Rongqio Chen,Chune Guo,Wubo Chu,Nn Jing,He Li,*

a Key Laboratory of Marine Materials and Related Technologies,CAS,Zhejiang Key Laboratory of Marine Materials and Protective Technologies,Ningbo Institute of Materials Technology and Engineering,Chinese Academy of Sciences,Ningbo 315201,China

b Nano Science and Technology Institute,University of Science and Technology of China,Suzhou 21500,China

Key words:FTIR-ATR Bacteria settlement Secondary structure Surface interaction

ABSTRACT Marine microorganism accumulated on the surface of ships or pipelines w ould accelerate fouling organisms,such as mussels and barnacles,adhered on the surface.It was signi fi cant to understand the bio-interaction between the microorganisms and the surface.Attenuated total re fl ection(ATR)Fourier transform infrared(FTIR)spectroscopy was used to study the initial stages of marine microorganism adhering to surfaces,because it could probe the microorganism interaction to the surface regardless of the w ater interference.Bacillus sp.and Escherichia coli were selected to study the initial attachment on different surfaces,because they were typical fouling microorganisms and showed opposite Gram stain results.The assays were conducted respectively in dried and settled bacteria on tw o different surfaces(Zn Se,Al2O3 coated on Zn Se).IRspectra of settled bacteria showed amide Iband red shift and amide II band blue shift in aqueous environment on both surfaces compared with the dry bacteria.The reasons of amide bands shift were investigated and it was discovered that the hydrogen bond between the water and the protein of the bacteria led to the protein secondary structure change.ATR-FTIR provided an approach to study the attachment process and showed dynamic changing process on the surface,and it could be an appropriate approach to study the interaction between proteins and chemicals.

Biofouling was de fined as undesired accumulation of fouling organisms on the surface,and usually occurred on the ships,pipelines,medical devices,which could lead to huge economic losses.When an object was im mersed in the sea w ater,a condition layer w ould form and cover the surface in the fi rst few minutes[1–3].During next few hours,marine microorganisms including bacteria and diatoms w ould settle on the surface as a reversible attachment[4],generating a friendly environment for the larval macroorganisms.As time went on,macroorganisms like barnacles and mussels started to colonize on the surface,and formed an irreversible biofouling community[5].DLVOtheory was applied to describe this process and according to it[5,6],the interaction between surface and microorganism was the summation of their electrostatic and van der Waals interaction[5].Initial adhesion of bacteria and diatoms influenced the settlement of macroorganism intensely and was a critical step to trigger marine biofouling.Extracellular polymeric substances(EPS)and cell w all material such aspolysaccharidesand bacterial nano fibersplayed signi fi cant roles for the development of biofouling[5].Coarse surface of the substrate was easier attached by marine organisms[7],and bacteria were likely to aggregate on a positive charged surface.In order to restrain the formation of marine biofouling,the mechanism of bacteria initial settlement needed to be fi gured out.

Many experiments had been designed to investigate the bacteria settlement process on the surface and the influence thereafter[8–11].Scanning electron microscopy(SEM)had been used to provide information of surface morphology and properties of the EPS[8].But it could not be used to monitor in situ settlement process in aqueous circumstance.Atomic force microscopy(AFM)had been employed to study the strength of attachment Ulva spore[9–11].Hydrodynamic methodshad also been used to measure the adhesion strength of Ulva spores using a w ater jet apparatus[9].However,AFM and hydrodynamic measurements information did not refer to the interfacial adhesion properties of the w hole adhesion system.Transmission electron microscopy(TEM)imaging had been used to look at algal spore ultrastructure during both sw imming freely and settled stages[12,13].Raman spectroscopy had also been used in study of microorganism,such as rapid detection analysis for free-cell,taxonomic comparison,characterization of microbes,differentiate the discrepancy between populations,and analyzing composition difference between cells grow n on different cultures[8,14–17].Recently,Raman spectroscopy was used to compare cells in bio fi lms and planktonic cells[8].Though TEM could really show the specific molecule interaction,it was obscure to reveal further reaction mechanism.Raman spectroscopy could offer the non-destructive analysis and could rapidly con fi rm cell properties,but it could only provide fi nal state sample information.

