Achievements

AN EXPERIMENTAL STUDY OF REMEDIATION OF NITROGEN POLLUTION IN GROUNDWATER WITH PERMEABLE REACTIVE COLUMNS ENHANCED BY MICROORGANISM

Updated :10,29,2012

Xiao Shangping*Li Yilian

School of Environmental Studies, China University of Geosciences, Wuhan 430074, China

*Corresponding author.Tel.:+86 027 63111966; E-mail address: xiaoshangping@126.com

 

Abstracts: Based on the permeable reactive barriers (PRB) system, sand filtration (SF) system and constructed rapid infiltration (CRI) system, a sectional type of permeable reactive column (PRC) was simulated and constructed in our laboratory to remedy nitrogen pollution in groundwater. Clay, fine sand, grit and marble were filled in the permeable reactive column in sequence in order to simulate aeration zone-saturated zone in underground environment. Microorganism resources were chose from fertile garden mould, the experiment domesticated and enriched nitrifying bacteria, aerobic denitrifying bacteria, anaerobic denitrifying bacteria in aerobic condition and anaerobic condition separately. Then the highly efficient nitrifying bacteria and aerobic denitrifying bacteria were inoculated into aerobic aeration zone on the top of the permeable reactive column, the anaerobic denitrifying bacteria which had good capability were inoculated into anaerobic saturated zone in the substrate of the permeable reactive column. After running for a period of time, the biofilm in the permeable reactive column had grown stably, the capability of nitrification and denitrification were improved highly in this system, the removal rate of NH4+-N and NO3--N achieved above 90%. This system was verified to be feasible to remedy nitrogen pollution in groundwater by modeling study.

Key Words: MicroorganismPermeable Reactive ColumnRemedygroundwaterNitrogen Pollution


1 Introduction

Nitrogen pollution of groundwater is a problem recognized throughout the world, many countries and areas exist this pollution problem with different degrees[1]. Some governments and scholars have attached importance to this problem, the research of

nitrogen pollution and control of groundwater has became the hotspot in the world now[2-6].

With the developing of agriculture, the usage of fertilizer has been growing steadily, the contamination of nitrogen in groundwater became more heavy. The soil of unsaturated zone acts as a natural protector to groundwater’ quality, the biological denitrification is one of the most important roles on denitrifying bacteria transform nitrogen in unsaturated zone of soil[7,8], nitrate concentrations in percolating water can be decreased , being reduced to N2O or N2. Today denitrificationis a highly advanced method both technically and scientifically[9-11].

The permeable reactive barriers (PRB) system is a recently developed in-situ remedy technology for contaminated groundwater. The concept of PRB is relatively simple. Reactive material is placed in the subsurface where a plume of contaminated groundwater flows through under its natural gradient, creating a passive treatment system, and treated water comes out from the other side after adsorption and reaction between contaminant and reactive material. After properly designed and installed, PRB are capable of remedying a number of contaminant with extremely low maintenance costs[12]. Sand filtrationSFis the flow of water through a bed of granular media, normally following settling basins in conventional water treatment trains. The purpose of this filtration is to remove any particulate matter left over after flocculation and settling. The filter process operates based on two principles, mechanical straining and physical adsorption. Sand filtration is a “physical-chemical process for separating suspended and colloidal impurities from water by passage through a bed of granular material. Water fills the pores of the filter medium, and the impurities are adsorbed on the surface of the grains or trapped in the openings.” The key to this process is the relative grain size of the filter medium. Constructed Rapid InfiltrationCRIsystem is a new type of land treating technology based on the traditional rapid infiltrationRIfor wastewater[13]. Land treatment system utilizes natural processes in soils and aquifers to produce high quality water as opposed to conventional technologies for wastewater reclamation. It is premised on the infiltration of treated wastewater into the soil and percolation through the vadosezone. Improvements in water quality can occur due to many different mechanisms including filtration , biological degradation , chemical reaction , physical adsorption , ion exchange and precipitation.

The pollutants of most concern depending on the groundwater properties were ammonium and nitrate. Then an efficient nitrogen removal from groundwater should include nitrification and denitrification[14]. Based on the permeable reactive barriers (PRB) system, sand filtration (SF) system and constructed rapid infiltration (CRI) system, a sectional type of permeable reactive column (PRC) was simulated and constructed in our laboratory to remedy nitrogen pollution in groundwater. Clay, fine sand, grit and marble were filled in the permeable reactive column in sequence in order to simulate aeration zone-saturated zone in underground environment. Microorganism resources were chose from fertile garden mould, the experiment domesticated and enriched nitrifying bacteria, aerobic denitrifying bacteria, anaerobic denitrifying bacteria in aerobic condition and anaerobic condition separately. Then the highly efficient nitrifying bacteria and aerobic denitrifying bacteria were inoculated into aerobic aeration zone on the top of the permeable reactive column, the anaerobic denitrifying bacteria which had good capability were inoculated into anaerobic saturated zone in the substrate of the permeable reactive column. After running for a period of time, the biofilm in the permeable reactive column will have grown stably, the capability of nitrification and denitrification will has been improved highly, this system will be verified to be feasible to remedy nitrogen pollution in groundwater by modeling study.

