Biosensors and Bioelectronics 21 (2006) 1703–1709

Novel BOD optical fiber biosensor based on co-immobilizedmicroorganisms in ormosils matrix

Ling Lin a, Lai-Long Xiaoa, Sha Huanga, Li Zhaoa, Jian-Shen Cuic,Xiao-Hui Wangc, Xi Chena,b,∗

a Department of Chemistry and Key Laboratory of Analytical Sciences of the Ministry of Education, College of Chemistryand Chemical Engineering, Xiamen University, Xiamen 361005, China

b State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen 361005, Chinac Department of Environmental Science, Hebei University of Science and Technology, Shijiazhuang 050000, China

Received 29 April 2005; received in revised form 3 August 2005; accepted 15 August 2005Available online 3 October 2005

Abstract

A biochemical oxygen demand (BOD) sensor has been developed, which is based on an immobilized mixed culture of microorganisms combinedw SIL) filme ilized on ap constantt d solution,a the BODs onal BODm©

K

1

wwbCton(aoa2

bio-trodement.r thectiverfor-t

aoa

ds-

sms

ann

et

0d

ith a dissolved oxygen (DO) optical fiber. The sensing film for BOD measurement consists of an organically-modified silicate (ORMOmbedded with tri(4,7-diphenyl-1,10-phenanthroline) ruthenium(II) perchlorate and three kinds of seawater microorganisms immobolyvinyl alcohol sol–gel matrix. The BOD measurements were carried out in the kinetic mode inside a light-proof cell and with

emperature. Measurements were taken for 3 min followed by 10 min recovery time in 10 mg/L glucose/glutamate (GGA) BOD standarnd the range of determination was from 0.2 to 40 mg/L GGA. The effects of temperature, pH and sodium chloride concentration onensing films were studied. BOD values estimated by this optical BOD sensing film correlate well with those determined by the conventi5ethod for seawater samples.2005 Elsevier B.V. All rights reserved.

eywords: Biochemical oxygen demand; BOD optical biosensor; Sol–gel; Ormosils

. Introduction

Biochemical oxygen demand (BOD) is one of the mostidely used and important parameters for the estimation ofater quality. The authorized method for BOD was adoptedy the American Public Health Association Standard Methodsommittee (APHA, 1989), which not only requires 5 days of

edious procedures, but also demands experience and skill tobtain reproducible results. Therefore, it is time-consuming andot suitable for in situ determinations or on-line measurementsJISC, 1989). SinceKarube et al. (1997)first developed a rapidnd reliable biosensor for BOD determination in 1977, severalther kinds of microbial BOD sensor have been applied. (Qiannd Tan, 1998; Tag et al., 2000; Kang et al., 2003; Chang et al.,004; Rastogi et al., 2003). In most cases, the BOD sensors con-

∗ Corresponding author. Tel.: +86 592 2184530; fax: +86 592 2186401.E-mail address: xichen@xmu.edu.cn (X. Chen).

sist of a synthetic membrane using microorganisms as thelogical recognition element, and a dissolved oxygen elec(Clark electrode) as a transducer for the oxygen measureDuring the past decade, fiber optical chemical sensors fodetermination of oxygen have appeared to be more attrathan conventional amperometric devices due to rapid pemance, no oxygen consumption and less toxicity (Hartmann eal., 1998; Klimant and Wolfbeis, 1995; Chan et al., 1999; Amet al., 2000). In the first report for BOD determination usingfiber optical microbial sensor (Preininger et al., 1994), a sin-gle microorganism,Trichosporon cutaneum, was selected animmobilized in polyvinyl alcohol (PVA) used for the BOD sening membrane. Until now, many assimilative microorganihave been applied and reported including:Arxulao adeninivo-rans LS3 (Riedel et al., 1998a,b; Gruendig et al., 2000; Lehmet al., 1999), Bacillus cereus (Sun et al., 1992), Bacillus sub-tilis (Riedel et al., 1998a,b; Xie et al., 2003), Klebsiella oxytocaAS1(Ohki et al., 1994), Pseudomonas putida (Chee et al., 1999)Trichosporon cutaneum (Preininger et al., 1994; Murakami

956-5663/$ – see front matter © 2005 Elsevier B.V. All rights reserved.

oi:10.1016/j.bios.2005.08.007

1704 L. Lin et al. / Biosensors and Bioelectronics 21 (2006) 1703–1709

al., 1998), Trichosporon brassicae (Xie et al., 2003), and yeast(Li et al., 2004; Chen et al., 2002). The BOD sensor is highlycapable of analyzing a sample of complex constituents with rel-atively low selectivity. The sensor should respond to multiplebiodegradable organic solutes in the sample, and give resultscomparable to those obtained using the conventional BOD meth-ods. Since each microbial species has its metabolic deficiencies,a single BOD film-immobilized microorganism is only able torespond to limited organic solutes. Therefore, biosensors withmixed microorganisms (including activated sludge) immobi-lized within a single membrane onto an oxygen sensor weredeveloped (Riedel et al., 1998a,b; Liu et al., 2000; Tan et al.,1993; KOnig et al., 2000; Jia et al., 2003).

