functional diversity changes of microbial communities along a soil aquifer for reclaimed water...

R E S EA RCH AR T I C L E

Functional diversity changes of microbial communities along asoil aquifer for reclaimed water recharge

Xue Zhang, Xuan Zhao & Meng Zhang

Laboratory of Environmental Technology, Institute of Nuclear and New Energy Technology, Tsinghua University, Beijing, China

Correspondence: Xuan Zhao, Laboratory of

Environmental Technology, Institute of

Nuclear and New Energy Technology,

Tsinghua University, Beijing 100084, China.

Tel.: +8610 62796428; fax: +8610

62771150; e-mail: [email protected]

Received 12 July 2011; revised 15 October

2011; accepted 14 November 2011.

Final version published online 11 January

2012.

DOI: 10.1111/j.1574-6941.2011.01263.x

Editor: Julian Marchesi

Keywords

groundwater recharge; soil aquifer

treatment; BIOLOG assay; soil-attached

microbial community; reclaimed water.

Abstract

The physiochemical and functional diversity of soil-attached microorganisms

was investigated using a stabilized laboratory-scale soil aquifer treatment (SAT)

system. In this system, reclaimed water after ozonation was used as the feed

water, and 60% dissolved organic carbon was removed by the unsaturated

vadose layer in 0.8 days. Soil biomass (volatile solids, phospholipid extraction)

and functional diversity significantly decreased from the unsaturated vadose

layer to the saturated aquifer, where they maintained the same level. Using

principal components analysis based on substrate utilization pattern, the vadose

layer soil sample was clearly separated from the saturated layer samples. Excep-

tionally, the oxidation rates of esters remained stable during SAT, indicating

the purification potential on certain recalcitrant organic compounds in the sat-

urated aquifer given an adequate retention time. Correlation analysis revealed

that organic carbon was the key limiting factor for microbial biomass and

activity, especially for tyrosine-like aromatic proteins and soluble microbial

byproduct-like materials.

Introduction

Water stress attributable to water scarcity and quality deg-

radation is occurring in many regions of the world.

Reclaimed water, considered a promising alternative water

resource, has as a result gained an increasing level of atten-

tion. Artificial groundwater recharge (AGR) used with

reclaimed water is an attractive option for water reuse

because of the additional advantages in seasonal and long-

term water storage and control of saltwater intrusion

(Miller, 2006). During AGR, soil aquifer treatment (SAT)

provides final purification of the reclaimed water and is

considered a sustainable, effective and economic method of

organic matter removal (Fox et al., 2005; Zhao et al.,

2009). In SAT, removal of organic matter is primarily

attributed to biodegradation, especially aerobic biodegrada-

tion (Quanrud et al., 2003; Kolehmainen et al., 2007; Xue

et al., 2009). Thus, the microbial community in SAT plays

an important role in the attenuation of organic pollutants.

The dynamics of microbial communities and their role

in pollutant removal is one of the hottest topics in SAT.

A large number of studies on microorganisms suspended

in pore water have revealed that during SAT, the bacterial

cell concentration/extracellular enzyme activities (EEAs),

e.g. a-D-glucosidase and phosphomonoesterase, were sig-

nificantly correlated with nutrient concentrations [e.g.

dissolved organic carbon (DOC)/biodegradable dissolved

organic carbon (BDOC)] of the water (Hendel et al.,

2001; Kolehmainen et al., 2007, 2009). Moreover, the bac-

terial community changed from an Actinobacteria-domin-

anted population in lake water to a diverse and then

primary proteobacterial community after travelling 0.6 m

in the sand column (Kolehmainen et al., 2008).

