Biofilms on abiotic surfaces – in situand in lab experiments

Yamini JangirMicrobial Diversity Course 2014

Abstract

Many microorganisms adhere to living and nonliving materials during the process of theirgrowth. The significance of surface attachment for growth was observed with Thiothrixgrowing on rocks along the mouth of a Trunk river. There are many new techniques ofculturing novel organisms where abiotic surfaces are used to provide an attachment surface fortheir growth. This project aims to understand the cell attachment (with respect to biofilmformation) and its further growth on various abiotic surfaces – polyurethane sponge, carboncloth and glass slide. Trunk river was chosen as the site for study due to the presence oforganisms which benefit from this ecological niche of attachment. The attachment wasobserved to be best in polyurethane sponge and least on glass slide proving the fact that surfaceroughness and porosity plays important role during attachment. No significant difference wasobserved in the microbial community colonizing various surfaces, analyzed using 16S rRNAclone libraries and CARD-FISH.

Introduction

A biofilm is an assemblage of irreversibly surface-associated microbial cells enclosed in anextracellular polymeric substance matrix [Donlan, 2002]. They have been shown to form inwide variety of surfaces and are present in various forms and structure. The biofilm growthcycle encompasses bacterial adhesion at various levels, including initial physical attraction ofbacteria to a substrate [Garrett, 2008]. Using this capability of some bacteria which prefer toattach to surfaces – various techniques including continuous flow reactor that comprises ofhanging polyurethane sponge have been developed to enrich for slow growing methanogenicarchaea [Imachi, 2011]. A fairly new technique of cultivating metal reducing and oxidizingbacterium with carbon electrodes poised at appropriate potential may or may not be dependenton the surface attachment of the microbes [Summers, 2013; Torres, 2009].

Trunk river was selected for incubating various abiotic surfaces. This river has been extensivelystudied for sulfur oxidizing bacteria, but a complete phylogenetic diversity in the river in stilllacking. Along with studying attachment on the surface of biofilm using microscopic techniquelike scanning electron microscopy and confocal microscopy, phylogenetic diversity of theorganisms which inhabits these surfaces was studied using 16S clone libraries and catalysedreporter deposition (CARD) fluorescence in situ hybridization (FISH) techniques.

Methods and Materials

Total of seven incubation were set up: four along the Trunk river and three in the lab (seatable). Polyurethane sponge cubes, carbon cloth and glass slide were fastened to each otherusing fishing line for in situ incubation at four sites (Site1, S1; Site 2, S2; Site3, S3) along theTrunk river (Figure 1). Sediment mat was collected in round grad beaker with cap (500 ml)from site 2 (ILS2), site 3 (ILS3) and site 4 (ILS4) for incubating the three surfaces in lab.Temperature, pH, salinity and water density was recorded with the help of thermometer, pHpaper and refractometer. Water samples were collected in 200 ml serum bottles to study waterchemistry.

The sediment sand brought back to the lab were used to incubate polyurethane sponge, carboncloth and glass slide in the lab and were kept at the sea table with open cap for ambientconditions. After 4 days the surfaces from various sites were collected and stored in cold roomfor further processing.

Figure 1: Incubation set up was deployed at four sites along the Trunk river to characterize the community withcan attach to abiotic surfaces: polyurethane sponge, carbon cloth and glass slide. The site was properly markedwith sign boards to inform the public about the ongoing experiment. b) Site 1 under the bridge with sponge (S1-sponge), carbon (S1-carbon) and glass (S1-glass), c) Site 2 approx 30 m into the river with sponge (S2-sponge),carbon (S2-carbon) and glass (S2-glass), d) Site 3 approx 60 m into the river with sponge (S3-sponge), carbon(S3-carbon) and glass (S3-glass), and e) Site 4 was in the middle of the pond with sponge (S4-sponge), carbon(S4-carbon) and glass (S4-glass).

Determination of chemical concentration

The overhead gas (250 l) in the serum bottle from the four sites was sampled for methaneconcentration using GC-2014 gas chromatograph (Shimadzu; Columbia, MD, USA). Lactate,acetate and formate were measured using LC-2010C HT liquid chromatograph (Shimadzu;Columbia, MD, USA). The liquid samples was acidified using 1 M H2SO4 to a finalconcentration of 100 mM H2SO4 and filter sterilized using HPLC certified 0.2 m nylonmembrane filters.

Micro-sensor measurements

To characterize the mat collected from the site for lab incubation and to understand themetabolic activity present in the mat, variation of oxygen, pH and sulfide was measured withdepth using micro-sensors (outside tip diameter - 500 m) from Unisense (Aarhus, Denmark).Depth profiles were measured in steps of 500 m, with waiting time of 1 sec and measuringperiod of 3 sec. Oxygen micro-sensor was calibrated using DI water bubbled with air – 21%oxygen, and DI water reduced by 0.1 M Ascorbic acid – 0% oxygen. For calibrating sulfidemicro-sensor, buffer solution was prepared using stap water base at pH < 3 (in 0.1 M citric acidbuffer). The buffer was made anaerobic by bubbling N2. Known volume of sulfide stocksolution (Na2S) was added to the buffer to calibrate with 100 M, 300 M, 500 M, 1000 Mand 2000 M H2S. The pH micro-sensor was calibrated using pH buffer 4, 7, 10.