The immobilization method for binding microorganisms isanother vital step for optical biosensor production. Silicones,Teflon®, plasticized polyvinyl chloride, cellulose and polyvinylacetate are considered to be appropriate polymer materials forimmobilization. Compared with many other organic polymers,organically-modified silicates (ormosils) are assimilated intosilicon-oxide networks; these have been attracting great inter-est in recent years as novel materials for optical oxygen sensors(Chen et al., 2002; Brinker and Scherer, 1990; Walcarius, 2001).They have proven to be a better solid matrix to bind the microor-ganisms and at the same time maximally maintain their activity.Since PVA and silica associate well with interactions such as ahydrogen-bonding (Nakane et al., 1999), ormosils-PVA materialh ulesf ea mob trixo

aveb ed,s satif geno ed ot izedi samp wateI lizeBf ensw earr loridc is td on ot

2

2

VA)w ).D omF[

used as the oxygen-sensing indicator was synthesized andpurified in the laboratory of the Department of AppliedBiology and Chemical Technology, Hong Kong PolytechnicUniversity. Glucose/glutamate (GGA) solution was preparedby adding 0.0750 g glucose and 0.0750 gl-glutamic acid to100 mL phosphate buffer solution (pH 7.4), and used as thestandard BOD solution (1000± 185 mg/L) (JISC, 1990). Theseawater samples were collected from the area around XiamenUniversity, Xiamen, China and filtered through 10�m isoporemembrane filters (Millipore, USA) before determination. Allother chemicals used in this study were analytical-reagentgrade and all solutions were prepared with deionized distilledwater.

A field-emission scanning electron microscopy (FE-SEM;JSM-7400F, JEOL, Japan) was employed to obtain the scan-ning electron microscopic (SEM) images of the BOD andoxygen-sensing films. The excitation and emission wavelengthsof oxygen-sensing film used in this work were 465 and 580 nm,respectively. The fluorescent spectra were acquired using aHITACHI-4500 spectrofluorimeter (Hitachi, Japan).

2.2. Oxygen-sensing film preparation

1.0 mL of TMOS and 1.6 mL of DiMe-DMOS were placedin an open vial. After the mixture was magnetically stirred fora cidw the6 ringt no uslys pre-p chh shedw leftu vid-u h at1 d tor , thet to be3

as excellent biocompatibility that can immobilize biomolecor fabricate biosensors.Dai et al. (2004)reported an effectivnd useful BOD sensor, in which a sol–gel acted as the imilizing material to immobilize yeast in the sol–gel host man an oxygen optical sensing film.

Although most of the BOD sensors previously reported heen designed for long-term use, and even commercializeems that the performance of these sensors is still notactory. Their limitations, such as (a) the depletion of oxyccurring during BOD measurement using a sensor bas

he Clark electrode; and (b) the microorganisms immobiln current BOD sensors are not able to be applied in theles containing a higher concentration of salt such as sea

n this work, ormosil-PVA was used as a matrix to immobiacillus licheniformis, Dietzia maris andMarinobacter marinus

rom seawater. A series of experiments characterizing the sere carried out including response time, reproducibility, lin

ange and the effects of temperature, pH and the sodium choncentration in the sample. The aim of the present studyevelop a biosensor for rapid and stable in situ determinati

he BOD in seawater.

. Experimental

.1. Chemicals and instrumentation

Tetramethoxysilane (TMOS) and polyvinyl acetate (Pere purchased from Aldrich (Milwaukee, WI, USAimethyldimethoxysilane (DiMe-DMOS) was purchased frluka AG (Buchs, Switzerland). The [Ru(Ph2phen)3](ClO4)2

Ph2phen = 4,7-diphenyl-1,10-phenanthroline, Ru(dpp)32+]

-

its-

n

-r.

or

eof

pproximately 1 min, 2.0 mL of 0.01 mol/L hydrochloric aas added to the mixture. The vial was then immersed in0◦C water bath and stirred for 3 h. An emulsion formed du

his step; 2 h later, 0.2 mL of Ru(dpp)32+ with a concentratio

f 1.5 g/L in THF was added. The mixture was then vigorotirred for 20 min to ensure a homogenization. Films wereared by pipetting 60�L of the mixture onto a glass slide, whiad been soaked in concentrated nitric acid for 12 h and waith distilled water and ethanol. The resulting films werendisturbed under ambient conditions for 12 h. Finally, indial films were thermally cured in an oven for another 1250◦C and were ready for analysis after the films cooleoom temperature. According measurements using SEMhickness of the films prepared in this way was estimated5�m (Fig. 1).