Fourier transform infrared spectroscopy(FTIR)was an ideal approach to study the alive bacteria without destroy their cells.However,w ater could strongly absorb infrared radiation and w ould highly interfere the collected IR spectra.Therefore,attenuated total re fl ection infrared(ATR-IR)spectroscopy was introduced in this study because there was limited penetration of IR radiation into the aqueous medium and it was also a nondestructive detection method which provided an in situ approach to monitor the bacteria settlement process on the surface in aqueous environment[12].Previously,ATR-FTIR spectroscopy had been used in rapidly detecting Bacillus globigii spores[11],exploring microbes adsorbing metal ions in industrial wastew ater[18,19],microbes adsorbing toxic ions and analyzing phosphate adsorption onto hematite[19].In this paper,ATR-FTIR spectroscopy was applied to investigate the bio-reaction continuously during gram-positive Bacillus sp.and gram-negative Escherichia coli settlement on bare and Al2O3coated Zn Se surface,respectively.

The bacteria samples were cultured as follow ing method.Gram-positive Bacillus sp.bacteria(MCCC1A00791)was supplied by Marine Culture Collection of China(MCCC)and cultured in sterile CM 0471-2216E media.The CM 0471-2216E media were prepared by dissolving 5 g peptone,1 g yeast extract,1 g beef extract and 0.01 g FePO4in 1000 m L ASW(Artificial sea w ater).Then 100 m LCM 0471-2216Emedia were sterilized in autoclave at 120?C for 20 min. A single bacterial colony was picked from nutrition agar plate to 100 m L sterile CM 0471-2216E media.The sterile media with the single bacterial strain were shaken at 30?C at 120 rpm for 24 h.To collect clean bacteria,Bacillus sp.suspension was rinsed with sterile deionized w ater for 3 times through centrifugation with a rotational speed of 3000 rpm for 5 min then re-suspended in sterile deionized water.Gram-negative Escherichia coli bacteria(ATCC25922)were chosen to compare adsorption of bacteriawith different cell w all properties.The nutrient medium was prepared by dissolving 10 g peptone,10 g NaCl and 5 g yeast extract in 1000 m Ldeionized water.The same w ay to sterilize and transfer bacteria to 100 m L sterile nutrient medium.The sterile media with Escherichia coli were shaken at 37?C at 120 rpm for 24 h.Escherichia coli suspension was rinsed by sterile deionized w ater to collect clean bacteria.The morphology of both bacteria was imaged by a metalloscope(Leica DM 2500M)with enlargement factor of 500 after they well settled on the surface.

Fig.1.Schematic of internal re fl ection of IR radiation in an internal re fl ection element(IRE).dp represents the penetration depth.

ATR-FTIRhad been selected to investigate the bacteria.The ATRFTIR spectra were collected using a standard ATR liquid trough plate(PN 022-19XX)arrangement from Pike equipped with Zn Se IRE(80?10?4 mm3,45?,17 re fl ections).ZnSe wasselected for this experiment because of its relatively high refractive index[57_TD DIFF](?2.4)and low solubility product constant in water(K sp=3.6?10?26).The refractive index of bacteria samples was usually less than Zn Se crystal,and the angle of incidence was 45?which was greater than the critical angle,therefore,the total re fl ection of infrared radiation occurred in the Zn Se IRE,and the interface reaction information was carried out by the evanescent w ave(Fig.1).Bare and Al2O3coated Zn Se were conducted to get kinetic ATR-FTIR spectra of Bacillus sp.and Escherichia coli attached on the surface.Zn Se surface almost carried no electric charges and alumina surface carries positive charges in w ater at p H 5–7,so we could discover mechanism of bacteria adhere on surfaces with different property.2 m L bacteria suspension with a concentration of 5?108cells/m L was dripped to the trough plate with bared Zn Se using a pipette.The concentration of bacteria was determined at the w avelength of 600 nm by a microplate reader SpectraMax190(Molecular Devices,U.S.A.).After that,the same procedure was repeated on a surface with 100 nm thickness alumina fi lm which was made by deposition of alumina suspension.The alumina suspension was prepared by adding 20 mg alumina to 30 m L ethanol and ultrasonicated for 30 min.A 200 m L aliquot of the suspension was deposited evenly on one side of the Zn Se crystal using a pipette and the solvent was then allowed to evaporate.After all solvent evaporated,coated IRE was fl ushed by sterile deionized w ater to remove loose alumina.Background spectrum was collected at the time zero after bacteria suspension was dripped to trough plate.The spectra of bacteria were collected kinetically with an interval of 15 min.All spectra were collected at 4 cm?1resolution with 100 co-adding scans.