2 Materials and Methods

2.1 Culture medium

The first culture medium (CM1) was used for enriching nitrifying bacteria and contained (g/L) (NH4)2SO2.0, K2HPO4 1.0, NaH2PO4 0.25, MgSO4·7H2O 0.03, MnSO4·4H2O 0.01, CaCO5.0 and distilled water1000mL[15]. The second culture medium (CM2) was used for enriching traditional denitrifying bacteria and contained (g/L) KNO2.0, K2HPO4 0.5, MgSO4·7H2O 0.2, C4H4O6KNa·4H2O 20.0, distilled water 1000mL and the pH of this medium was adjusted to 7.2[15]. CM1 and CM2 were the culture medium for enriching, they would induce nitrifying bacteria and traditional denitrifying bacteria to become the predominant microorganisms in the biofilm in separate domesticated container.

The screening medium (SM) was used to screen and isolate aerobic denitrifying bacteria and contained (g/L) KNO2.0, K2HPO4 0.5, MgSO4·7H2O 0.2, C4H4O6KNa·4H2O 20.0, a few drops of bromothymol blue (BTB), distilled water 1000mL and the pH of this medium was adjusted to 7.2. BTB is the critical component in SM, as a pH indicator, it will turn yellow to blue when pH value changes from 6.2 to 7.6. It will indicatedenitrification for isolating aerobic denitrifying bacteria because the pH of SM is between 6.2 and 7.6, and denitrification will increase the pH of the medium. But it was toxic to bacteria grown on the culture medium if it was dropped too enough, so we should only drop 1-2 drops of BTB into the Petri dish for indication.

2.2 Enriching nitrifying bacteria and traditional denitrifying bacteria

The bacteria resource, aerobic activated sludge and anaerobic activated sludge were all brought from the Sha Hu sewage treatment plant in Wuhan. A 2.0 L volume beaker with an aerator on the bottom was used for enriching nitrifying bacteria from the aerobic activated sludge in aerobic conditions. A 500 mL volume airproof plastic bottle with a shaker was used to enrich traditional denitrifying bacteria from the anaerobic activated sludge in anaerobic conditions.

During the oxidation of organic material coupling to reduction, oxygen is commonly accepted to be the first choice as electron acceptor because reduction of oxygen leads to a higher energy yield than reduction ofnitrate[16]. Therefore, denitrification is generally thought to occur only under anaerobic condition, which is different from nitrification occurring under aerobic condition. In this study, we created aerobic and anaerobic (micro-aerobic) conditions for the growing of nitrifying bacteria and traditional denitrifying bacteria.

First, the aerobic activated sludge and anaerobic activated sludge were inoculated into separate domesticated container according to 10% of effective capacity. Then the liquid culture medium was added separately after being diluted. With the pass of time, the dilution multiplier of the added liquid culture medium changed from 30 to 1, one times was reduced in turn everyday. After a month, we would improve the concentration of NH4+-N and NO3--N properly in order to induce nitrifying bacteria and traditional denitrifying bacteria becoming the predominant microorganism in separate biofilm in their domesticated container. We changed liquid above the sludge and tested the removal rate of NH4+-N and NO3--N everyday.

2.3 Screening and isolation of aerobic denitrifying bacteria

In the last decades, some aerobic denitrifying bacteria were reported, such as Thiosphaera Pantotropha, Alcaligenes faecalis, Pseudomonas nautical, Thaurea mechernichensis, Microvirgula aerodenitrificans and so on[17-21]. Aerobic denitrification, as a new way to remove nitrogen from wastewater, makes it possible that denitrification and nitrification occur at the same time in one reactor, which greatly reduces the operation cost. There are plenty of aerobic denitrifying bacteria in nature[22].

We thought for screening and isolating aerobic denitrifying bacteria from nature in order to improve denitrification in our experimental system. We chose the fertile garden mould brought from a vegetable plot on the campus of China University of Geosciences as the bacteria resource. Then the fertile garden mould was washed with 50 mL tap water and centrifugated, and then the feculent liquid was inoculated into a 2.0 Lsterilized beaker with an aerator on the bottom, and 500 mL liquid SM was added into the beaker. We changed the liquid SM everyday for the culture of aerobic denitrifying bacteria in order to cause them becoming the predominant microorganism in the beaker.