Fig. 1. FE-SEM images of oxygen-sensing film.

L. Lin et al. / Biosensors and Bioelectronics 21 (2006) 1703–1709 1705

Pore distributions of ormosils were detected by Tristar 3000(Micrometritics Instrument Co., USA). A 5.00 g sample wascrushed rubbed, and then heated at 120◦C for 24 h under thepressure of 0.0267 Pa. Nitrogen (99.9%) (Linde Co. China) wasemployed as the adsorbent gas.

2.3. Microorganisms

Three microorganisms,B. licheniformis, D. maris and M.marinus, were selected and applied for BOD sensing film.B.licheniformis was obtained from freshwater and cultured in sea-water.D. maris, andM. marinus were selected and obtaineddirectly from seawater of Yantai, north of China.D. maris andM. marinus were cultured in a medium containing 0.5% beefextract, 1.0% peptone and 0.1% glucose in seawater, while theculture forB. licheniformis contained 0.8% beef extract, 1.0%peptone and 0.5% amylum in seawater. All microorganisms weregrown under standard aerobic conditions in a rotating shaker at35◦C for 24 h, then harvested by centrifugation at 6000 rpm for10 min at room temperature, washed twice with phosphate bufferof pH 7.4 and resuspended in the same buffer.

2.4. Immobilization of microbial cells

Ormosils were prepared by mixing TMOS, DiMe-DMOS and0 reda ms( -to icaloe er-ab

2

or iss dmm hene asd ughc ns-

ferred by an optical fiber to the GD-1 glimmer measure (Xi’anReike Electronic Instrument Co. Ltd., China) equipped with aR928 PMT (Hamamatsu, Japan). The temperature was kept con-stant by a temperature controller with a precision of±0.2◦C.The experimental results were analyzed by an Echrom98 chro-matogram workstation (Dalian Elite Scientific Instruments Co.Ltd., China).

2.6. Experimental procedure

Generally, two modes including the dynamic balanceable andthe kinetic method were employed for BOD sensing measure-ments (Liu et al., 2000). Compared with the dynamic balance-able mode, BOD sensing measurements in the kinetic modeswere much faster, and thus more suitable for rapid monitor-ing application. In this study, the BOD sensing measurementswere performed in the kinetic mode. The BOD sensing filmwas inserted into a black and airtight detection cell in whichsample solutions were kept motionless during all measure-ments. The temperature of the detection cell was maintainedat 35◦C. When the fluorescence intensity reached a steadystate, the sample solution (20 mL) was added to the detectioncell. The output of the sensing film increased gradually andthen reached a steady state after a few minutes. Experimentalresults showed that there was a linear relationship between themaximal changing rate of fluorescence intensity (dI/dt) and theB

3

3

l( osilso wast withs out4b ofR atest orsc sizef ionb y a

stem

.01 mol/L HCl (1:1.2:1.5, v/v). The precursors were stirt 60◦C for 1 h. Two hundred microlitres of microorganisB. licheniformis: D. maris: M. marinus, 1:2:1, w/w) in soluion was mixed with 200�L 8% (w/w) PVA and 200�Lrmosil. After the mixture of sol was spread onto an opt1xygen-sensing film produced as described previously (Jiangt al., 2001), the microbial films were dried at room tempture for 24 h and stored in 100 mg/L GGA solution at 4◦Cefore use.

.5. Construction of the BOD sensor

The structure of an in-house optical chemical BOD senshown schematically inFig. 2. The microorganism-immobilizeembrane was placed in the detection cell at a 45◦ angle toaximally detect the changes of fluorescence intensity w

xcitation light (wavelength of 465 nm from a blue LED) wirected onto it. The emission fluorescence was passed throut-off filter with a half bandwidth of 10 nm at 580 nm, and tra

Fig. 2. Optical fiber sy

a

OD value.

. Results and discussion

.1. Microstructure of ormosils

Typically, TMOS, DiMe-DMOS and 0.01 mol/L HC1:1.2:2, v/v) were selected for the preparation of the ormxygen-sensing films. The color of the ormosils obtained

ransparent and the texture rubbery. The percent of poresizes ranging from 0.8 to 2.0 nm for the ormosil film was ab7.8% (vol.%, same as below,Chen et al., 2004). Calculationy HYPERCHEM software showed the molecular sizeu(dpp)32+ reagent had a diameter of about 2.0 nm. It indic

hat the ormosils film using TMOS–DiMe-DMOS precursontained more than 47.8% silica cages with a suitableor the Ru(dpp)32+ embedding. The overall chemical reactetween TMOS and DiMe-DMOS can be presented b

for BOD measurement.