Bacillus sp.bacteria suspension 50 m Lwasdripped onto the bare Zn Se IREfollowed by fl ow ing dry air at 30?Cuntil w ater completely evaporated.Then,the transmittance IRspectrum of dried bacteria on Zn Se was collected using the bare Zn Se IREas a reference.20 m L high concentration Bacillus sp.suspension was dripped on a 13 mm diameter Zn Se w indow,another Zn Se w indow was using to cover on,then ZnSe w indow wasput on the FTIRholder to collect spectra.The transmittance IRspectrum of the wet bacteria was recorded to get the live bacteria in aqueous environment,and an open beam spectrum was used as a background.All spectra were collected at 4 cm?1resolution with 100 co-adding scans without baseline correction.

Peptone,beef extract and ethanol were obtained from Sinopharm Chemical Reagent limited corporation.Yeast extract and alumina pow der(30 nm)were supplied by Aladdin.Artificial sea w ater was prepared according to ASTMD 1141-98,all involved reagents in ASW were obtained from Aladdin.Zn Se w indow s were acquired from PIKE Technologies Incorporation.All the solvents and reagents were used as received without any further puri fi cation.All spectra were collected on an ABB MB 3000 series Fourier transform infrared(FTIR)spectrometer equipped with a DTGS detector.

The morphology of bacteria was captured by an optical microscope.Fig.2 demonstrated the status of Bacillus sp.and Escherichia coli after they settled on bare Zn Se surface.Both Bacillus sp.and Escherichia coli were rod-shaped bacteria.Their lengths were both around 2.5–3 m m,and both of them were agglomerate w hen they adhered on the surface.

Fig.2.The status of Bacillus sp.and Escherichia coli on the ZnSe internal re fl ection element.

Fig.3.Transmittance IR spectra of Bacillus sp.KBr pellet and Escherichia coli KBr pellet.

Table 1 Major IR absorption peaks assignments of bacteria.

Fig.4.ATR-FTIRspectra of bacteria on surfaces:(a)Dried Bacillus sp.on bare Zn Se,(b)Bacillus sp.in aqueous environment on Al2O3 coated Zn Se surface,(c)Bacillus sp.in aqueous environment on bare Zn Se,(d)Dried Escherichia coli on bare Zn Se,(e)Escherichia coli in aqueous environment on Al2O3 coated ZnSe surface,(f)Escherichia coli in aqueous environment on bare ZnSe.

Fig.3 demonstrated the transmittance IR spectra of both bacteria,and the major peaks of bacteria were assigned and listed in Table 1[18,20].The distinguished peaks were at around 1640 cm?1,1550 cm?1and bands between 900 cm?1and 1150 cm?1.The peaks at 1640 cm?1and 1550 cm?1were amide I and amide II vibrations of proteins,respectively,and the peaks from 900 cm?1to 1150 cm?1were complex composite of various overlapping peaks including C??O,C??O??P,and C??O??Cstretching vibration,which were from the functional groups of cell polysaccharides and phosphatide.Though it was difficult to make a distinction between them,the intensity change of the peak was still used to monitor the settlement progress of the bacteria.The peak at 1735 cm?1was assigned to C?O stretching in carboxylic acid group at the Cterminal of the protein,carboxylic acid in the cytoplasm,and lipids in the bacteria membrane.