However, the presence of soil-attached microbial bio-

masses cannot be disregarded. Numerous studies on

aquatic ecosystems have shown higher cell-specific activi-

ties of attached vs. suspended bacteria and have provided

evidence that the majority of biodegradation occurs

through soil-attached microbial communities (Harvey

et al., 1984; Middelboe et al., 1995; Grossart & Simon,

1998). Some indicators, such as EEAs and soil biomass,

have been used to characterize the dynamics of soil

microbial communities during SAT. A strong positive

correlation was found between total viable soil biomass

and organic carbon removal in SAT (Rauch & Drewes,

2005; Rauch-Williams & Drewes, 2006). In using EEAs,

FEMS Microbiol Ecol 80 (2012) 9–18 ª 2011 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

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no major differences were discerned between the surface

and bottom sediments (Kolehmainen et al., 2009). How-

ever, Schutz et al. (2010) found a significant decrease in

EEAs with soil depth. Different results may be attribut-

able to different sites or to the relatively low sensitivity of

the methods. Although soil biomass and enzyme activities

provide some data, the dynamics and the role of the

microbial community during SAT remain relatively

unknown. Novel sensitive methods need to be applied to

characterize microbial communities.

Generally, functional diversity is essential in understand-

ing the role of microbial communities in different environ-

ments (Preston-Mafham et al., 2002). The BIOLOG assay

has proved to be a useful and sensitive method in distin-

guishing functional differences among microbial commu-

nities from various habitats (Garland & Mills, 1991;

Firestone et al., 1998; Weber et al., 2008; Al-Mutairi,

2009). Therefore, the BIOLOG assay is helpful in the inter-

pretation of biodegradation capabilities during SAT.

In the present study, the BIOLOG assay is used to

distinguish the functional diversity of soil-attached micro-

bial communities in laboratory-simulated SAT systems,

wherein secondary effluent after ozonation was supplied as

feed water. The appearance, distributions and biomasses of

soil-attached microbial communities were detected using

environmental scanning electron microscopy (ESEM),

analysis of volatile solids (VS) and phospholipid extraction

(PLE). Thereafter, correlations between the characteristics

of the microbial communities (biomass, functional diver-

sity, metabolic activity) and the nutrient availability of feed

water were analysed to identify the role of microbes in SAT.

Materials and methods

The laboratory-scale SAT system

The SAT system was simulated using five soil columns

(Fig. 1). The internal diameters were 12 cm for the first

column (C1) and 24 cm for the other four columns

(C2–C5). All five columns were 200 cm in height, with a

packed-bed height of 180 cm. These columns were filled

with sandy powdery soil (grain size 0.4–0.8 mm) with

porosity of 0.39 collected from a 9- to 17-m-deep aquifer

in a suburb of Beijing. The C1 column was operated

under unsaturated conditions by pumping reclaimed

water into the top of the column, with the cycle compris-

ing 3 days of flooding and 1 day of drying. This column

was used to simulate the vadose soil layer. The other four

columns had sealed lids and were operated under satu-

rated conditions (constant flooding), representing the

saturated aquifer layer. All columns were kept at room

temperature (20 ± 2 °C) in the dark.

The secondary effluent from the Gaobeidian Wastewa-

ter Treatment Plant (WWTP), which used a traditional

activated sludge treatment process, was supplied as the

feed water of C1 after the addition of 5 mg L�1 ozone.

The ozone dosage was optimized in a previous study to

achieve a higher BDOC/DOC ratio and enhanced biode-

gradability (Liu, 2003). The effluent of C1 was stored in a

10-L tank, and a portion of this effluent was pumped in

sequence through columns C2–C5. The cumulative

hydraulic retention times of columns C1–C5 were 0.8,

7.8, 14.8, 21.8 and 28.8 days, respectively. The system was

biologically adapted and ready for sampling approxi-

mately after 1 year of acclimatization, with stable quality

effluents monitored at least once a week.

Water sample collection and physiochemical

analysis

The secondary effluent from the Gaobeidian WWTP (R0)

and the effluents after ozonation (R1), as well as all

the effluents from the bottom of the five soil columns

(C1–C5) were identified as WS1, WS2, WS3, WS4 and

WS5, respectively, and were sampled at least once a week.

All water samples were first filtered using a 0.45-lm filer,

and the following parameters were then measured:

DOC, absorbance at 254 nm (UV254), nitrate (NO3-N),

ammonia (NH4-N), phosphate (PO4-P) and three-dimen-

sional excitation-emission matrix (EEM) spectra. DOC

Tank

C1

2

C2 C3 C5 C4

Ozone detector

Ozonizor

Ozone decomposer

Tank for R0 Ozone reactor

Fig. 1. Schematic diagram of the laboratory-

scale AGR system.

