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Microbial diversity in anaerobic baffled reactorstreating nitrobenzene wastewaterYingzi Lin a b , Mingxin Huo a , Jianhui Wang c & Hai Lu ca School of Urban and Environmental Sciences, Northeast Normal University , Changchun ,130024 , PR Chinab Key Laboratory of Songliao Aquatic Environment, Ministry of Education, Jilin Architecturaland Civil Engineering Institute , Changchun , 130118 , PR Chinac Jinlin Province Laboratory of Urban Water Resource and Environmental RestorationEngineering , Changchun , 130118 , PR ChinaPublished online: 08 Aug 2013.

To cite this article: Yingzi Lin , Mingxin Huo , Jianhui Wang & Hai Lu (2013): Microbial diversity in anaerobic baffled reactorstreating nitrobenzene wastewater, Desalination and Water Treatment, DOI: 10.1080/19443994.2013.827778

To link to this article: http://dx.doi.org/10.1080/19443994.2013.827778

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Microbial diversity in anaerobic baffled reactors treatingnitrobenzene wastewater

Yingzi Lina,b,*, Mingxin Huoa, Jianhui Wangc, Hai Luc

aSchool of Urban and Environmental Sciences, Northeast Normal University, Changchun 130024, PR ChinaEmail: linyingzi1000@163.combKey Laboratory of Songliao Aquatic Environment, Ministry of Education, Jilin Architectural and CivilEngineering Institute, Changchun 130118, PR ChinacJinlin Province Laboratory of Urban Water Resource and Environmental Restoration Engineering,Changchun 130118, PR China

Received 25 June 2012; Accepted 10 July 2013

ABSTRACT

The microorganism diversity in the five compartments of an anaerobic baffled reactor (ABR)treating nitrobenzene wastewater was investigated. Fluorescence in situ hybridization wasused to test the amounts of archaea and eubacteria in the different compartments. The resultsshowed that the microbial diversity decreased longitudinally along the five compartments.The microbial diversity before acclimation was higher than after. However, the evennessindices in the different compartments were similar. The amounts of eubacteria were higherthan the archaea, the amounts of archaea and eubacteria in the reactor before acclimationwere greater than after. These results indicated that the microbial diversity and thepopulation in the reactor were greatly influenced by the nitrobenzene. The study providesinformation on microbial diversity for the ABR treatment of nitrobenzene wastewater.

Keywords: Nitrobenzene; Anaerobic baffled reactor; Eubacteria; Archaea; Diversity

1. Introduction

Nitrobenzene has been designated as “prioritypollutant” by USEPA on the basis of its known orsuspected carcinogenicity, mutagenicity, teratogenicity,or high acute toxicity [1] that NB wastewater causedby improper handling might contaminate the surfaceand groundwater and impact human health. Amongvarious physical, chemical, and biological methods forthe removal of nitrobenzene, the anaerobic biologicaltreatment option is the most cost-effective [2,3].The anaerobic baffled reactor (ABR) is a new third-

generation anaerobic technology which integratesupflow anaerobic sludge bed (UASB) with stagedmultiphase anaerobic reactor [4]. ABRs were used totreat traditional medicine industrial wastewaters, highsulfate wastewaters, and low-strength soluble waste-waters [5–9]. ABR has potential advantages in thetreatment of toxic wastewater [10–13].

A wide variety of micro-organisms thrive in ABRs.Their types change with the different substrates.Because the micro-organisms in an ABR reactor arefacultative and anaerobic, many micro-organisms aredifficult to culture or unculturable. This characteristicmeans that the micro-organisms cannot fully andaccurately express the microbial population in the*Corresponding author.

1944-3994/1944-3986 � 2013 Balaban Desalination Publications. All rights reserved.

Desalination and Water Treatmentwww.deswater.com

doi: 10.1080/19443994.2013.827778

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reactor through conventional culture methods,however, molecular biologic methods overcome thedisadvantages of conventional culture methods[14–17], and reflect the microbial population and thespatial distribution in ABRs more comprehensively.Fluorescence in situ hybridization (FISH) integratesthe fluorescently labeled oligonucleotide probe withthe RNA (rRNA) or DNA in morphologically intactcells. This technique combines precise molecularbiologic technology and visual microscopic observa-tion technology [18,19]. It is also a new biologicaltechnology for analyzing and identifying the spatialdistribution of a characteristic micro-organism [20,21].However, studies on the degradation efficiency ofnitrobenzene in different ABR compartments usingthe FISH technique and on microbial diversity havenot yet been reported. Fully understanding of themicrobial diversity of ABRs can provide a theoreticalbasis for improving the processing efficiency.