1706 L. Lin et al. / Biosensors and Bioelectronics 21 (2006) 1703–1709

three-step process. In the hydrolysis of TMOS, a pure oxide gelof SiO2 is formed, which contributes numerous, hard, plumpspots on the ormosils surface after drying. The remaining–OH groups on the Si atoms of the PDMS product continueto form hydrogen bonds with other PDMS chains and/or withother SiOH groups. Although a lower response time can beachieved when spin-coating is applied to oxygen-sensing filmpreparation, the weaker fluorescence response of the coatedoxygen film leads to lower detection limits for BOD due toits smaller capacity for Ru(dpp)3

2+. In this experiment, anormosils film with a thickness of about 35�m was prepared bydip-coating so that a higher fluorescent intensity change couldbe obtained in the BOD measurement (Fig. 1).

PVA has been applied to immobilize microbial cells as it is anontoxic and hydrophilic material, and is capable of maintainingmaximum microbial activity. PVA/silica complexes have beenstudied by Suzuki et al. (Nakane et al., 1999). It is consideredthat the three-dimensional network structure of PVA/silica canbe formed with more than 30 wt.% of silica in PVA. The diam-eter (dp) of silica is 3–4 nm. InFig. 3, PVA/ormosils are evenlyspread in a network on an oxygen ormosil film, and severalquadrate plump spots with a length of 1–2�m can be found onthe surface of PVA/ormosils. The thickness of the PVA/ormosilslayer is estimated to be 1.2�m. A crosslinking of DiMe-DOMSand TMOS is considered to be dispersed homogeneously in thePVA matrix through hydrogen bonds (Schmidt, 1989). The lows s fort hasa rgan-i cane nsingl uar-a nda con-d Sw con-c onsei ucesa 10%( ay

Table 1Comparison of the characteristics of BOD sensing films with single or multi-microorganism immobilization

Parameter Film ofB. licheniformis

Film ofmulti-microorganisms

R2 0.9783 0.9866Detection limit (mg/L) 0.9 0.2Response time (min)a 30 3.2Curve slope 0.31 2.33Reproducible responses 2% 2%Optimum pH 7.4 7.9Optimum temperature (◦C) 30 50Optimum NaCl % concentration 3% 3.2%

a Concentration of 5.0 mg/L BOD.

from oxygen film, so a concentration of 8% PVA in ormosilswas selected.

3.2. Effect of different cell concentrations of the microbiallayer

When an individual microorganism,B. licheniformis isapplied to the microbial layer alone, it presents admissibleresponses for BOD sensor (Dai et al., 2004). However, a remark-able improvement can be obtained when the layer is immobilizedby a mixed culture of microorganisms. As shown inTable 1, interms ofR2, response time, sensitivity, and curve slope, the filmof mixed culture microorganisms is superior to the film formedby individual microorganisms (B. licheniformis), but the repro-ducibility and the effects of pH on the response of the sensingfilms are very similar. The maximal allowed concentration of thechloride anion for the two kinds of films is 3.0 and 3.2%, respec-tively. However, for the determination of low BOD, BOD filmimmobilized by mixed culture of microorganisms is superiorwith respect to sensitivity and is promising for further develop-ment. For example, a higher cell density in a microbial layer canbe applied to achieve a better signal response even for very lowBOD. However, it is obvious that sensing film containing highercell concentrations is more sensitive, but has a smaller dynamicrange and a longer response time when the volume of multiple

n-sen

hrinkage rate of ormosils and PVA produces larger porehe immobilization of microorganisms. The content of PVAgreat effect on the physical characteristics of the microo

sm films. The increase of the DiMe-DMOS concentrationnhance the response and the flexibility of the microbial-se

ayer. However, further improved hydrophobicity cannot gntee enough solubility for PVA in the ormosils matrix also lengthen the response time. Under our preparationitions, the optimized volume ratio of DiMe-DMOS to TMOas 1.2 to 1. Based on the experimental results, as theentration of PVA in BOD sensing film increases, the respntensity of microbial film raises and the response time redccordingly. However, when the percentage of PVA reachesPVA/ormosils, v/v), the film begins to dilate and flake aw

Fig. 3. FE-SEM images of an PVA/ormosils on oxyge

sing film at (a) low magnification and (b) high magnification.