ª 2011 Federation of European Microbiological Societies FEMS Microbiol Ecol 80 (2012) 9–18Published by Blackwell Publishing Ltd. All rights reserved

10 X. Zhang et al.

was measured using a Shimadzu Total Organic Carbon

Analyzer (TOC-VWP, Shimadzu Corp., Japan). UV254 was

analysed using a Shimadzu UV-3100 UV-visible spectro-

photometer (Shimadzu) at 254 nm. Nitrate (NO3-N),

ammonia (NH4-N) and phosphate (PO4-P) were measured

according to the specifications of the Ministry of Environ-

mental Protection PR China (2002). The average water

quality parameters of 5 weeks’ worth of records (2 weeks

prior, 2 weeks after and the week during soil sampling) are

listed in the Results. EEM spectra were measured using an

F-7000 FL spectrophotometer (Hitachi, Japan) to identify

the dissolved organic matter (DOM) composition changes.

The EEM spectra were a collection of corresponding scan-

ning emission spectra (Em) from 280 to 550 nm at 2-nm

increments through variation of the excitation wavelength

(Ex) from 220 to 450 nm at 5-nm sampling intervals. The

excitation and emission slits were maintained at 5 nm, and

the scanning speed was set at 1200 nm min�1. The spec-

trum of super-Q water was recorded as the blank control

and subtracted from the EEM spectra of all samples. The

normalized region-specific excitation–emission area vol-

ume (ui,n) and the percentage fluorescence response (Pi,n)

of region i were calculated using the fluorescence regional

integration (FRI) technique (Chen et al., 2003).

Soil sample collection and preparation

After 1 year of operation, the physicochemical parameters

of feed water and all effluents in the SAT were stable,

implying that mature and steady microbial communities

were formed during SAT. Four soil samples (equal to

50 g dry soil weight each) were scratched from the top

(depth of 5–10 cm) of columns C1–C4 using a clean steel

scoop. The four soil samples were identified as S1, S2, S3

and S4, respectively, representing soil samples from differ-

ent soil depths. All samples were stored in sterile blue-

capped bottles at 4 °C before analysis.

ESEM examination

Micrographs of the four soil samples were obtained using

ESEM (FEI Quanta 200; FEI, Czech Republic) without

any pretreatments. One clean sand sample prepared from

1 mL S4 and was used as the blank control. This control

sample was first washed with 1% NaOH to remove

microorganisms before being observed by ESEM.

Soil biomass quantification

Total biomass accumulation in the soil was identified as

VS and phospholipids. VS were identified according to

standard APHA 2540 (2005) through sand combustion at

550 °C. PLE quantifies the amount of phospholipids in

the cell walls of viable cells biomass using a colour reac-

tion with ammonium molybdate and malachite green, as

described by Rauch-Williams & Drewes (2006). Each soil

sample was measured in triplicate.

BIOLOG assay

The microbial community was first extracted from the

soil samples. Each soil sample (equal to 45 g dry weight)

was suspended in 35 mL sterile saline solution (0.85%

NaCl) on a rotary shaker at 240 r.p.m. for 30 min at

25 °C and then shaken in an ultrasonic cleaning machine

(Shanghai Lvyu Biotech Co., KQ-500E, 40 kHz, 100 W)

for 3 min. The microorganisms were proven to be effec-

tively detached from the soil without lysis during the pro-

cesses (Joyce et al., 2003). The suspensions were allowed

to settle for 5 min. The supernatants were transferred to

50-mL tubes and centrifuged at 1400 g for 5 min. The

supernatants were again transferred to new tubes and

diluted with sterile saline solution, resulting in a uniform

OD600 nm of 0.06 cm�1 (Wang et al., 2009). Microbial

suspensions of 150 lL were added to each well of the

BIOLOG microplate, and were incubated under aerobic

conditions at 30 °C in the dark.