After a certain time, we might observe some activated sludge growing in the beaker, and with the pass of time, the activated sludge became more, tinged with gray and downy. Then we inoculated it onto the Petri dish filled with a layer of sterilized solid SM, and the Petri dish was kept in the cultivated chest with 30, it was favourable to the growing of bacteria colonies on the solid SM. We chose the blue colony from bacterial colonies in the Petri dish, then purified it using the dilution-plate technique. The isolated strains were then tested for their ability to denitrify under aerobic conditions.

2.4 The experiment enhanced by microorganism inoculation

The lab-scale plant used in this experimental study consisted of a sectional column (100 cm high and 20 cm diameter) made from polymethyl methacrylate plastics. Clay, fine sand, grit and marble were filled in the permeable reactive column in sequence in order to simulate aeration zone-saturated zone in underground environment.

Then the selected microorganisms were inoculated, the highly efficient nitrifying bacteria and aerobic denitrifying bacteria were inoculated into aerobic aeration zone on the top of the permeable reactive column, the anaerobic denitrifying bacteria which had good capability were inoculated into anaerobic saturated zone in the substrate of the permeable reactive column. The column system created a good nitrification and denitrificationenvironment.

In this study, groundwater characteristics were the following: NH4+-N 50 mg/L, NO3-N 50 mg/L, COD (glucose) 1000 mg/L, pH was adjusted to about 7.0. The permeable reactive column operated with a downward flow of above groundwater pumped onto the top of the column. After a period of time, the selected microorganisms attached to different layers in the column will have been grown more and stably and formed biofilm. We tested the concentration of NH4+-N and NO3--N in the outflow from the system everyday for knowing the effect of this system.

3 Results and Discussion

3.1Nitrification capability of nitrifying bacteria

Nitrification is the ability of nitrifying bacteria oxidize NH4+-N to NO3--N under aerobic conditions, and it is a oxidation process.


Fig.1 The removal rate of NH4+-N in the enriching system under aerobic condition

Stage 1 (1-7 days), the aerobic activated sludge had a certain number of nitrifying bacteria and showed a degreed of nitrification, the removal rate of  NH4+-N was about 40%. Stage 2 (8-18 days), the nitrifying bacteria grown gradually in the activated sludge and became the predominant microorganism of the biofilm, meanwhile the removal rate of  NH4+-N rose. Stage 3 (after 18 days), the nitrifying bacteria had become the predominant microorganism of the biofilm in the beaker, the removal rate of  NH4+-N kept about 90%.

3.2Denitrification capability of traditional denitrifying bacteria

Denitrification is the ability of traditional denitrifying bacteria using nitrogen oxides (NO3--N) as electron acceptors to produce gaseous nitrogen, mainly N2, and it is a reducing process.


Fig.2 The removal rate of NO3--N in the enriching system under anaerobic condition

Stage 1 (1-10 days), the anaerobic activated sludge had a certain number of denitrifying bacteria and showed a degreed of denitrification, the removal rate of  NO3--N was about 30%. Stage 2 (11-26 days), the denitrifying bacteria grown gradually in the activated sludge and became the predominant microorganism of the biofilm, meanwhile the removal rate of  NO3--N rose gradually. Stage 3 (after 26 days), the denitrifying bacteria had become the predominant microorganism of the biofilm in the beaker, the removal rate of  NO3--N kept about 80%. Apparently, the denitrifying bacteria grown more slowly than the nitrifying bacteria.

3.3 Aerobic denitrifying bacteria isolation

All the isolated and purified strains from biofilms were tested for denitrifying activity (capacity of reducing NO3--N to N2O or N2). Every single isolated strain was inoculated in a sterilized test-tube with 5-6 mLliquid SM, then each test-tube closed with cotton plugs was stirred at 120±2 r/min and 30±1 on a rotary shaker under aerobic conditions. After 24 hours, bacteria suspension formed in the test-tube. Subsequently, 3mL bacteria suspension was transferred into another sterilized flask containing about 30mL fresh liquid SM. Each flask closed with cotton plugs was stirred at 120±2 r/min and 30±1 on a rotary shaker under aerobic conditions. After 24 hours, we tested the removal rate of NO3--N for deciding the denitrifying activity of every single isolated strain.

Table.1 The denitrifying activity of 40 strains isolated from the fertile garden mould sludge

Name of strain

NO3--N removal rate (%)

Name of strain

NO3--N removal rate (%)

X1

25.6

X21

63.2

X2

32.9

X22

19.6

X3

17.8

X23

25.9

X4

35.8

X24

32.8

X5

56.8

X25

48.5

X6

32.8

X26

37.8

X7

64.7

X27

24.9

X8

52.3

X28

19.2

X9

81.6

X29

28.6

X10

42.6

X30

52.6

X11

36.6

X31

17.8

X12

70.6

X32

22.9

X13

35.4

X33

24.7

X14

60.3

X34

42.6

X15

35.6

X35

38.6

X16

18.9

X36

62.3

X17

72.9

X37

56.8

X18

58.6

X38

49.8

X19

24.8

X39

26.5

X20

17.4

X40

69.8

The data showed, the removal rate of NO3--N above 50% had 13 strains, and the assimilation of microorganism wasn’t over 30%[23], in other words, the 13 strains all had some degree of denitrifying activity under aerobic condition. The strain X9 had the highest removal rate during them, 81.6%. Bacterium strain X9 was chosen to be inoculated in the column owing to its high denitrifying activity.