Scanning Electron Microscopy

The samples were fixed with 4% Paraformaldehyde (PFA) overnight, and imaged using HitachiTM3030 tabletop scanning electron microscope.

Confocal Microscopy

The carbon cloth (S1- carbon) was cut into pieces (1 cm x 1 cm) using sterilized scissors.Sample was fixed using 4% Paraformaldehyde (PFA) at room temperature for 2 hours,followed by staining in 1 g/ml DAPI solution for 10 min at 37°C. Single strands of carboncloth was put on a glass slide and were mounted using VECTASHIELD® mounting media(Burlingame, CA, USA). The slipcover was sealed using nail polish to avoid drying of thesample. Zeiss LSM700 confocal laser scanning microscope was used to image DAPI stainedcells attached to the carbon cloth using an excitation wavelength of 405 nm. An area of 160x160 m2 was scanned along z-axis at a step size of 1 m to cover a total height of 160 m.

Clone Library

DNA was extracted from the samples using PowerSoil® DNA Isolation kit following themanufacturers' protocol. The extracted DNA was quantified using QuantusTM Flourometer(Promega; Madison, WI, USA). This as followed by PCR amplification of the extracted DNA

using bacterial (8F and 1391R) and archaeal primers (4FA and 1391R) for 16S analysis. Mastermix recipe comprised of Promega Go-Taq Green 2X Mix (25 ml), forward primer (4 ml),reverse primer (4 ml) and Nuclease-free water (13 ml) to give a total reaction volume of 46 mlper sample. PCR procedure consisting of a denaturing step of 2 minutes at 95°C, then 30 cycleswith 1) 30s denaturing at temperature of 95°C, 2) annealing at 46°C for 30s, 3) elongation stepfor 90 s at 72°C, with a final extension at 72°C for 10 min was applied. The thermocycler waskept on hold at 4°C until the PCR products were taken out of the system. The PCR productswere analyzed for length by using gel electrophoresis.

Further processing was done on four samples (S3 sponge, S3 carbon, ILS3 sponge and ILS3carbon) with promising DNA extraction results. pGEM®-T Easy vector system whichcontained ampicillin resistance was used for ligation. This ligation buffer was used totransform bacterial strain JM109. All these steps were performed using manufacturers'instructions. The transformed cells were plated on LB plates with Ampicillin (100 g/ml), X-gal (0.1 g/ml) and IPTG (0.5 mM) and incubated at 37°C overnight. Forty eight colonies werepicked from each library and incubated in 96-well plate containing LB (with Ampicillinresistance) at 37°C. The samples were further processed for plasmid extraction and sent for 16Ssequencing.

Catalysed reporter deposition florescence in situ hybridization (CARD-FISH)

Samples were fixed in 4% PFA for 24 hours at 4°C. Followed by washing twice in 1XPhosphate-buffered saline (PBS), pH 7.6. The sample were then transferred in 1:1 mix of PBSand ethanol. Sonication at 30% intensity with 20 pulses for 20 sec was performed to loosencells attached to the surfaces (Sonopuls HD 70, Bandelin, Berlin, Germany). Sample wasvortexed and 100 l of the mixture was mixed with 10 ml of 1X PBS. This was further filteredon a 16 mm (diameter) membrane polycarbonate filter. Filter was washed with more 1X PBSwhile it sat on the filtering apparatus. Now the filter was left for air drying on a whatman paperwhile covering the top (to avoid contaminants sticking to the filter).

After the filter were dried the cells were embedded using low gelling agarose (0.1% gelstrength). The agarose was heated in the microwave for 2 mins, cooled down to 37°C and usinga spray dispenser it was sprayed on the filter. Again the filter was let to air dry on whatmanpaper at room temperature.

Permeabilization step which is necessary to allow diffusion of probe inside the cell differed forbacteria and archaea. Bacteria were permeabilized with lysozyme whereas for archaeaproteinase K was used. Each filter (total 4; from S3 sponge, S3 carbon, ILS3 sponge, ILS3carbon) were labeled accordingly with pencil and part of each filter was cut using razor bladefor separate treatment. Bacterial permeabilization step involved incubaing the filter in freshlysozyme buffer (100000 U/ml in 0.05 M EDTA, pH 8.0; 0.1 M tris-Hcl, pH 8.0) for 60 min at37°C in dark. Archaeal permeabilization step involved incubating the filter in fresh ProteinaseK solution (15 g/ml in 0.05 M EDTA, pH 8.0; 0.1 M tris-HCl, pH 8.0, 0.5 M NaCl) for 5 minat room temperature in dark. Both the filter were washed in excess MilliQ water and left for airdrying for further processing.

