Introduction:
Nitrate entrance to water bodies can cause eutrophication and decrease quality of them, furthermore it can effect on human health so concentration of nitrate must be at defined standard level. There are several methods for reduction or removal of nitrate in water such as ion exchange, reverse osmosis, electrodialysis and denitrification. Microbial denitrification of nitrate in anoxic condition can be a cost effective and high efficiency miner in that nitrate was reduced to N2 Gas. There are two microbial denitrifications which are autotrophic and heterotrophic. The heterotrophic bacteria need to organic source while in autotrophic process the bacteria need to inorganic source like sulfur components, CO2, bicarbonate, carbonate as well as H2.These bacteria have a slow growth rate therefore they produce low sludge .A few Studies about autotrophic denitrification has conducted in Iran, so the aim of this study was to investigate the autotrophic denitrification process and compare the achieved results in different energy sources for bacteria.
Material and method :
In this research study 9 reactors in 3 series were designed and operated. The water was prepared with different concentrations of nitrate and sulfur sources synthetically. The energy sources were sulfide, thiosulfate and elemental sulfur. For investigating of autotrophic denitrification, firstly N2 gas injected to water for 30 minutes to reduce DO to zero. Then the operational status was defined according to table 1 and operation of reactors was started. The studied variables were mole ratio of nitrate to sulfur source, alkalinity consumption per every mg/l of reduced nitrate as nitrogen, produced sulfate per every mg/l of reduced nitrate as nitrogen, hardness concentration changes and removal percent of nitrate. The optimum ratios of nitrate to sulfur source achieved from studied articles and considered in first series of reactors. In second series tried to reverse the previous optimum ratios to see the effect of mole ratio on process, and in third series of reactors the ratios of nitrate to sulfur source were equal 1.All examinations were according to Standard methods for water and wastewater examinations. The operational temperature was 22±1 ºC. Nitrate detection conducted whit Spectrophotometer at 220 nm wave length, sulfate was determined gravimetric and other parameters like alkalinity, hardness as well as sulfide were determined according titration methods. Autotrophic denitrifier bacteria was prepared from anoxic sludge of domestic wastewater treatment in south of Tehran. After adding macro and micro elements needed for bacteria, the operation of reactors started. All of examinations repeated for 3 times to validate data and the Statistics analysis of data done by SPSS program.
Table.1.the operational status of reactors







Discussion of results and conclusion:
The results of this study have been shown in table no.2.
In denitrification process with sulfur as electron donor, H+ ions produce and alkalinity consumed although it would be different in various sulfur components. In this study the alkalinity consumption in all reactors were comprised to each other. The results show that when the electron donor is sulfide and the mole ratio of NO-3 to sulfide is 8:5, alkalinity consumption rate is 1.39 mg/l as caco3 per mg/l removed nitrate as nitrogen. When the mole ratio of NO-3 to sulfide was 1:1 alkalinity consumption rate was 1.45 mg/l as caco3 per mg/l removed nitrate as nitrogen. These amounts are lower than the other energy sources. On the other hand when the electron donor is sulfide the alkalinity consumption is lower than other components of sulfur.
As shown in table no.2 the added hardness when the elemental sulfur is electron donor is more than other two mentioned energy sources.
Table.2. the results of hardness, alkalinity, sulfate and nitrate variations in autotrophic denitrification



when sulfid was electron donor this addition was lower.
There is a stoichiometry reaction about autotrophic denitrification that is shown below:
NO3-+1.1S0+0.76H2O+0.4CO2+0.08NH4+ 0.5N2+1.1SO42-+1.28H++0.08C5H7O2
According to this reaction when elemental sulfur is electron donor , the alkalinity consumption is 4.57 mg/l CaCO3 and the added sulfate is 7.54mg/l per mg/l removed nitrate as nitrogen. The results of this study show that alkalinity consumption in reactors series1 was 5 in series2 was 5.21 and in series 3 was 5.38 mg/l as caco3 per removed nitrate as nitrogen.sulfate addition in reactors series 1 was 8 in rectors series2 was 8.12 and in reactors series 3 was 8.46 mg/l as CaCO3 per removed nitrate as nitrogen. Statistics analyses of data show that there is not a significant difference between the stoichiometry amounts and the achieved data from this study.(P>0.05).There are some other stoichiometry reactions about alkalinity consumption with HS- and S2O3 2- are electron donor. These reactions are shown below:
0.844 S2O32- + NO3- + 0.347 CO2 + 0.086 HCO3- + 0.086 NH4+ + 0.434 H2O→ 1.689 SO42- + 0.5 N2 + 0.086 C5H7O2N + 0.697 H+
0.421 H2S + 0.421 HS- + NO3- + 0.346 CO2 + 0.086 HCO3- + 0.086 NH4+→ 0.842 SO42- + 0.5 N2 + 0.086 C5H7O2N + 0.434 H2O + 0.262 H+
According to these reactions when the electron donor is hydrogen sulfide the alkalinity consumption is 0.93 mg/l as CaCO3 per removed nitrate as nitrogen and when thiosulfate is energy source this amount is 2.49 mg/l as CaCO3 per removed nitrate as nitrogen. Statistics analyses of data show that there is a significant difference between the stoichiometry amount and the achieved data from this study about sulfide (P<0.05) but there is not a significant difference between the stoichiometry amount and the achieved results about thiosulfate(P>0.05).
The results of nitrate removing shows that in raectors seri 1 with elemental sulfure as electron donor the nitrate removal was complete.In rectors seri 2 with tiosulfate as electron donor the result was like.In other rectors with diffrent mole ratio the results were different so we can see the effect of mole ratio on autotrophic denitrification.Autotrophic denitrification seems to be an effective process for removal of nitrate from water.If the source of energy and mole ratio of nitrate to energy sourec be suitable the efficiency of process will be grate.In this study sulfid was the best source since the alkalinity consumption and hardness addition and nitrate removal percent all were acceptable.