Then we tested the removal rate of NO3--N by strain X9 for different concentration of NO3--N. The strain X9 had a high denitrifying activity, about 80% removal of NO3--N, while the concentration of NO3--N was less than 1000mg/L. But when the concentration of NO3--N was above 1000mg/L, the removal rate would decline, maybe plenty of NO3--N caused the NO2--N accumulated owing to denitrification, and NO2--N was toxic to  the microorganisms.

And the bacterium strain X9 will be identified by analysis of the sequence of the encoding 16S rRNA (16S rDNA) in order to decide whether it is a new one or not and research furtherly.

3.4 The removal of NH4+-N and NO3--N in the system enhanced by microorganism

Once the column system had been inoculated, treatment of the groundwater (NH4+-N 50 mg/L, NO3-N 50 mg/L) began.


Fig.3 The concentration of NH4+-N in outlet water from the system enhanced by microorganism

The tranformation of ammonium ion may involve adsorption, cation exchange, incorporation into microbial biomass, nitrification or release to the atmosphere in the gas form. In the first week, adsorption andcation exchange were probably the major mechanism of removal of NH4+ in this system., where positively charged ammonium ion is readily adsorbed onto negatively charged soil particles, and the concentration ofNH4+-N in outlet water was about 15mg/L. After a week, the adsorption arrived saturated, meanwhile the nitrifying bacteria didn’t form a biofilm one the top layer, in other words, the system had a little nitrification, so that the concentration of NH4+-N in outlet water increased and reached a maximum (about 35 mg/L) after 12 days. Then the nitrifying bacteria became more and the nitrification was notable, the concentration of NH4+-N in outlet water reduced gradually, and after 28 days, the concentration of NH4+-N in outlet water was about 5mg/L basically, it went into the stable stage.


Fig.4 The concentration of NO3-N in outlet water from the system enhanced by microorganism

The transformation of NO3-N may involve the assimilation of microorganism and denitrification in this system. In the first week, some assimilation maybe the mechanism of removal of NO3-N, the concentration of NO3-N in outlet water was about 35mg/L. Subsequently, the aerobic denitrifying bacteria and traditional denitrifying bacteria grown gradually on different layers in the column, meanwhile the nitrification of the system was stronger than the denitrification so that the concentration of NO3--N in outlet water increased and were above 50mg/L soon. After a month, maybe the aerobic denitrifying bacteria and traditional denitrifying bacteria became more on their layers, the denitrification of the system became stronger, the concentration of NO3-N in outlet water reduced. After one and half months, the denitrification of the system went into the stable stage, and the concentration of NO3-N in outlet water was about 8 mg/L basically,

4 Conclusions

A major drawback of groundwater nitrification and denitrification is the fact that formation of biofilm from its own microbiota in the influent is necessarily an extremely slow process, owing to the low presence of microorganisms in the water. And this study presents a possible solution lying in selective inoculation using bacteria with high nitrifying and denitrifying activity as inocula, meanwhile carbon source should be added properly.

Based on the permeable reactive barriers (PRB) system, sand filtration (SF) system and constructed rapid infiltration (CRI) system, selective inoculation of this permeable reactive column (PRC) system applied to treat nitrogen pollution of groundwater is very successful, owing to the inoculated bacteria are the predominant microorganisms in different layers in the column. In the light of these results we may state that the capability of nitrification and denitrification were improved highly in this system, the removal rate of NH4+-N and NO3--N achieves above 90% so that nitrogen pollution will be solved basically. This system was verified to be feasible to remedy nitrogen pollution in groundwater by modeling study.

We should carry out field experiment to improve this system under different conditions to control a series of different parameters such as pH, temperature and dissolved oxygen concentration, because this lab-scale plant was performed in a simple condition.



 

Acknowledgements

This study was supported by funds of “2006AA305A01” (the item number), graduate students’ academic exploration and innovation in China University of Geosciences (2006), and college students’ scientific research in their spare time in China University of Geosciences (2006). And it was carried out in the major laboratory of ministry of education, biogeology and environmental geology, China University of Geosciences,Wuhan, China.

Our research group, Zhang wei, Chen huaqing, Yang zhu, Cheng huilin and Zhou xiaojuan are thanked for their help in the experimental study. Professor Li yilian, Teacher Luo zhaohui and Professor Liu hui are thanked for their help and constructive suggestions.

 

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