Each BIOLOG ECO plate (Biolog Inc., Hayward, CA)

consists of three replicates of 96 wells, each comprising

31 sole carbon sources and one water blank control.

Metabolism of the substrate in particular wells results in

a colour change in the tetrazolium dye. Absorbance read-

ings of the plates were taken at 595 nm every 4 h for

168 h, using a microtitre plate reader (MD SpectraMax

MS, MD Inc.).

Data analysis

The well absorbance values of the BIOLOG plate were

adjusted through subtraction of the blank control well.

The average well colour development (AWCD), calculated

as the average adjusted absorbance of all wells per plate,

was used as an indicator of general microbial activity

(Garland & Mills, 1991). The absorbance data for the 31

individual carbon substrates at 72.6 h was used to quan-

tify C-source utilization using principal components anal-

ysis (PCA) because the AWCD value at 72.6 h preserved

the greatest variance between well responses while keeping

the maximum number of wells within the linear absor-

bance range (Weber et al., 2008). PCA was performed

using SPSS software with standardized absorbance data

(Wang et al., 2009). Differences in microbial functional

diversity among soil samples were also compared using the

mean Shannon–Weaver indices. Following the formula of

Al-Mutairi (2009), richness (R, the number of oxidized

carbon substrates) and the Shannon–Weaver index (i.e.


Microbial dynamic during SAT for water recharge 11

diversity H′ and evenness index J′) were calculated using

an OD of 0.25 as the threshold for positive response (Go-

mez et al., 2006). The substrate oxidation rate (cm�1 h�1)

was calculated as the slope of the colour development

curve (e.g. AWCD curve) during its linear phase.

Pearson product-moment correlations among the

parameters were conducted using SPSS 14.0 software (SPSS

Inc.).

Results and discussion

Physiochemical characteristics of water during

the laboratory-scale SAT

The physicochemical parameters of water quality at each

sampling point are listed in Table 1. The organic matter

content was presented as DOC, UV254 and SUVA (spe-

cific UVA, which is equal to UV254/DOC). Robust

removal of DOC in laboratory-scale SAT was certified

during the 1 year of operation. DOC did not decrease

after ozonation, whereas more than 60% of the DOC was

removed within a 1.8-m unsaturated vadose soil layer

(column C1), and an additional 19% was removed after

7 days travel within the first 1.8 m of saturated aquifer

treatment (column C2). These results are consistent with

previous laboratory and field studies (Quanrud et al.,

2003; Vanderzalm et al., 2006; Zhao et al., 2009). The

long-term operation of SAT with high and constant

removal efficiency of organic pollutants indicates that

biodegradation, rather than adsorption, is the primary

process, as has also been shown using abiotic control in

previous studies (Quanrud et al., 2003). Based on the

high removal efficiency of DOC by the unsaturated

vadose layer, aerobic degradation was considered to be

primarily responsible for DOC removal.

UV254 decreased by 60% after ozonation, whereas 13%

and 10% removal was achieved along columns C1 and

C2, respectively. However, both DOC and UV254 changed

slightly in later saturated aquifer treatments (columns C3

–C5). SUVA, which represents the relative aromaticity of

the bulk organic compounds, decreased dramatically after

ozonation, but increased during SAT. These results indi-

cated that aromatic compounds are preferentially

destroyed by ozonation, whereas more aliphatic com-

pounds are removed by the sand columns. The increase

in SUVA during SAT may be attributable to enrichment

of aromatic compounds through biodegradation (Xue

et al., 2009).

Most NH4-N was removed during ozonation in the

unsaturated vadose layer, whereas slight denitrification

occurred in the saturated aquifer, and total nitrogen

could be reduced by 1.5–2.0 mg L�1. Most PO4-P was

removed in the first 7 days of travel in the saturated aqui-

fer (column C2). SAT ran steadily with good purification

performance for over 1 year, indicating its stability.