In this work, ABRs were employed to treat nitro-benzene wastewater and the molecular biotechnologywas applied to study the microbial diversity and thespatial distribution of granular sludges in each com-partment which provided information on the micro-bial diversity.

2. Materials and methods

2.1. Experimental equipment

Fig. 1 shows a flow chart of ABR in this study.Continuous operation was carried out during thewhole experiments. The volume of ABR reactor was12.8 L (415mm� 100mm� 308mm). Temperature con-trol was accomplished by temperature controller. Thestudy was conducted at mesophilic condition at 30(±0.1)˚C. The seed sludge was inoculated from a full-scale UASB. NaOH was added to make influent pHvalue at 6.5. Elastic packing made of polyethyleneplastic was filled into every compartments of ABR.

Gradually increasing the concentration of NB inwater during the acclimation process of sludge

ensures that the micro-organisms can adapt to the tox-icity of NB and then degrade it. The initial concentra-tion of nitrobenzene was from 2 to 67.35mg/L. Thehydraulic retention time was 24h. The nitrobenzeneload was increased step-by-step according to the efflu-ent concentration of nitrobenzene and COD removal.The detailed information about the experiment isdescribed by Lin et al. [22].

2.2. Sample collection and analysis

2.2.1. Conventional parameters

NB was measured by gas chromatography-massspectrometry (6890GC/5973MSD; Agilent). The col-umn is an HP-5MS (5% phenyl and 95% dim-ethylpolysiloxane phase), with the size 30m� 0.32mmI.D.� 0.25lm film thickness. The temperature pro-gram for the primary stage began at 35˚C, held for2min, and then rose to 150˚C at 1˚C/min. For the sec-ondary stage, the temperature was initially held at 20˚Cand then raised to 280˚C, and the final temperaturewas kept at 300˚C, held for 10min. The temperaturefor injector and detector were both 250˚C.

Samples were withdrawn from the liquid media atthe beginning and at the end of each treatment period.CODCr is measured with COD fast determinatormodel 5B-1; clear supernatants were analyzed forCOD; samples were analyzed in triplicate, and finallyaverage values were reported.

2.2.2. Calculation of diversity index

The two diversity indices, namely the Shannon–Wiener index (H) and the evenness index (E) [23]were commonly used. Diversity was measured usingthe Shannon index (H´) as follows:

H0 ¼ �X

pilog2pi ð1Þ

where pi is the proportion of individuals of the ithspecies given by ni/N, ni represents the number ofindividuals of the ith species, N is the total sampleabundance, and S is the total number of species in thesample. This index of species diversity is widely usedto incorporate both the species richness and equitabil-ity components of diversity.

Equitability was measured using the evennessindex (E) which is calculated as follows:

E ¼ H0=log2S ð2Þ

The two diversity indices, namely the Shannon–Wiener index (H) and the evenness index (E) [23]Fig. 1. Setup of ABRs.

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were commonly used. The Shannon-Wiener index (H)is calculated as follows:

H0 ¼ �X

pi � Inpi ð3Þ

where pi is the proportion of the clone number to thewhole clone number at the ith OUT, pi= ni/N, ni isthe whole clone number at the ith OTU, and N is thewhole clone number in the library.

The evenness index (E) is calculated as follows:

E ¼ H0=Ini ð4Þ

where i is the total number of micro-organisms in thesample (Emax = 1.0).

2.2.3. Fluorescence in situ hybridization (FISH)

The hybridization probe for the experiment con-sisted of eubacteria EUB338 (Cy3) and archaeaARC915 (FITC) probes. Approximately, 9 lL ofhybridization solution was added into the pretreat-ment sample. Then, 2lL probe solution was mixedevenly. Absorbent paper, saturated with 2� SSChybridization solution, was introduced into the cas-sette to maintain the humidity. The sample washybridized for 2.5 h at 46˚C. After hybridization,washing liquid was at 48˚C used for 20min, and theprobe was removed [24]. Then, distilled water wasused to wash away the washing liquor residue, andthe sample was dried naturally. Fluorescence micros-copy was used to test and observe the FISH results,and the proportion of the target bacteria to the totalbacteria was determined using the bundled software.The quantity of bacteria was computed as:

A ¼ B�M�D� V�1 ð5Þ

where A is the number of bacteria per unit volume; Bis the average number of bacteria in the microscopicfield; M is the ratio of the sample volume to thevolume of the microscopic field; D is the dilutionfactor of the sample; and V is the volume of thehybrid sample.

3. Results and discussion

3.1. Degradation characteristics of nitrobenzene in the fivecompartments of ABR

The HRT was maintained at 24 h, and the concen-tration of influent nitrobenzene was 67.35mg/L. Thewater quality was detected, three parallel samples

were chosen each time, and the average was com-puted to obtain the test data.

