Biyoreaktörler Için Mükemmel Bir Biyofilm Desteği Olarak Nano-hidroksiapatit Kaplamalı Karbon Fiberler


Please download to get full document.

View again

of 11
All materials on our website are shared by users. If you have any questions about copyright issues, please report us to resolve them. We are always happy to assist you.


Publish on:

Views: 2 | Pages: 11

Extension: PDF | Download: 0

  Full Length Article Carbon fibers with a nano-hydroxyapatite coating as an excellent biofilmsupport for bioreactors Qijie Liu a , Chao Zhang a , Yanling Bao b , Guangze Dai a, ⇑ a School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, PR China b  Aerospace Composites Research Institute, Xi’an 710000, PR China a r t i c l e i n f o  Article history: Received 26 October 2017Revised 27 January 2018Accepted 11 February 2018Available online 12 February 2018 Keywords: HydroxyapatiteCarbon fibersBiofilm supportsDLVO theoryBacteria adhesionBiocompatibility a b s t r a c t Abiofilmsupport withhighbiocompatibilityisneededforbioreactors. Anano-hydroxyapatite (HA)coat-ing on carbon fibers (CFs) was prepared by electrochemical deposition (ECD). The sludge immobilizationassays, bacterial cells adhesion assays and Derjaguin–Landau–Verwey–Overbeek (DLVO) theory wereusedtoevaluatethecapacityofCFsupportstoimmobilizeactivatedsludgeandbacterialcells.ThesludgeimmobilizationandbacterialcellsadhesionassaysillustratedthatHAcoatingcouldenhancethecapacityof CFs to immobilize microorganisms. SEM images showed that HA and bacterial cells formed a densefilm on CFs surface. In addition, HA, acting as a glue, could combine CFs with bacterial cells or betweencells,whichhelpedCFscapturemorebacterialcells.DLVOtheoryillustratedthatCFswithHAcoatinghadalowertotal interactionenergythanCFs withouthandling, explainingthehighercapacityofCFs withHAcoatingtoimmobilizebacterialcells.Thisresultwasowningtothelessnegativezetapotentialandhigherhydrophilicity of CFs with HA coating, and the hydrophilicity made a greater contribution to the lowertotal interaction energy. Experiments and theory reveal that HA coating could enhance the biocompati-bility of CFs, and CFs with HA coating could be used as an excellent biofilm support for bioreactors.   2018 Elsevier B.V. All rights reserved. 1. Introduction Biofilm supports for microorganisms are widely used inbioreactors for wastewater treatment [1,2], beer production [3], methanogenesis [4], etc. Biofilm supports could prevent the out-flow of microorganisms and decrease the damage to microorgan-isms caused by changes in environment [5]. The support materialwith excellent biocompatibility could improve efficiency of biore-actors. Accordingly, it is important to optimize the design of sup-ports for the immobilization and growth of microorganisms.Activated carbon and synthetic resins were used as support mate-rial for methanogenic phenol-degrading consortia and possessedthe highest adsorption capacity for  Pseudomonas aeruginosa  cells[6]. Tsekova and Llieva [7] used polyurethane foam as support material for immobilizing  Aspergillus niger   B-77, and the efficiencyof copper removal reached more than 99%. Zeolite [8] has beenwidely used as support material for the removal of ammonium inanaerobic digestion because of its favorable characteristics formicroorganism adhesion.Carbonfiberiswidelyusedasbiofilmsupportsowingtoitshighchemical durability and good capacity to immobilize microorgan-isms. MatsumotoandOhtaki [9] provedthat CFis anexcellentbio-filmsupportfor wastewater treatment byexperimentsandtheory.However, CFs without handling has smooth surface, great inertia,low surface energy and low reactivity, which could not cater forthe growing needs of bioreactor. Consequently, it is significantfor CFs to improve biocompatibility for immobilizing microorgan-isms.Someresearcherswerefocusedonimprovingthebiocompat-ibility