ESEM micrographs and biomass qualification of

the soil samples

ESEM micrographs of sand samples S1–S4 were obtained

at various magnifications (Fig. 2). Obvious biofilms were

found on the surface of S1 and S2, where bacteria sur-

rounded by extracellular mucilages appeared to be the

primary colonizers. The appearance of S3 and S4 was sig-

nificantly similar to clean sand (blank control), indicating

the existence of fewer microorganisms in the deeper satu-

rated aquifer after 7 days of travel. Some unicellular

eukaryotes (algae/fungi/prozotoa) were also found in sand

samples S1 and S2, but were sparse in samples S3 and S4.

Based on the ESEM micrographs, an obvious decrease

in biofilm formation was found during SAT. The biomass

contents of soils were determined quantitatively as VS

and phospholipids. The VS content, representing the total

biomass, was clearly higher in S1 (7.8 mg g�1) than in

the other three samples, whereas S2, S3 and S4 had simi-

lar VS contents, ranging from 5.3 to 5.8 mg g�1

(Table 2). Similar results were determined based on PLE,

the phospholipids representing the viable biomass. The

phospholipid content in S1 (32.6 nmol PO3�4 g�1) was

significantly higher than in the other three soil samples,

Table 1. Physicochemical water quality at each sampling point

Sample R0* R1* WS1* WS2* WS3* WS4* WS5*

DOC (mg L�1) 4.35 (0.25) 4.31 (0.30) 1.73 (0.62) 0.91 (0.24) 0.87 (0.07) 0.82 (0.10) 0.91 (0.14)

UV254 (m�1) 11.50 (0.34) 4.44 (1.39) 2.92 (1.06) 1.84 (0.44) 1.88 (0.29) 1.72 (0.33) 2.26 (0.21)

SUVA [L (mg m)�1] 2.64 1.03 1.69 2.03 2.15 2.11 2.49

NH4-N (mg L�1) 1.02 (0.75) 1.08 (0.69) 0.18 (0.03) 0.14 (0.02) 0.13 (0.01) 0.13 (0.02) 0.13 (0.01)

NO3-N (mg L�1) 21.92 (4.44) 21.88 (4.23) 23.17 (2.84) 22.86 (1.89) 20.73 (0.98) 20.61 (0.71) 20.88 (0.76)

PO4-P (mg L�1) 2.49 (0.35) 2.43 (0.35) 1.68 (0.99) 0.73 (0.01) 0.91 (0.05) 1.09 (0.10) 0.88 (0.03)

pH 7.27 (0.21) 7.34 (0.18) 7.64 (0.31) 7.94 (0.28) 7.70 (0.30) 7.73 (0.26) 8.03 (0.24)

Data are presented as the mean (SD) of 5-week records.

*Water samples: R0, secondary effluent from Gaobeidian WWTP; R1, water sample after ozonation; WS1, WS2, WS3, WS4 and WS5, effluents

obtained from bottom of the five soil columns (C1–C5), respectively.


12 X. Zhang et al.

Fig. 2. ESEM micrographs of the sand from

the top of different sand columns and one

washed sand sample (S1–S4).

Table 2. Soil biomass and microbial functional diversity of the four soil samples*

Sample VS (mg g�1)†PLE (nmol

PO3�4 g�1)† H′ R J′

S1** 7.5 (0.8) 32.6 (3.8) 2.97 (0.05) 30 (1.1) 0.87 (0.01)

S2** 5.8 (0.6) 13.0 (2.6) 0.95 (0.46) 8 (4.0) 0.45 (0.10)

S3** 5.3 (0.9) 13.3 (5.1) 1.04 (0.26) 6 (2.1) 0.58 (0.04)

S4** 5.7 (0.7) 13.9 (4.7) 1.78 (0.02) 11 (0) 0.74 (0.01)

*Data are presented as the mean (SD) of triplicate experiments. H′, Shannon–Weaver diversity index; R, number of oxidized carbon substrates; J′,

Shannon–Weaver evenness index.

**S1, S2, S3, S4 were four soil samples taken from the top of columns C1–C4, respectively.†Given as the amount per gram of dry soil.



which ranged from 13.0 to 13.9 nmol PO3�4 g�1. Both VS

and PLE data indicate that biomass in the soil sharply

decreased from the unsaturated vadose soil layer to the

saturated aquifer layer, whereas biomass remained at the

same level in the saturated layer (S2–S4). The decrease in

soil biomass co-occurred with the decrease in DOC

concentration during SAT.