As shown in Fig. 2, when the influent nitrobenzeneconcentration was 67.35mg/L and that of the influentCOD was 1,064mg/L, the removal efficiency of CODin the first, second, and third compartments were 29,43 and 76%, respectively. The removal efficiency ofCOD in the first three compartments was higher. Therates in the last two compartments were 77 and90.4%, respectively, which indicate that the removalefficiency was low. The removal efficiency of nitroben-zene in the first three compartments was higher.When the influent nitrobenzene concentration was67.35mg/L, the nitrobenzene concentrations were51.51, 11.05, and 0.58mg/L in the first, second, andthird compartments, respectively. The degradation ofnitrobenzene in the compartments occurred mainly inthe first three compartments. The nitrobenzene con-centrations in the fourth and fifth compartments were0.42 and 0.46mg/L, respectively, indicating that thedegradation was stable and that the concentrationremains the same in the fourth and fifth compart-ments. The fastest nitrobenzene degradation wasoccurred in the first and second compartments.

3.2. Microbial diversity index analysis of each ABRcompartment

The distribution of the Shannon–Wiener index (H´)in the anaerobic granular sludge samples in thereactor before and after acclimation using the Biodapsoftware is shown in Table 1. In the two systems, themicrobial diversity (H´) in the anaerobic granularsludge samples clearly changed among the fivecompartments before and after acclimation, but theirevenness indexes (E) were close. This result indicated

0 1 2 3 4 50.1

1

10

100

1000

Influ

ent c

once

ntra

tion

(mg/

L)

Compartment

COD NBCOD removal efficiency NB removal efficiency

0

20

40

60

80

100

120R

emov

al e

ffici

ency

(%)

Fig. 2. The NB and COD concentration and removalefficiency in five compartments of ABR.

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Tab

le1

Thediversity

analysisofbacteriacommunitybefore

andafteracclim

ationin

ABR

Before

acclim

ation

After

acclim

ation

Microorgan

ism

First

compartm

entSecond

compartm

entThird

compartm

entFourth

compartm

entFifth

compartm

entFirst

compartm

entSecond

compartm

entThird

compartm

entFourth

compartm

entFifth

compartm

ent

CK1

CK2

CK3

CK4

CK5

NB1

NB2

NB3

NB4

NB5

H´

3.64

3.56

3.35

3.30

3.14

3.47

3.33

3.11

3.06

3.01

E0.93

0.93

0.92

0.92

0.92

0.93

0.93

0.92

0.91

0.93

CK:before

acclim

ationdep

artm

ent;NB:afteracclim

ationdep

artm

ent.

Tab

le2

Amountofeu

bacteriaan

darch

aeain

fiveABRscompartm

ents

After

acclim

ation

Before

acclim

ation

Microorgan

ism

type

First

compartm

entSecond

compartm

entThird

compartm

entFourth

compartm

entFifth

compartm

entFirst

compartm

entSecond

compartm

entThird

compartm

entFourth

compartm

entFifth

compartm

ent

Bacteria

(cells/mL

sample�10

9)

0.24

0.32

0.46

0.52

0.66

0.79

1.96

2.36

3.53

2.55

Archaea

(cells/mL

sample�10

9)

0.08

0.12

0.24

0.42

0.35

1.25

1.13

1.57

1.96

1.34

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that although the diversity of the microbial populationchanged greatly, the amount and spatial distributionin each compartment were uniform.

Before acclimation, the microbial diversity (H´)sequence in the reactor was CK1>CK2>CK3>CK4>CK5, which indicated that microbial diversitygradually decreased longitudinally down the reactor.As shown in Table 1, the diversity in microbial popu-lation clearly changed in each compartment beforeacclimation. The H´ in the CK1 sludge samples was

3.64 in the first compartment, the highest among allsamples. Then, the value decreased to 3.14 in the fifthcompartment along the direction of the water flow.The main reasons for the higher microbial diversityindices in the first two compartments were the aero-bic, facultative, and anaerobic groups that comprisedthe microbial population. The microbial populationgradually transitions to facultative and anaerobicbacteria along the direction of the flow. Therefore,anaerobic microbial populations were mainly in the

Fig. 3. Spatial distribution of eubacteria and archaea in each ABR compartmen after acclimation.

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subsequent compartments, and the microbial diversitygradually decreased. Given the high organic matterconcentration in the front compartments, microbialnutrients were adequate for the micro-organisms,which was the reason for the higher microbial diver-sity in the front compartments. With the progressionof the treatment along the compartments, the organiccarbon sources contents in the water decreases and

many microbial populations were eliminated. Anaero-bic micro-organisms replaced the facultative microbialpopulations to become the main force in the degrada-tion of pollutants, and the diversity of the microbialpopulation decreased.