of CF. Bao and Dai [10] used nitric acid oxidized CF as abiofilm support material, and CF after nitric acid oxidation has agood biocompatibility. Wang and Liu [11] revealed that nitricacid-treated CF has enhanced hydrophilicity and could immobilizemore  Candida tropicalis  cells in xylitol fermentation. In our previ-ous work [12], carbon nanotubes/carbon fiber hybrid materialwas evaluated as a biofilm support material for wastewater treat-ment. Carbon nanotubes deposited on CF surface could enhancethe biocompatibility of CF to immobilize  E. coli  and  S. aureus  cells.Hydroxyapatite(HA,Ca 10 (PO 4 ) 6 (OH) 2 )existswidelyinnatureasan inorganic component of bones and teeth [13]. HA has excellentbiocompatibility, andithas apotentialtobeaoutstandingsupportmaterial for microorganisms [14]. Kamitakahara and Takahashi [5] found that HA has a good capacity for the adhesion of   E. coli because of the high hydrophilicity. Fibers with HA coating havebeen applied for tissue engineering application [15,16]. However,   2018 Elsevier B.V. All rights reserved. ⇑ Corresponding author. E-mail address: (G. Dai).Applied Surface Science 443 (2018) 255–265 Contents lists available at ScienceDirect Applied Surface Science journal homepage:  no studies so far have investigated CFs with HA coating as biofilmsupport material for bioreactor.In this study, a nano-hydroxyapatite coating on carbon fiberswas prepared by electrochemical deposition (ECD). HA-coatedCFs with different deposition times (60min, 120min and 180min) compared with CFs without handling to evaluate them assupport materials for microorganisms. The sludge immobilizationand bacterial cells adhesion tests were used to investigate theproperties of CFs as support material for microorganisms in prac-tice. We adopted  Escherichia coli  ( E. coli ) and  Staphylococcus aureus ( S. aureus ) here, due to their common representative strains of Gram-negative bacteria and Gram-positive bacteria, respectively.Derjaguin–Landau–Verwey–Overbeek (DLVO) theory was used tocalculate interaction energy profiles between support materialsand bacteria cells. The interaction energy profiles would be theimportant factor to determinate the properties of CFs as supportsfor microorganisms in theory. The results implied that HA coatingon CFs could significantly improve the capacity of CFs to immobi-lize microorganisms. 2. Materials and methods  2.1. CF supports PAN-CFs (Commercial T300, diameter:   7 l m, purity: >92%)was usedas CFsupportsinthisstudy. To removea hydrophilic siz-ingagent (mainly hydrophilic surfactant and polyvinyl alcohol), CFbundles (0.2g) were immersed into acetone (500mL), and thesolution was heated to reflux at 60  C (thermostatic water bath)for 72h. Then CF bundles were washed with distilled water anddried at 120  C (electric vacuum drying oven) for 4h (denoted asCF-0).For depositing HA coating, CFs (0.05g) and graphite electrodesacted as cathode and the parallel anode in ECD, respectively. Theelectrolyte solution consisted of 3.80  10  4 mol/L NH 4 H 2 PO 4 ,6.35  10  4 mol/L Ca(NO 3 ) 2  and 0.1mol/L NaNO 3 , with Ca/P ratiobeing 1.67 [17]. 0.1mol/NaNO 3  was added to improve the conduc-tivity of the electrolytes. 3.80  10  4 mol/L NH 4 H 2 PO 4  and 6.35  10  4 mol/L Ca(NO) 3  were used corresponding to the pH value of 6[18].DCPower(M8853,Maynuoelectronics)wasappliedtosupplya constant current. The temperature of electrolyte solution wasrefinedto98.4  Cinawaterbath.Tofindtheoptimalconstantcur-rent,CFsweredepositedfor60minatdifferentdepositingcurrents(1.0mA, 3.0mA, 5.0mA, 7.0mA and 9.0mA). After deposition, CFswere rinsed with distilled water and dried at room temperature.CFs were deposited for 60min, 120min or 180min (denoted asCF-HA 60 , CF-HA 120  and CF-HA 180 , respectively) at 5.0mA deposit-ing current. The