L. Lin et al. / Biosensors and Bioelectronics 21 (2006) 1703–1709 1707

Table 2Effect of coexisting metal ions on response of BOD sensor

Mn+ �F (%) Mn+ �F (%) Mn+ �F (%) Mn+ �F (%) Mn+ �F (%)

Al(III) −4.58 Fe(III) +4.36 Mg(II) −3.80 Ba(II) −6.03 Ca(II) +3.85Cu(II) −1.45 Zn(II) −5.07 As(III) −9.06 Bi(III) +0.85 Mn(II) +3.57Sr(II) +1.43 W(VI) +3.16 V(V) +3.05 Ni(II) +2.82 Sn(II) −3.51Co(II) −5.10 Cd(II) −12.50 Mo(VI) +0.93 Se(VI) −6.65 Li(I) +2.35Cr(III) −0.25 Ti(IV) −5.88 Pb(II) −15.01 Ag(I) −15.43

Keep the concentration of each ion 1 mg/L, BOD 10 mg/L, in KH2PO4–Na2HPO4 buffer solution (pH 7.9).�F (%) = [dI/dt−(dI/dt)0]/(dI/dt)0 × 100%. dI/dt: maximalmutative velocity of fluorescent intensity after coexisting substance added, (dI/dt)0: initial maximal mutative velocity of fluorescent intensity.

microorganisms is above 200�L. This is due to the higher oxy-gen consumption by the cells through endogenous respiration(Klimant and Wolfbeis, 1995).

3.3. Effect of measurement temperature

An appropriate temperature can promote the respirationand the activation of the microorganisms and also reduce theresponse time. Unfortunately, under this temperature, bacteriamay undergo changes in conformation and metabolism modeand even experience protein aggregation, which leads to deathof the bacteria. The optimum temperature studied ranged from20 to 55◦C. The response intensity increased rapidly and theresponse time dropped sharply up to 50◦C, but the lifetimeof the BOD sensing film was shortened, since the microor-ganisms became inactive at a higher temperature. To expandthe lifetime of the film, the optimum temperature of 35◦C waschosen; response time for 10 mg/L GGA is about 3 min in thisstudy.

3.4. Effect of pH

The mobilized microorganisms are cultured or sieved by sea-water. They present high assimilation efficiency to BOD andadapt the application to seawater. Since the respiration activityof the microorganisms depend on pH (Chee et al., 2000), thei pH5 ntil p7 ed bt thep loyet heref justet

3

aterw ntra-t ffi-c or-g ther chlor ,t chlor ndi-

cates that our BOD sensor is able to tolerate the inhibitioneffect of high salt concentrations in seawater on microorganismrespiration.

Other coexisting compounds and ions are the main interfer-ence sources to be considered for BOD sensing films. Fortu-nately, our results show that there are no obvious changes inresponse intensity when the samples are saturated by nitrogen,ammonia, or hydrogen sulfide. Similar work was performed totest the effect of metal ions, which commonly exist in seawa-ter at a concentration of 1 mg/L via the measurement of thefluorescent intensity. As shown inTable 2, the prepared BODsensing films are almost unaffected by most metal ions exceptAg+, Cd2+, and Pb2+ owing to their toxicity to the microorgan-isms, which leads to a decrease in response by 15.43, 12.5 and15.01%.Jia et al. (2003)have suggested that the most effectiveway to overcome the presence of inhibitory or toxic cations insamples is by chelation or sample dilution to facilitate the for-mation of stable complexes in the presence of metal ions or indiluted sample solutions. Fortunately, the concentration of toxiccations is generally lower in seawater.

3.6. Stability and service life

Typically, BOD sensing film is stored in GGA solutions at4◦C. Generally, the film needs to be reactivated before BODmeasurement. The film is immersed in a 50 mg/L GGA solu-t ismso sixs nal isr 0s thefi -i teps,w itialfl paredw s thes ntalr ed upt lmsh e theb

3

soru e the

nfluences of pH on BOD response were investigated from.2–8.5 in seawater. The fluorescent response increases uand remains constant above that. This is probably caus

he inactivation of the microorganism at lower pH values. AsH of seawater is around 7–8, the BOD sensor can be emp

o detect the BOD value of different seawater samples. Tore, in subsequent experiments, the pH of samples was ado 7.9 using phosphate buffer.

