects of metal ions on ALAS and ALAD activities

Previous studies showed that many metal ions (Fe2+, Mg2+ and Ni2+) were used as PNSB growth medium components. Metal ions (Mo2+, Mg2+ and Ni2+) were critical elements in various enzymes such as hydrogenase and nitrogenase. They were also activators to some enzymes (Wang & Wan 2008; Liu et al. 2009; Eroglu et al. 2011). Studies also revealed that some metal ions (Fe2+, Cu2+ and Co2+) had important effects on ALAS activity and they could regulate the ALA biosynthesis process (Sasaki et al. 1989; Sasikala et al. 1994; Tangprasittipap et al. 2007). ALAS and ALAD are key enzymes in the ALA biosynthesis pathway. In this pathway, ALA is formed by ALAS catalysis, and ALAS activity has a positive effect on ALA production. On the contrary, ALAD can catalyze conversion of two molecules of ALA into one molecule of PBG, resulting in a decrease of ALA production (Liu et al. 2014). Hence, in order to investigate the effects of metal ions on the key enzymes in ALA biosynthesis, ALAS and ALAD activities with different metal ion additions were examined. The results are shown in Figure 3.
Figure 3

Effects of metal ions on (a) ALA synthetase (ALAS) activity and (b) ALA dehydratase (ALAD) activity. The activity of the control in the assay was arbitrarily set as 100%. Bar represents the standard error of means for replicates. Means sharing no common letter (above bar ± s.e.m.) are significantly different (P < 0.05).

Figure 3

Effects of metal ions on (a) ALA synthetase (ALAS) activity and (b) ALA dehydratase (ALAD) activity. The activity of the control in the assay was arbitrarily set as 100%. Bar represents the standard error of means for replicates. Means sharing no common letter (above bar ± s.e.m.) are significantly different (P < 0.05).

Figure 3(a) shows that metal ions of Fe2+, Mg2+, Co2+, Ni2+ and Zn2+ increased ALAS activities, and further improved the ALA yield. This agreed with the results in Figure 2(c) where those metal ions increased the ALA yield. DMRT analysis showed that ALAS activities varied differentially under metal ions in the following order: Fe2+ group > Mg2+ group > Co2+ group = Ni2+ group ≥ Zn2+ group. Choi et al. (2004) demonstrated that the ALAS activity of Rp. palustris KUGB306 was strongly inhibited by Fe2+, Co2+ and Zn2+ at 1 mmol/L, but it was only slightly affected by Mg2+. In this study, metal ions of different concentrations such as 400 μmol/L for Fe2+, 15 mmol/L for Mg2+, 4 μmol/L for Co2+, 8 μmol/L for Ni2+ and 4 μmol/L for Zn2+ increased the ALAS activities of Rp. palustris strain ACCC10649. This finding showed that different metal ions had different effects on ALAS of different species. Moreover, different species displayed differential responses to metal ions of specific concentration.

As another key enzyme in ALA biosynthesis pathway, ALAD can catalyze the degradation of ALA; therefore, the inhibition of the activity of ALAD is desirable for higher ALA yield (Liu et al. 2014). Figure 3(b) shows that Mg2+, Co2+, Zn2+ and Mn2+ increased the ALAD activities, which consequently increased ALA degradation. The possible reason was that these metal ions might be activators to the enzymes. A previous study reported that Mg2+, K+ and Zn2+ maintained the catalytic activity of ALAD (Sasikala et al. 1994). It is worth underlining that Mn2+ also increased ALAS activity, but low ALA yield was achieved (Figure 2(c)). The reason was that Mn2+ promoted ALAD activity to degrade more ALA.

In addition, the highest ALA yield was achieved with 400 μmol/L of Fe2+. From Figure 3(a) and 3(b), it is found that there were two possible reasons. On the one hand, Fe2+ increased ALA yield by increasing ALAS activity. One the other hand, Fe2+ also decreased ALA degradation by inhibiting ALAD activity. A similar study reveals that 30 μmol/L for Fe2+ enhanced ALA production by increasing ALAS activity (Tangprasittipap et al. 2007). Hence, proper Fe2+ addition would increase ALA yield by changing ALAS and ALAD activities.

Taking into account that too much metal ion in the effluent could be a new pollution, we detected the concentrations of residual metal ions in the effluent with a Plasma-Atomic Emission Spectrometry System (Otima 5300 DV ICP, Perkin-Elmer Inc., Boston, USA). The results in Table 2 show that metal ion additions in this study could meet a common sewage discharge standard (GB 18918-2002) by the Chinese Government, so it was accepted to add proper metal ions.

Table 2

The effluent concentrations of metal ions in Rp. palustris wastewater treatment

Metal ion Fe2+ Mg2+ Co2+ Ni2+ Zn2+ Mo2+ Cu2+ Mn2+ 
Discharging concentration threshold in GB 18918–2002 (mg/L) – – – – – 
Average effluent concentration in this study (mg/L) 1.5 2.9 0.275 0.201 0.136 0.404 0.304 0.318 
Metal ion Fe2+ Mg2+ Co2+ Ni2+ Zn2+ Mo2+ Cu2+ Mn2+ 
Discharging concentration threshold in GB 18918–2002 (mg/L) – – – – – 
Average effluent concentration in this study (mg/L) 1.5 2.9 0.275 0.201 0.136 0.404 0.304 0.318 

CONCLUSIONS AND PERSPECTIVES

This study showed that metal ion addition could improve the biomass and ALA production in Rp. palustris wastewater treatment. Optimal dosages of metal ions were 400 μmol/L for Fe2+, 15 mmol/L for Mg2+, 4 μmol/L for Co2+, 8 μmol/L for Ni2+ and 4 μmol/L for Zn2+, respectively. Four hundred micromoles per liter for Fe2+ led to the highest ALA yield of 13.1 mg/g-biomass. The maximum COD removal and biomass production were achieved at 15 mmol/L for Mg2+. Mechanism analysis showed that different metal ions had different influencing mechanisms on ALA yield by regulating ALAS or ALAD activities.

Based on the results above, proper metal ion addition improving biomass and ALA production will be a simple and green biotechnology in the field of wastewater treatment. Producing valuable ALA might be applied in other fields. This biotechnology will promote wastewater treatment recycling, and it also will provide some guidance for ALA production in agriculture, medical or other fields.

ACKNOWLEDGEMENT

This work was financially supported by the National Natural Science Foundation of China (51278489).

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