electrolyte solution was renewed every 60min.And HA-coated CFs were used as cathode under the same condi-tions. After deposition, HA-coated CFs were rinsed with distilledwater and dried at room temperature. CF supports were weightedbefore and after deposition to calculate the mass percentage of HAcoating.Afield-emissionscanningelectronmicroscopy(FE-SEM,QuantaFEG 250, FEI) was used to observe the surface morphology of CFsupports. Thechemical properties of CFswerepresentedbyfouriertransforminfrared(FTIR)spectrometry. AfullycomputerizedNico-let5700spectrometerwasadoptedtorecordFTIRspectra(KBrdis-persed pellets) in the range of 400–4000cm  1 at a resolution of 4cm  1 . Energy-dispersive X-ray analysis (EDS, X-Max 50, OXFORD)was applied to analyze the elemental content of CFs surface. Thecrystal structure of the HA coating was carried out by a X-raydiffraction (XRD, D8 Advance, BRUKER) instrument. The instru-ment was operated with a Cu K a  radiation source at 40kV and40mA.  2.2. Sludge immobilization Activated sludge was obtained from a municipal wastewatertreatment plant. The activated sludge was cultivated in a thermo-static container with organic synthetic wastewater (peptone, 0.48g  L   1 ; meet extract, 0.32g  L   1 ; urea, 0.08g  L   1 ; NaCl, 0.024g  L   1 ;NaHPO 4 , 0.08g  L   1 ; KCl, 0.011g  L   1 ; CaCl 2 , 0.011g  L   1 ; MgSO 4 ,0.008g  L   1 ) [9] under aeration at 25  C for one month.The mixed liquor suspended solids (MLSS) concentration in theactivated sludge suspension was adjusted to 4000mg/L. CF sup-ports were cut into segments of 10cm in length and grouped into2gbundles. Thirtybundlesofasupportwerefixedononesideofastick (50cmlong) and immersedinto the activated sludge suspen-sion. At every sampling time point, three bundles of CF supportswere taken out and slightly rinsed with deionized water. Then,CF supports were dried and weighed. The mean mass of sludgeimmobilized on one gram CF supports was calculated.  2.3. Bacterial cells adhesion testsEscherichia coli  and  Staphylococcus aureus  were purchased fromChina General Microbiological Culture Collection Center (CGMCC).The strains were grownin Beef extract medium(10g/L peptone, 3g/Lbeefextract,5g/LNaCl)at37  Cundershakingat120rpmovernight. Bacteria cells in exponential phase were harvested by cen-trifugation (2500g, 15min) and then were suspended in 10-foldserial dilution phosphate-buffered saline (pH=7.2) to obtain bac-teria suspension with an initial optical density (OD) of 0.65 at600nm (1.3  10 9 CFU/mL). 0.02g of CF supports were immersedinto 10mL of bacteria suspension to immobilize bacterial cells.And the suspension was shaken at 200rpm by a universal shaker.A visible spectrophotometer was used to measure OD 600  value of thesuspensionat everytime point. For CFU(colony-formingunits)formation assays, the bacteria suspension (1mL) after 2h adsorp-tionwasemployed.Aftergradientdilution,thedilutedsuspensions(20 l L each in triplicate) were dispersed evenly on nutrient agarculture medium plates for aerobic incubation at 37  C for 3days,and then the colonies were counted.For FE-SEMobservation of bacteria cells on CFs surface, CF sup-ports were taken out after 1h adhesion and soaked in 2.5% glu-taraldehyde for 12h to fix bacterial cells. Then CF supports weredehydrated in graded ethanol (30%, 50%, 70%, 85%, 95%, 99.5%,20min for each concentration) and immersed twice into isoamylacetate for 20min. Subsequently, the specimens were treated bylyophilization and spray-gold.  2.4. Bacterial cells viability tests Thebacterialcellsviabilitytestswereconductedtomeasurethebacterialviabilityafteradsorption.Thebacterialcellswereshakingcultivatedovernightunder37  Cinbeef extractmedium.Thenthebacteria cells were harvested by centrifugation and suspended inphosphate buffered saline with an OD of 0.3 at 600nm (6  10 8 CFU/ml). CF supports