Fig.5.Secondary derivative IR spectra of Bacillus sp.and Escherichia coli under different conditions.

FTIR-ATRspectra of both Bacillus sp.and Escherichia coli under different conditions showed distinguished shift of amide bands(Fig.4).The amide Ibands of Bacillus sp.in aqueous environment on bare Zn Se surface and Al2O3coated Zn Se surface appeared at 1633 cm?1and 1634 cm?1,and the amide II bands appeared at 1548 cm?1and 1551 cm?1,respectively.Compared with dried Bacillus sp.on bare Zn Se,the amide I bands red shifted about 7–8 cm?1to the low w avenumber,but the amide II bands blue shifted about 8–11 cm?1to the high w avenumber.The similar case happened on Escherichia coli.Because there were few proteins in the bacteria cell w alls and cell membranes,the amide bands shift indicated that the w ater passed through the cell w alls and cell membranes of the bacteria and led to the secondary structure of proteins changed by forming new hydrogen bonds[21].The secondary structure change of the protein was reversible,because the amide bands went back to their original position w hen the w ater evaporated.The band at 1735 cm?1in the dry statusalso blue shifted 8 cm?1w hen Bacillus sp.were in aqueous environment on both bare and Al2O3coated Zn Se surface,because of ionization of the carboxylic acid group at the Cterminal of the protein.However,no carbonyl IR bands were observed in the spectra of Escherichia coli.under both ATR mode and transmittance mode.The“disappeared”carbonyl band could not be simply attributed the less amount of the protein in the cell w all to the bacteria,because the transmittance mode of IRw ould give the complete information of the bacteria.

The second derivative of IRspectra was used to distinguish the secondary structure of protein under different conditions(Fig.5)[21],and the calculated resultsshowed in Tables2 and 3.In order to eliminate w ater bands affect,each spectrum above subtract a certain amount of w ater spectrum until the bands 1750–2000 cm?1become a straight line before all spectra were conducted to the second derivative of IR spectra[22].A straight base line in the bandsfrom 1750 cm?1to 2000 cm?1isa criterion to make sure that w ater affection was removed[22].The calculation demonstrated that the protein of both Bacillus sp.and Escherichia coli had similar ratio of their secondary structure w hen they were on bare Zn Se surface in dry.However,w hen the bacteria settled on bare Zn Se surface in aqueous environment,α-helix and β-turn’s ratio dropped rapidly andβ-sheet increased,random coil could be distinguished.When the bacteria settled on Al2O3coated Zn Se surface, β-turn’s ratio of Bacillus sp.increased,but its ratio of Escherichia coli decreased.And theirα-helix and β-sheet showed similar trend as on bare Zn Se.It was noticed that the secondarystructure’s ratio change was relevant to the amide bands shift in their IR spectra.Fig.6 demonstrated the w avenumber change of amide I bands w hen both bacteria settled progress on bare Zn Se and Al2O3coated Zn Se surface.The trend of the change of the secondary structure was coincident with the shift of the amide bands.Therefore,the shift of the amide bands was caused by the proteins’secondary structure change,but w hat caused the proteins’secondary structure change,the hydrogen bond or the interaction with the surface?The IR spectra of Bacillus sp.in transmittance mode were used to investigate the main reason to cause the amide bands shift.In Fig.7,the spectra clearly showed the amide bands shift w hen the bacteria were in w ater.Therefore,the band shift could be attributed to the new hydrogen bonds between the protein and w ater.However,the amide Iand IIbands of dry Bacillus sp.were located at 1647 cm?1and 1535 cm?1in the transmittance mode,and they were located at 1641 cm?1and 1540 cm?1in ATR mode.The amide bands of the bacteria in aqueous environment were located at 1642 cm?1and 1546 cm?1in transmittance mode.As show n in Fig.4,the amide Iand IIbands position in ATR mode were at 1634 cm?1and 1551 cm?1on bare Zn Se,but they were at 1633 cm?1and 1548 cm?1on Al2O3coated Zn Se surface,respectively.It implied that not only the hydrogen bonds interacted with the protein of the bacteria,but also the surface.Another evidence was the different positions of carbonyl bands in Figs.4 and 7.In transmittance mode,the carbonyl bands appeared at 1735 cm?1,no matter the bacteria were in dry or wet status,but in ATRmode,the carbonyl bandsappeared at 1743 cm?1w hen the bacteria settled on the surface.The band shift of carbonyl bands was triggered by the interaction between the bacteria and the surface.