.5. Effects of chloride ion and coexisting ions

Chloride ion is the most common component of seawith an average concentration of 3.2%; chloride conce

ion greatly affects the activity of microorganisms. The eiency of most biosensors immobilized with limnetic microanisms is negatively influenced by chloride anion andesponse intensity decreases sharply with the increase ofide ion concentration by 0.6% (Jia et al., 2003). In our studyhe response intensity only decreases by 20% when theide ion concentration is higher than 4%. The result i

Hy

d-d

-

-

ion and then exposed to air to reactivate the microorgann the films. The sensor is ready for use after obtainingignals with a S.D. less than 2%. The measurement sigeproducible within a mean value of±1.2% in a series of 1amples in 10 mg/L BOD standard GGA solution. Even iflm is stored in GGA solution at 4◦C for 1 month, the activty of the film can be recovered after several reactivation shich takes less than 30 min. After 6 month’s storage, the inuorescence response of the films decreased by 5% comith the newly made films, but the response to BOD remainame if the sensing film is reactivated sufficiently. Experimeesults demonstrate that the BOD sensing films can be storo 1 year without significant deterioration. However, the fiave to be reactivated for 1 day in GGA solutions to achievest performance before use.

.7. Response and calibration of the BOD sensor

All the calibration curves established for the BOD sensed the GGA solution as standard solution. To determin

1708 L. Lin et al. / Biosensors and Bioelectronics 21 (2006) 1703–1709

Fig. 4. Responses of BOD sensing films to different BOD concentrations. BOD value increased: 0, 2, 5, 10, 15, 20, and 30 mg/L. Experimental condition: constanttemperature, 35◦C; pH of buffer, 7.4; concentration of NaCl, 3.2%.

potential application on the BOD measurement of seawater, theresponses of the sensor were measured at various BOD values. Alinear relationship between BOD standard GGA concentrationand change of velocity of the fluorescence intensity (dI/dt) wasobserved in the range of 0.2–40 mg/L GGA solution. The detec-tion limit of the sensor is 0.2 mg/L. As shown inFig. 4, whenthe concentration of GGA solution is increased, the responsetime decreases and the maximal dI/dt increases and establishesa good linear correlation between GGA concentration and dI/dt.A typical response time for 5 mg/L BOD standard GGA solutionis about 3.2 min.

3.8. Application of the BOD sensor

Usually, the BOD value of the seawater not seriously pol-luted is lower than 5 mg/L. The BOD sensor has been usedto determine the BOD values of seawater from around Xia-men University, China, and the results were compared withthe conventional 5-day BOD. Seawater samples from differentsampling sites were filtered through a membrane filter (10�m).Before testing, the water sample was pre-heated at 35◦C, and20 mL processed water sample was then placed in the detectioncell to be analyzed.

In Table 3, there is about a 20% subtractive error of BODvalues between BOD measured by the biosensor and the standardB int

TR

S

123456

consideration. The first is the limitation of the GGA solution,containing glucose and glutamic acid only. In GGA solution,the metabolism pathways are much simple than those conductedin actual environmental samples, which may contain variouscarbon and nitrogen sources (Chee et al., 1999). Another reasonis insoluble organic components in seawater can be partiallydegraded by bacteria during the incubation in the conventional 5-day BOD method, which can contribute to the BOD5 values butnot to those measured by BOD sensors. Although the measuringprocess can be simplified without the filtering step, it causesmore noise and less reproducibility owing to the light dispersionof the insoluble substances in seawater. Furthermore, there aredifferences of BOD values between refluent and tide seawateraround Xiamen University area. The BOD of refluent seawateris higher than that of tide seawater, indicating that the increaseof the BOD value could be caused by the wastewater from theliving area of Xiamen University.

4. Conclusions

We have presented a novel optical BOD sensing film immo-bilizing sieved bacteria from seawater. In a matrix based onPVA-ormosils derived composite materials, the co-immobilizedmicroorganisms can keep their activity even if they are kept upto one year at 4◦C. The stored sensing film can be employedf umr and3 ther erei (e n ofs tudyr s itsa ablet beingd Thed stratea ODa

ernw are

OD5 method. There are two reasons that should be taken

able 3esults of BOD determination of seawater samples (n = 5)

amples BOD (mg/L)

Sensor BOD5

a 1.21± 0.2 1.77± 0.3a 1.94± 0.3 2.21± 0.2b 1.62± 0.3 2.30± 0.3b 1.72± 0.4 1.94± 0.5c 2.04± 0.2 2.43± 0.3d 2.71± 0.3 2.97± 0.3

a Tide seawater, sampling date, 8 March 2004.b Refluent seawater, sampling date, 8 March 2004.c Tide seawater, sampling date, 24 March 2004.d Refluent seawater, sampling date, 24 June 2004.

oor BOD measurement after 1 day’s reactivation. The optimesponse of the BOD sensing film was obtained at pH 7.95◦C. The minimum measurable BOD was 0.2 mg/L, andesponse time was only 3 min for 10 mg/L BOD, which wmproved compared to the previously described BOD probeDait al., 2004). Seawater contains low BOD, high concentratioodium chloride and various heavy metal ions, but the sesults showed that the optical BOD sensing film exhibitdvantages in the determination of low BOD, and is suit

o evaluate the complicated seawater samples withoutramatically affected by sodium chloride and other ions.etermination results obtained from actual seawater demongood correlation with those obtained from conventional B5nalysis.