were soaked into the bacterial suspension.And the suspension was shaken at 200rpm for 1h. The cells werestained with 5 l M PI and 5 l M SYTO 9 for 1h in the dark. A laserscanningconfocalmicroscope(LSCM510,CarlZeiss,Germany)wasused to obtain fluorescence images. All samples were tested forthree times, and five images were taken for each test. The Image J software was used to counted total cells and dead cells. The per-centage of live bacterial cells was calculated from the quantity 256  Q. Liu et al./Applied Surface Science 443 (2018) 255–265  ratio of live cells to total cells. The results were averaged out andthe standard deviations were calculated. 3. Calculation of interaction energy profile Interaction energy profiles between bacteria cells and CF sup-ports were calculated from the following equation [19]. Total interaction energy ¼ repulsion energy þ attraction energy G Total  ¼ p e r  e 0 a ½ð w 1 þ w 2 Þ 2 ln ð 1 þ e  j h Þþð w 1  w 2 Þ 2 ln ð 1  e  j h Þ  A a = 6 h ð 1 Þ where e r  istherelativepermittivityofthesuspensionmedium, e 0  isthe vacuum permittivity,  a  is the radius of the bacteria cell, W 1  isthe surface potential of the CFs, W 2  is the surface potential of thebacterial cell,  j  is the inverse Debye –Hückel length, A is thehamakerconstantandhisthedistanceofclosestapproachbetweenCFs and the bacteria cell. At 20  C,  j  could be calculated accordingto the relation [20]: j  1 ð nm Þ¼ 0 : 304 =  ffiffi I p  ð 2 Þ where I is the ionic strength expressed in molar. The value of I is202mM, when the bacteria cells are suspended in 10-fold serialdilution phosphate buffered [21]. According to Young’s equation and Fowkes [22], we obtain c L  ð 1 þ cos h Þ¼ 2  ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi c LW S   c LW L q   ;  ð 3 Þ Fig. 1.  SEM images of CFs after 60min deposition at a constant current of (a) 1.0mA, (b) 3.0mA, (c) 5.0mA, (d) 7.0mA and (e) 9.0mA. Q. Liu et al./Applied Surface Science 443 (2018) 255–265  257  where c L   isthetotalsurfacetensionoftheliquid,  c LW  S  istheLifshitz-vanderWaalssurfacetensionof solidsurface,  c LW L  is heLifshitz-vanderWaalssurfacetensionofliquidand h isthecontactangleofsolidsurface. For water, at 20  C, c LW  L   =21.8  10  3 N/m and  c L   =72.8  10  3 N/m. Therefore,  c LW  S  could be obtained by measuring the con-tact angle  h  of CF supports. The hamaker constant (A SLS ) for theinteraction between CFs immersed in water is [23]  A SLS   ¼ 1 : 44  10  18  ffiffiffiffiffiffiffiffi c LW S  q     ffiffiffiffiffiffiffiffi c LW L q    ð  J Þ :  ð 4 Þ The hamaker constant (A SWS ) for CFs immersed in water couldbe also written as [24]  A SWS  ¼  ffiffiffi  A p  SS    ffiffiffiffiffiffiffiffiffiffi  A WW  p   2 :  ð 5 Þ When bacterial cells are interacting with CFs surface in water,the hamaker constant (A BWS ) can be obtained according to [25]  A BWS   ¼  ffiffiffiffiffiffiffi  A BB p     ffiffiffiffiffiffiffiffiffiffi  A WW  p   ffiffiffiffiffiffiffi  A SS  p     ffiffiffiffiffiffiffiffiffiffi  A WW  p   ;  ð 6 Þ where A ij  is the Hamaker constant of between materials i and j (i, j=B, W, S) and subscripts B, W and S represent bacteria, water andCFs surface, respectively. For water and  E. coli , A WW  =4.0  10  20  J[26] and A SS  =4.173  10  20  J [27]. According to Eq. (3)–(6), the hamaker constant (A BWS ) can be obtained by measuringthe contactangle of CF supports. The contact angle of carbon fibers cannot be directly measureddue to the small diameter. Therefore, a dynamic contact angle ten-siometer (DCAT 15, Dataphysics, Germany) was adopted to mea-sure the contact angle. The deionized water was conducted astestliquid. Thereportedcontactanglesweretheaverageofatleast10 repeated measurements. An electrokinetic analyser SurPASS 3(AntonPaarKG,Graz,Austria)wasusedtomeasurethezetapoten-tials of CF supports. CF supports were cut into pieces (2mm inlength and 0.5g in weight) and dispersed in 100mL deionizedwater with ultrasonic technique. The suspension was poured intothe measurement cell. A hydraulic pressure was provided andthe resulting electric potentials were measured by two electrodes.More details are described elsewhere [9]. 