Table 2 Calculated percentage of protein secondary structure of Bacillus sp.

Fig.6.Amide I bands shift progress of Bacillus sp.and Escherichia coli on bare Zn Se and Al2O3 coated ZnSe surfaces.

Table 3 Calculated percentage of protein secondary structure of Escherichia coli.

Fig.7.IRspectra of dry and wet Bacillus sp.

In the settlement progress on the surfaces,except gravity and buoyancy on the bacteria,the main forces w ould be the interactions between the bacteria and surfaces.There were few functional groups on Zn Se surface,but there were a lot of hydroxyl groups on Al2O3surface.Hence,because the isoelectric point of Al2O3wasabout p H 9,w hen the Al2O3exposed to the suspension of the bacteria,the Al2O3coated Zn Se surface was expected to carry positive charges which were opposite to the charges on the bacteria.In a w ord,the interaction between the bacteria and Al2O3was greater than it between the bacteria and Zn Se.The broad bands abound 1085 cm?1,which were assigned to the stretching vibrations of C??O,C??O??P,C??O??C and P??O in bacteria cell w alls,were used to monitor the relative amount of the bacteria on the surfaces.Fig.8 showed the kinetic progress of the both bacteria settling on the surfaces.Bacillus sp.appeared on the both surfaces rapidly and the amount of the bacteria saturated under ATRmode in about 2 h.The maximum amount of Bacillus sp.on Al2O3coated Zn Se surface was about 20%higher than on Zn Se surface.Escherichia coli showed the similar trend as Bacillus sp.on the both surfaces,but the maximum amount of the bacteria on Al2O3coated Zn Se surface wasabout 3 timeshigher than on Zn Se surface.Because Al2O3carried opposite charges to the bacteria,it was understandable that the bacteria preferred to settle on Al2O3surface.However,the maximum amount of the bacteria on the surfaces suggested that the cell w all property of Escherichia coli was different from that of Bacillus sp.The externus structure of Bacillus sp.contained about 90%peptidoglycan and 10%teichoic acid,and Escherichia coli had peptidoglycan,lipopolysaccharide,phospholipids with saturated fatty acid and some enzymes[23,24].Because of the thicker peptidoglycan in Bacillus sp.cell w all,the settlement behavior on Al2O3coated Zn Se surface was similar with on bare ZnSe surface.Escherichia coli had very thin peptidoglycan layer,so the other active componentsin the cell w all played signi fi cant roles and were inclined to bind with Al2O3.

Fig.8.Kinetic progress of Bacillus sp.and Escherichia coli settling on bare Zn Se and Al2O3 coated Zn Se surfaces.

In conclusion,ATR-FTIR was used to study the settlement process of gram-positive bacteria Bacillus sp.and gram-negative bacteria Escherichia coli on bare Zn Se surface and Al2O3coated Zn Se surface.When the both bacteria were in aqueous environment,their amide Iand amide IIbands in the ATR-FTIRspectra were red shifted and blue shifted comparing with the dry bacteria,respectively.The hydrogen bonds between the w ater and the protein in the bacteria led to the protein secondary structure change,which cause the amide bands shift.The interaction between the surface and the bacteria also played signi fi cant role in the band shift.Due to the different components of the cell w all of Bacillus sp.and Escherichia coli,Escherichia coli was inclined to settle on Al2O3coated ZnSe surface,but Bacillus sp.did not show the clear trend to settle on both surface.And the research indicated that ATR-FTIR could be an appropriate approach in protein secondary structure study and it could be also used to monitor the protein interaction with other chemicals.

Ack now led gm ent

This research was supported by[58_TD DIFF]2IF]the National Natural Science Foundation of China(No.21675165).