Rapid and on-line monitoring of BOD is of major conchen surveying water pollution. Although BOD sensors

L. Lin et al. / Biosensors and Bioelectronics 21 (2006) 1703–1709 1709

mainly based on the Clark-type oxygen electrode, new tech-nologies using optical fiber will lead to improved BOD sensors.Despite the development of new BOD measurement modes andprinciples, there are still a number of choices of which microor-ganisms can be used. Moreover, miniaturization technologies(the application of integrated systems that include probes, sam-ples, and detectors), and the design of full-automated analyticalsystems, etc., are other challenging areas of research. Finally, aBOD system’s success and commercial viability will depend onits robustness, simplicity, rapidity, and cost considerations.

Acknowledgements

This research work was financially supported by the NaturalScientific Foundation of Fujian (D0410001) and the NationalHigh Technical Development Project (863 project) Founda-tions (2001AA635100, 2003AA635100), which are gratefullyacknowledged. We express our sincere thanks to Miss Peiwei Li,Department of Biology, Indiana University, USA, for her kindrevisions.

References

APHA, 1989. American Public Health Association Standard Methods, 17thed. American Public Health Association, Washington, DC.

Amao, Y., Asai, K., Okura, I., 2000. Fluorescence quenching oxygen sensorActa

B , New

C per-silica

C im,and07–

C on of

C sen-ens.

C ased.

C onsehim.

D ar-isms15,

G . 15

H ruthen by

J anda

J rd As

Jia, J.B., Tang, M.Y., Chen, X., Qi, L., Dong, S.J., 2003. Co-immobilizedmicrobial biosensor for BOD estimation based on sol–gel derived com-posite material. Biosens. Bioelectron. 18, 1023–1029.

Jiang, Y.Q., Li, Z., Zhong, Z.M., Chen, X., Wang, X.R., Wong, K.Y., 2001.Luminescence quenching behavior of oxygen-sensing ormosil films basedon ruthenium complex. Chem. Res. Chin. University 17, 374–379.

Kang, K.H., Jang, J.K., Pham, T.H., Moon, H., Chang, I.S., Kim, B.H., 2003.A microbial fuel cell with improved cathode reaction as a low biochemicaloxygen demand sensor. Biotechnol. Lett. 25, 1357–1361.

Karube, I., Matsunage, T., Mitsuda, S., Suzuki, S., 1997. Microbial electrodeBOD sensors. Biotechnol. Bioeng. 19, 1535–1547.

Klimant, I., Wolfbeis, O.S., 1995. Oxygen-sensitive luminescent materialsbased on silicone-soluble ruthenium diimine complexes. Anal. Chem. 67,3160–3166.

KOnig, A., Reul, T., Harmeling, C., Spener, F., Knoll, M., Zaborosch,C., 2000. Multimicrobial sensor using microstructured three-dimensionalelectrodes based on silicon technology. Anal. Chem. 72, 2022–2028.

Lehmann, M., Chan, C.Y., Lo, A., Lung, M., Tag, K., Kunze, G., Riedel,K., Gruendig, B., Renneberg, R., 1999. Measurement of biodegradablesubstances using the salt-tolerant yeast Arxula adeninivorans for a micro-bial sensor immobilized with poly(carbamoyl) sulfonate (PCS). Part II.Application of the novel biosensor to real samples from coastal and islandregions. Biosens. Bioelectron. 14, 295–302.

Li, H.Z., Zhang, Y., Shi, H.C., Wang, J.L., 2004. Application of BOD biosen-sor for marine monitoring. Marine Environ. Sci. 21, 14–17.

Liu, J., Bjornsson, L., Mattiasson, B., 2000. Immobilised activated sludgebased biosensor for biochemical oxygen demand measurement. Biosens.Bioelectron. 14, 883–893.

Murakami, Y., Kikuchi, T., Yamamura, A., Sakaguchi, T., Yokoyama, K., Ito,Y., Takiue, M., Uchida, H., Katsube, T., Tamiya, E., 1998. An organicpollution sensor based on surface photovoltage. Sens. Actuators B 53,

N andSci.

O , H.,toca

P r for

Q ensor.

R an-ovelical

n. 18,

R Anal.

R biol.

