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: Microorganism；Permeable Reactive Column；Remedy；groundwater；Nitrogen 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 filtration（SF）is 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 Infiltration（CRI）system is a new type of land treating technology based on the traditional rapid infiltration（RI）for 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)₂SO₄ 2.0, K₂HPO₄ 1.0, NaH₂PO₄ 0.25, MgSO₄·7H₂O 0.03, MnSO₄·4H₂O 0.01, CaCO₃ 5.0 and distilled water1000mL^[15]. The second culture medium (CM2) was used for enriching traditional denitrifying bacteria and contained (g/L) KNO₃ 2.0, 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) KNO₃ 2.0, 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

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.

 

References

[1]      Williams A E, Lund L J, Johanson J A, et al. Natural and Anthropogenic Nitrate Contamination of Groundwater in a Rural Community, California. Env. Sci .Tech ,1998 ,32(1) :32 -39.

[2]      Zhao Lin,Wang Rongshu,Lin Xueyu. Existing form of nitrogen and its pollution control by microorgnism in the groundwater. ACTA SCIENTIAE CIRCUMSTANTIAE. July,1999 Vol.19,No.4:443-447.

[3]      Hiscock K M ,Lloyd J W,Lerner N. An engineering solution to the nitrate pollution of borehole at Swaffham ,Norfolk ,U. K.Journal of Hydrology ,1989 ,107 :267-281.

[4]      Andersen L J ,Kristansen H. Nitrate in groundwater related to landuse in the Karup Basin ,Denmark. Environmental Geology ,1984 ,5 (4) :207-212.

[5]      Shen Zhaoli ,Zhong Zuoxin ,Wang Min et al. Nitrogen pollution and transformation in groundwater system in urban area. The Proceedings of Scope Workshop on Groundwater Contamination in China.Beijing,1995.

[6]      John V. Occurrence and distribution of pharmaceutical organic compound in groundwater down gradient of a landfill. Environmental Science and Technology ,1995 ,29 (5) :1415-1420.

[7]      Zhang Sheng,Zhang Cuiyun,Xiao Yu. Effect Denitrification on Transformation in the Unsaturated Zone of Soil. Agro-environmental protection. 2002,21(3):254-256.

[8]      Wu Yaoguo. Denitrification in groundwater systems. Techniques and Equipment for Environmental Pollution Control. Mar.,2002 Vol.3,No.3:27-31.

[9]      A.Mohseni-Bandpi, D.J.Elliot, Groundwater denitrification with alternative carbon sources, Water Sci.Technol. 38 (6) (1998)237-243.

[10]  B.O.Mansell, E.D.Schroeder, Biological denitrification in a continuous flow membrane reactor, Water Res. 33 (8) (1999) 1845-1850.

[11]  B.Moreno, M.A.Go’mez, A.Ramos, J.Gonza’lez-Lo’pez, E.Hontoria, Influence of inocula over start up of a denitrifying submerged filter applied to nitrate contaminated groundwater treatment, Journal of Hazardous Materials B127 (2005) 180-186.

[12]  Hu Liming. Permeable Reactive Barriers for Remedy of Contaminated Groundwater Water. Resources and Hydropower Engineering. Vol.34 No.7,2003:11-13.

[13]  Li Zhengyu,He Tengbing,Pan Caiping,Yang Xiaomao. Apply in Renovation of Wastewater by Constructed Rapid Infiltration. Journal of Soil and Water Conservation. Dec.,2004 Vol.18 No.6:41-44.

[14]  Li Ping, Zheng Yongliang, Chen Shuli, et.al. Identification of An Aerobic Denitrifying Bacterium and Its Potential Application in Wastewater Treatment. Chin J Appl Environ Biol, 2005,11(5):600-603.

[15]  Zheng Ping, Laboratory Manual for Environmental Microbiology[M] Zhejiang: Zhejiang University Press, 2005.

[16]  Frette L, GejlsbjergB, Westermann P. Aerobic denitrifiers isolated from an alternating active sludge system. FEMS Microbiol Ecol, 1997, 24:363-370.

[17]  Robert son L A, VAN Niel ED W J, ROB A M. Torremans et al. Simultaneous nitrification and denitrification in aerobic chemostat cultures of Thiosphaeera Pantotropha. Applied and Environmental Microbiology, 1988, 54 (11):2812-2818.

[18]  Gupta A B. Thiosphaeera Pantotropha a sulphur bacterium capable of simultaneous heterotrophic nitrification and aerobic denitrification. Enzyme and Microbile Technology, 1997, 21 (8):589-595.

[19]  Chen F, Xia Q, Ju LK. Aerobic denitrification of Pseudomonas aeruginosa monitored by online NAD (P) H fluorescence. Appl & Environ M icrobiol, 2003, 69 (11):6715-6722.

[20]  Takaya N, Catalan-Sakairi MAB, Sakaguchi Y, Kato I, Zhou ZM, Shoun H. Aerobic denitrification bacteria that produced low levels of nitrous oxide. Appl & Environ Microbiol, 2003, 69 (6):3152-3157.

[21]  Hu LL, Wang JL, Wen XH, Qian Y. Improvements on biological nitrogen removal under low dissolved oxygen concentration. Chin J Appl Environ B iol, 2003, 9 (4):444-447.

[22]  Paturead D, Zumstein E, Delgenes JP, Moletta R. Aerobic denitrifiers isolated from diverse natural and managed ecosystems. Microb Ecol,2000, 39: 145-152.

[23]  Ma Fang, Wang Hongyu, Zhou Dandan. Selection and enrichment of aerobic denitrifier in activated sludge system. Journal of Hunan University of Science & Technology (Natural Science Edition). Jun.,2005, Vol.20, No.2:80-83.