BIOLOG assay of the soil samples

Using BIOLOG Eco plates, the metabolic activities of the

soil-attached microbial community were studied during

SAT. AWCD, as a measure of total microbial metabolic

activity, generally followed similar patterns with incuba-

tion time (Fig. 3). Clearly, a higher AWCD value was

detected in sample S1 than in the other three soil sam-

ples. The average substrate oxidation rate of S1 was

0.025 cm�1 h�1, approximately four times higher than

those of the other samples (0.005–0.007 cm�1 h�1). These

results indicate that significantly higher metabolic activity

was detected in the microbial community associated with

unsaturated vadose soil. Moreover, the lag time prior to

colour development was significantly shorter for S1

(25 h) than for the other three soil samples (over 50 h).

The lag time is determined by the initial inoculum den-

sity and relative growth rates of the species that are capa-

ble of utilizing the carbon source within each well

(Preston-Mafham et al., 2002). The four suspensions were

incubated at the same cell densities on the BIOLOG

plates, so the variance in lag time resulted from the dif-

ferent growth rates of the microbial communities. Thus,

the microbial community in sample S1 actively respired

and grew faster than those in the other samples. The

obviously higher metabolic activity in the top unsaturated

soil layer can explain the bulk organic carbon removal

within the upper vadose layer of 1.0–2.0 m (Drewes &

Fox, 1999; Rauch-Williams & Drewes, 2006; Vanderzalm

et al., 2006).

More specifically, the carbon sources in the BIOLOG

Eco plate were divided into seven groups (Jena et al.,

2006). A comparison of the substrate oxidation rates of

individual carbon source groups was then conducted

(Fig. 4). Most of the substrate (except for esters) oxida-

tion rates in sample S1 were significantly higher (approxi-

mately 2.3–9.5 times higher) than those in S2–S4,whereas those in the latter three (S2–S4) samples were at

roughly the same level. These results are consistent with

the average metabolic rates, which revealed a significant

decrease in metabolic activities from the unsaturated

column to the saturated aquifer. Exceptionally, the

oxidation rates of esters were all approximately 0.020

–0.028 cm�1 h�1, revealing their uniform metabolic capa-

bilities during SAT. Additionally, the lag times for ester

oxidation were 22 and 31 h for S1 and S2–S4, respec-

tively. The differences in lag time were significantly smal-

ler for esters than for AWCD. The ester-degrading

microorganisms were distributed evenly and remained

active during SAT, in dramatic contrast to other microor-

ganisms.

Regarding the unique distribution of ester-degrading

bacteria, there are two aspects to consider. First, the con-

tinuous presence of certain carbon sources is required in

the recharged water, meaning that such carbon sources

are refractory. Humic acids, fluvic acids and certain trace

pollutants (i.e. sulfamethoxazole and EDTA) are reported

as refractory compounds, possibly producing these results

(Drewes & Jekel, 1998; Conn et al., 2010; Maeng et al.,

Fig. 3. AWCD of the BIOLOG Eco plates for microbial samples from

different sand columns.

Fig. 4. Substrate oxidation rate of each carbon source group with

different microbial communities (CH, carbohydrates; CA, carboxylic

acids; AA, amino acids; PC, phosphorylated compounds).


14 X. Zhang et al.

2011). Secondly, the uniform metabolic activity of certain

microorganisms indicated the deeper purification of cer-

tain recalcitrant organic compounds, and this process

would occur in the saturated aquifer layer. Therefore, an

adequate retention time is especially important for the

removal of refractory materials, which is consistent with

previous laboratory and field studies that monitored the

removal characteristics of organic matter (Grunheid et al.,

2005; Zhang et al., 2007).

Differences in microbial functional diversity among the

soil samples were compared using mean Shannon–Weaver

indices (Table 2). All three indices (diversity H′, richnessR and evenness J′) of sample S1 were significantly higher

than those of samples S2–S4, whereas values for S2–S4were similar. These results clearly indicate that the micro-

bial community associated with S1 had a higher func-

tional diversity than the other microbial communities.