S es or

S time

T 2000.sed onB 67,

T e of

W sol-

X mical

using an aluminum phthalocyanine-polystyrene film. Anal. Chim.407, 41–44.

rinker, C.J., Scherer, G.W., 1990. Sol–Gel Science. Academic PressYork.

han, C.M., Chan, M.Y., Zhang, M., Lo, W., Wong, K.Y., 1999. Theformance of oxygen-sensing films with ruthenium-adsorbed fumeddispersed in silicone rubber. Analyst 124, 691–694.

hang, I.S., Jang, J.K., Gil, G.C., Kim, M., Kim, H.J., Cho, B.W., KB.H., 2004. Continuous determination of biochemical oxygen demusing microbial fuel cell type biosensor. Biosen. Bioelectron. 19, 6613.

hee, G.J., Nomura, Y., Karube, I., 1999. Biosensor for the estimatilow biochemical oxygen demand. Anal. Chim. Acta 379, 185–191.

hee, G.J., Nomura, Y., Ikebukuro, K., Karube, I., 2000. Optical biosor for the determination of low biochemical oxygen demand. BiosBioelectron. 15, 371–376.

hen, D.D., Cao, Y.B., Liu, B.H., Kong, J.L., 2002. A BOD biosensor bon a microorganism immobilized on an Al2O3 sol–gel matrix. AnalBioanal. Chem. 372, 737–739.

hen, X., Lin, L., Li, P.W., Dai, Y.J., Wang, X.R., 2004. Fluorescent respof sol–gel derived ormosils for optical ammonia sensing film. Anal. CActa 506, 9–15.

ai, Y.J., Lin, L., Li, P.W., Chen, X., Wang, X.R., Wong, K.Y., 2004. Compison of BOD optical fiber biosensors based on different microorganimmobilized in ormosils matrix. Intern. J. Environ. Anal. Chem.607–617.

ruendig, B., Kunze, G., Renneberg, R., 2000. Biosens. Bioelectron343–353.

artmann, P., Leiner, M.J.P., Kohlbacher, P., 1998. Photobleaching of anium complex in polymers used for oxygen optodes and its inhibitiosinglet oxygen quenchiers. Sens. Actuators B 51, 196–202.

apanese Industrial Standards committee, JISK 0102, Japanese StAssociation, Tokyo, 1989.

apanese Industrial Standard Committee, JISK 3602, Japanese Standaciation, Tokyo, 1990.

,

-

rds

so-

163–172.akane, K., Yamashita, T., Iwakora, K., Suzuki, F., 1999. Properties

structure of poly(vinyl alcohol)/silica composites. J. Appl. Polym.74, 133–138.

hki, A., Shinohara, K., Ito, O., Naka, K., Maeda, S., Sato, T., AkanoKato, N., Kawamura, Y., 1994. A BOD sensor using Klebsiella oxyAS1. Int. J. Environ. Anal. Chem. 56, 261–269.

reininger, C., Klimant, I., Wolfbeis, O.S., 1994. Optical fiber sensobiological oxygen demand. Anal. Chem. 66, 1841–1846.

ian, Z., Tan, T.C., 1998. Response characteristics of a dead-cell BOD sWater Res. 32, 801–807.

astogi, S., Kumar, A., Mehra, N.K., Makhijani, S.D., Manoharan, A., Ggal, V., Kumar, R., 2003. Development and characterization of a nimmobilized microbial membrane for rapid determination of biochemoxygen demand load in industrial waste-waters. Biosens. Bioelectro23–29.

iedel, K., Lehmann, M., Tag, K., Renneberg, R., Kunze, G., 1998a.Lett. 31, 1–12.

iedel, K., Renneberg, R., Kuehn, M., Scheller, F., 1998b. Appl. MicroBiotechnol. 28, 316–318.

chmidt, H., 1989. Organic modification of glass structure New glassnew polymers? J. Non-cyst. Solids 112, 418–423.

un, Y.S., Liu, X.M., Cui, J.S., Qi, J.Z., 1992. Study of long serviceBOD microorganism sensor. Environ. Sci. 13, 59–63.

ag, K., Lehmann, M., Chan, C., Renneberg, R., Riedel, K., Kunze, G.,Measurement of biodegradable substances with a mycelia-sensor bathe salt tolerant yeast Arxula adeninivorans LS3. Sens. Actuators142–148.

an, T.C., Li, F., Neoh, K.G., 1993. Measurement of BOD by initialratresponse of a microbial sensor. Sens. Actuators B 10, 137–142.

alcarius, A., 2001. Electroanalysis with pure chemically modified andgel derived silica-based materials. Electroanalysis 13, 701–718.

ie, J.B., et al., 2003. The progress of microbial biosensors for biocheoxygen demand. Chin. J. Anal. Chem. 31, 742–748.