4. Results and discussion 4.1. CF supports To optimize the electrophoresis conditions, HA crystals weredeposited on CFs surface at the different constant currents (1.0mA, 3.0mA, 5.0mA, 7.0mA and 9.0mA). Fig. 1 shows SEM imagesofCFswithHAcoatingafter60mindepositionatthedifferentcon-stant currents. Only a few HA crystals appeared on the surface of CFs at 1.0mA depositing current (Fig. 1(a)). A large number of rod-shaped HA crystals were deposited on CFs surface at 3.0mAdepositing current (Fig. 1(b)). But there were still some areas of CFs surface which was not covered by HA crystals because of thelow depositing current. CFs were covered with HA crystals evenlyafter deposition at 5.0mA, 7.0mA and 9.0mA depositing current(Fig. 1(c), (d) and (e)). HA crystals at 5.0mA depositing currentformed a better three dimensional (3D) structure in comparisonwith7.0mAand9.0mA,whichcouldincreasethecell-surfacecon-tact area. In addition, HA crystals were easy to formclusters under9.0mAdepositingcurrent(Fig. 1(e)).Theresultsindicatedthat5.0mA current was suitable for HA crystals formation and depositionon CFs surface.Fig. 2(a)illustratesthetypicaldepositionvoltage-timecurvesof CFs after 0–180min deposition at 5.0mA. Because the electrolytesolutionwasrenewedevery60min,theelectrophoresisdepositionrepeated every 60min. The voltage showed an upward trend withthe deposition time and remained relatively stable at 2.37V for 0–60mindeposition,2.45Vfor60–120mindepositionand2.62Vfor120–180min deposition. The relatively constant voltage increasedwith the repeated times of deposition, because the increase of HAcoating thickness resulted in the intense electric resistance. Fig. 2(b) shows the mass percentage of HA coating on CFs. With theincreasing deposition time, the mass percentage of HA coatingincreased from 9.43% for 60min to 26.15% for 180min.Fig. 3 shows SEM images of CFs without handling and CFs withHA coating with different deposition time (60min, 120min and180min). CF-0 had smooth surface and shallow ridges along thelongitudinal direction (Fig. 3(a)). A uniform and dense HA coatingon CFs surface could be obtained by ECD (Fig. 3(b), (c) and (d)).TheHAcoatingonCFssurfacegotthickerwiththedepositiontime.The crystal morphology of HA also transformed with the deposi-tion time. The crystals was needle-like in the first 60min deposi-tion. Then, the crystals transformed into rod after 120mindeposition. Finally, the crystals changed into hexagonal prismsafter 180min deposition. During 0–180min deposition, the aver-age diameter of HA crystals was about 60nm, 150nm and 170nm for 60min, 120min and 180min, respectively. The increasinggrain size indicated that crystals grew in both the longitudinaland radial directions. The results were consistent with some otherstudies [17,28]. The supersaturation around CFs surface was thedriving force which governed the formation of HA crystals [18].In the initial stage, HA coating was directly prepared on CFs sur-face, which resulted in the increasing density of HA crystals. And Fig. 2.  (a) Deposition voltage–time curves for the ECD of HA coating on CFs and (b)the mass percentage of HA coating on CFs.258  Q. Liu et al./Applied Surface Science 443 (2018) 255–265
Related Search
We Need Your Support
Thank you for visiting our website and your interest in our free products and services. We are nonprofit website to share and download documents. To the running of this website, we need your help to support us.

Thanks to everyone for your continued support.

No, Thanks