The functional differences among the four communi-

ties were analysed in detail using PCA based on the

absorbance data for the 31 individual carbon substrates at

72.6 h (Fig. 5 and Table S1). Clear separation of S1 and

the other three soil samples (S2, S3 and S4) were detected

based on PC1, which explains 45.0% of the variance.

According to the study of Garland & Mills (1991), the

most important carbon sources in differentiating among

the communities were defined as those that had at least

half of their variance explained by PCs (Table 3). On the

basis of PC1, S1 had positive coordinates, whereas S2–S4had negative coordinates. The microbial community in S1

utilized several carbohydrates (D-xylose, D-cellobiose,

i-erythritol, methyl b-D-glucoside, a-D-lactose, D-mannitol

and N-acetyl-D-glucosamine), carboxylic acid (2-hydroxy-

benzoic acid), amino acids (L-serine), polymers (glycogen,

a-cyclodextrin) and amines (phenylethylamine) to a

greater degree, and several carboxylic acids (c-hydroxybu-tyric acid, a-ketobutyric acid, D-galactonic acid lactone

and itaconic acid), phosphorylated chemicals (DL-a-glyc-erol phosphate), amino acids (L-threonine, L-phenylala-

nine) and esters (pyruvic acid methyl ester) to a lesser

degree compared with microbial communities associated

with S2–S4. PC2 explained 19.5% of the variance, and

coordinate values increased in the saturated aquifer along

the flow path (S2–S4). Separation of these soils largely

resulted from the greater utilization of esters (pyruvic

acid methyl ester), carboxylic acids (D-glucosaminic acid),

phosphorylated chemicals (glucose 1-phosphate) and

amino acids (glycyl L-glutamic acid) and lesser utilization

of amino acids (L-asparagine), carboxylic acids

(4-hydroxy benzoic acid, and itaconic acid), polymers

(tween 40) and amines (putrescine). These results indicate

that the functional structures of the microbial community

remained steady in the saturated aquifer, consistent withFig. 5. Score plots of the PCA based on 72.6 h absorbance data.

Table 3. Correlation of carbon source variables to PCs

PC1 PC2

Carbon source r* Carbon source r*

Amines Amines

Phenylethylamine 0.92 Putrescine �0.54

Amino acids Amino acids

L-Serine 0.69 Glycyl L-glutamic

acid

0.87

L-Threonine �0.89 L-Asparagine �0.90

L-Phenylalanine �0.65 Carboxylic acids

Carbohydrates D-Glucosaminic acid 0.72

D-Cellobiose 0.92 4-Hydroxy benzoic

acid

�0.88

D-Xylose 0.90 Itaconic acid �0.61

i-Erythritol 0.85 Esters

Methyl b-D-Glucoside 0.73 Pyruvic acid methyl

ester

0.74

a-D-Lactose 0.67 Phosphorylated chemicals

D-Mannitol 0.58 Glucose

1-phosphate

0.74

N-Acetyl-D-glucosamine 0.50 Polymers

Carboxylic acid Tween 40 �0.75

2-Hydroxybenzoic acid 0.84

c-Hydroxybutyric acid �0.99

a-Ketobutyric acid �0.97

D-Galactonic acid

lactone

�0.79

Itaconic acid �0.69

Esters

Pyruvic acid methyl

ester

�0.63

Phosphorylated chemicals

DL-a-Glycerol phosphate �0.90

Polymers

Glycogen 0.86

a-Cyclodextrin 0.82

*Pearson’s regression coefficient.



the result showing slight variation of bulk organic com-

pounds found in the recharged water of the saturated soil

aquifer.