After the acclimation of nitrobenzene, the micro-bial diversity sequence in each ABR compartment wasNB1>NB2>NB3>NB4>NB5. This sequence showed

Fig. 4. Spatial distribution of eubacteria and archaea in each ABR compartmen before acclimation.

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that the microbial population diversity in the toxicwastewater treatment process significantly decreasedfrom the first compartment to the fifth compartment.Table 1. The population diversity in each compart-ment was lower after than before acclimation. In thefirst compartment, the H´ was 3.64 before acclimationand 3.47 after acclimation. In the fifth compartment,the H´ was 3.14 before acclimation and 3.01 afteracclimation; the diversity index was higher beforeacclimation. After the long-acclimation period, thenitrobenzene played a screening role in ABR. Afteracclimation, some populations were eliminated,whereas the remaining micro-organisms adapted tothe toxic environment and gradually established theirdominant position.

3.3. FISH analysis of micro-organisms in eachABR compartment

3.3.1. The amount of eubacteria and archaea in differentcompartments

Florescence in situ Hybridization (FISH) was usedto analyze the amount and spatial distribution in thegranular sludge samples of ABR before and afternitrobenzene acclimation using eubacteria EUB338and archaea ARC915 probes. This test quantitativelydetected the samples in each compartment of the reac-tor and then analyzed the quantities of eubacteria andarchaea. The results are shown in Table 2, which indi-cates that the number of eubacteria is greater than thatof the archaea in the granular sludge samples. Inaddition, the amount of eubacteria and archaea wasgreater before nitrobenzene acclimation than that afteracclimation. Before acclimation, the archaeal contentof the granular sludge sample in the fourth compart-ment was highest, up to 3.53� 109 cells/mL, which is6.7 times greater than that after acclimation. On theother hand, the highest eubacterial content was also inthe fourth compartment before acclimation. However,because the micro-organisms were shocked by thenitrobenzene toxicity in the ABRs, the populationstructure of the microbes in the sludge was greatlyinfluenced, resulting in an inhibitory effect on the bac-teria. The amount of bacteria was lower after acclima-tion. The number of bacteria decreased in the fifthcompartment because of the consumption of nutrients.

3.3.2. FISH monitoring of the spatial distribution ofgranular sludge in each compartment

The FISH results of the granular sludge samples inthe reactor before and after acclimation using theeubacteria EUB338 (red) and archaea ARC915 (yellow)

probe are shown in Figs. 3 and 4 based on theintensity and amount of fluorescence, the eubacterialcontent was significantly higher than the archaeal con-tent. Before and after acclimation in the ABR, theeubacterial and archaeal content gradually increasedfrom the first to the fourth compartments. In thesubsequent compartments, the environment wasrelatively stable, and the large molecules were easilydegraded into smaller molecules that were easilydegraded and absorbed by the micro-organisms.

Along the direction of the water flow across thecompartments of the reactor, the spatial distributionof archaea in the granular sludge expanded from thecenter in the front compartment to the outer layer inthe subsequent compartments. The hierarchical distri-bution of microbial populations in the granular sludgewas obvious because of the characteristics of thesetwo types of bacteria. Eubacteria are aerobic and fac-ultative; thus, they occupy the areas with higher dis-solved oxygen. In contrast, archaea are anaerobic andthey mainly use the products of macromolecule degra-dation in the sewage. Given that the dissolved oxygengradually decreases in the subsequent compartments,archaea in the outer layer of the granular sludge grad-ually increased in the third and fourth compartments.

4. Conclusions

The nitrobenzene concentration in the five ABRcompartments decreased accordingly. The nitroben-zene removal efficiency was 90.4% in ABRs. Themicrobial diversity in the five ABR compartmentsdecreased. The evenness indices were close. FISHshowed that the eubacterial content in the sampleswas significantly higher than the archaeal content. Inthe reactor compartments, the number of eubacteriaand archaea before nitrobenzene acclimation wasgreater than that after acclimation. From the first com-partment to the fifth compartment, the spatial distri-bution of the archaea expanded from the center of thegranular sludge in the front compartment to the outerlayer in the subsequent compartments.

Acknowledgments

The authors gratefully acknowledge the financialsupport from the National Natural Science Foundationof China (No. 50978118), National water pollution con-trol and management technology major projects (No.2012ZX07408001-07), Education Department of theJilin Province Scientific and Technological ResearchProject (No. 2013216) and the Ministry of Housingand Urban Development Project (No. 2009-K7-13) fortheir financial support.

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