EEM spectra of the water samples

The different compositions of DOMs in the water sam-

ples were identified in the EEM spectra and were quanti-

fied as the normalized region-specific excitation–emission

area volumes (ui,n) using FRI (Chen et al., 2003). The

fluorescence intensity in all five regions decreased signifi-

cantly after ozonation, with approximately 60% removal

of total ui,n. In particular, removal of fulvic acid-like

(Region III, Ex < 250 nm, Em > 380 nm) and humic

acid-like compounds (Region V, Ex > 280 nm, Em >380 nm) was relatively higher (approximately 70%). By

contrast, around 30% of total ui,n was removed during

SAT, with significantly higher removal of 60% on aro-

matic proteins (Region I, Ex < 250 nm, Em < 330 nm)

and 40% on soluble microbial byproduct-like compounds

(Region IV, Ex = 250–280 nm, Em < 380 nm). Clearly,

differences in the removal preferences of fluorescent

DOMs were determined between ozonation and SAT,

indicating complementary effects in this treatment system.

Correlation between metabolic activity and

nutrients

Microbial functional diversity (e.g. H′, R, J′) and biomass

(e.g. VS, phospholipids) of soil are considered to be

related to the water quality (e.g. DOC, UV254) of the

respective feed water. These correlations were investigated

using the Pearson product-moment correlation. DOC was

correlated significantly (P < 0.05) with average metabolic

rates, R, VS and PLE results. Other indicators of water

quality (i.e. UV254, N, P) showed no significant correla-

tion with microbial biomass or functional diversity

(P > 0.05). This result indicates that organic carbon con-

tent is the key limiting factor for development of micro-

organisms, which can be explained by C/N/P ratios.

Specifically, aliphatic compounds contributed more to

microbial community development than aromatic com-

pounds. Moreover, microbial biomass (VS and phospho-

lipids), diversity (R) and metabolic rate were significantly

correlated with each other, thus confirming the reliability

of the BIOLOG methods.

To gain deeper insight into the effects of DOM compo-

sitions on the microbial metabolic activity during SAT,

correlations between the metabolic rates of various carbon

substrates and Pi,n of various DOMs in the respective feed

water were analysed using the Pearson product-moment

correlation. Tyrosine-like aromatic proteins in Region I

and soluble microbial byproduct-like material in Region

IV were significantly correlated with the oxidation rates of

most carbon source groups (i.e. polymers, carbohydrates,

amino acids, amines and phosphorylated chemicals)

(P < 0.05). These findings agree with the results above,

showing that the preferential removal of Regions I and IV

was observed during SAT. However, the oxidation rates

of carboxylic acids and esters were not significantly corre-

lated with any of the fluorescent materials in the feed

water (P > 0.05), which may be attributable to the non-

fluorescent characteristics of responsible substrates. Fulvic

acid-like (Region III) and humic acid-like compounds

(Region V) were negatively correlated with all the oxida-

tion rates, which correspond to their refractory character-

istics in SAT (Drewes & Jekel, 1998; Maeng et al., 2011).

In conclusion, biomass analysis and BIOLOG assay,

coupled with ESEM, enable the dynamics of microbial

communities during SAT to be studied. Regarding the

fate of contaminants, soil biomass and functional diver-

sity in the unsaturated vadose layer were significantly

higher than those in the saturated aquifer, whereas they

remained at the same level along the saturated aquifer.

The vadose layer soil sample was demonstrated to be

clearly separated from the saturated layer samples using

PCA based on substrate utilization patterns. Exception-

ally, the oxidation rates of esters remained steady during

SAT, indicating the purification potential of the saturated

aquifer on certain recalcitrant organic compounds given

an adequate retention time.

Acknowledgements

This research was supported by Major Science and Tech-

nology Program for Water Pollution Control and Treat-

ment (Grant no. 2008ZX07314-008-04) and the National

Natural Science Foundation of China (Grant nos.

50878115 and 51078211).

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Supporting Information

Additional Supporting Information may be found in the

online version of this article:

Table S1. Carbon sources that could be utilized by differ-

ent column soil samples.

Please note: Wiley-Blackwell is not responsible for the

content or functionality of any supporting materials sup-

plied by the authors. Any queries (other than missing

material) should be directed to the corresponding author

for the article.


18 X. Zhang et al.

functional diversity changes of microbial communities along a soil aquifer for reclaimed water...

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