It is important to perform inactivation of peroxidase activity for some organisms which maycarry peroxidases or protein with psuedoperoxidase activity responsible for high backgroundsignal. The filters were incubated in 0.01 M HCl and 3% H2O2 for 20 min at room temperature.Filters were washed in excess MilliQ water, followed by 96% ethanol and let air dry onwhatman paper.

After above step, all the following protocol should be done continuously. The filters now needto be hybridized with the probe of interest. Hybridization buffer (300 l) was prepared for eachprobe according to the table provided in the manual. The probes used for analysis wereNON338, ARCH915, EUB338-I-III, ALF968, BET42a (unlabeled GAM42a), GAM42a(unlabeled BET42a), DELTA495a-c, EPSY914, DSS658 and JTB1275 c1-4 which targetnonsense hybridization, archaea, bacteria, alphaproteobacteria, betaproteobacteria,gammaproteobacteria, deltaproteobacteria, epsilonproteobacteria, desulfosarcina (sulfurreducing bacteria) and various clades (may be responsible for sulfur oxidation bacteria). 1 l ofthe probe solution (and competitor probe wherever required) was mixed with 300 l ofhybridization buffer in an eppendorf tube. The filter sections were incubated in thehybridization solution at 46°C for 3 hours.

The filter were then washed in pre-warmed (48°C) washing buffer for 10 min, followed byincubating them to 1X PBS for 15 min at room temperature. The excess liquid was removedbefore amplification step. 300 l of amplification buffer was mixed with 0.15% H2O2 solutionin a ratio of 100:1. 1 l of fluorescently labeled tyramide (excitation at 594 nm, 1 mg/ml) wasmixed in amplification buffer. The filter sectioned were incubated in the amplification solutionfor 30 min at 46°C in dark. Next, the filter section were washed in 1X PBS for 10 min in darkat room temperature. Further washing was done using MilliQ water and 96% ethanol for 1 minand let for air drying.

Counterstaining with DAPI with 1 g/ml was performed after the filter sections were dried at37°C for 10 mins. The filter sections were again washed with MilliQ water and 96% ethanol forfew seconds. The filter section were air dried before mounting on glass slide with Vectashieldmounting media. The filter sections were imaged using Ziess Axio Imager.A2 under 100Xusing DAPI, Alexa Flour488, DsRed filter to look for DAPI stained cells, Co-enzyme F-420fluorescence (important for methanogenic pathway in methanogenic archaea) and respectiveprobes.

Results and Discussion

The basic site characterization showed the change in salinity and temperature along the Trunkriver. The pH did not seem to differ much, but this could also be attributed to the sensitivity ofthe pH paper (Table 1).

Site pH Salinity (‰) Density (g/ml) Temperature (°C)S1 7 40 1.005 31

S2 7 30 1.004 31

S3 7 20 1.0025 27

S4 7 20 1.002 27

Table 1: Basic Characterization of the site along the Trunk river

While setting up the in situ experiment bubbling was observed (possibly due to methaneproduction). Gas samples however did not show any presence of methane. This is attributed toincorrect gas sampling. However, site 3 and site 4 shows a slightly higher methaneconcentration compared to site 1 and site 2 (Table 2). For future gas sampling, it is advisable toemploy gas collection tube.

Incubations Methane Concentration (mM)

S1 1.93

S2 1.93

S3 1.95

S4 1.93

ILS2 1.93

ILS3 1.95

ILS4 2.04

Table 2: Methane concentration at various incubations: Site1 (S1), Site2 (S2), Site3 (S3), Site4 (S4), In lab site2 (ILS2), In lab site 3 (ILS3), In lab site4 (ILS4)

No measurable formate, acetate, lactate was detected in the samples. This may be attributed to the factthat the organisms present at this site use complex carbon sources. To verify this hypothesis furtherexperiments are required to be performed.

The depth profile of oxygen and sulfide provided some indications of the possible organisms residingin the sediment which may attach and form biofilms on the provided abiotic surfaces. Oxygen isdepleted in the first 2 mm into the sediment. However, if probed deeper the oxygen concentrationincreased caused by pockets of air introduced by sponge, carbon cloth which are porous in nature. Also,a lot of heterogeneity was observed with replicates with some sediment mat. Sulfide measurementsshows presence of a source of sulfide below 5 mm depth. This could be attributed to sulfur-reducingbacteria (distributed within the class deltaproteobacteria and epsilonproteobacteria). The reduction ofsulfide at the air sediment interface hints towards presence of sulfide oxidizing bacteria which useoxygen as the terminal electron acceptor (Figure 2).

The sulfide gas measured using microsensor does not account for speciation of sulfide gas intoHS- and S2- according to pH. The total sulfide was calculated using the following equation:

∑ [H 2 S ]=[H 2 S]×(1+ 10−pK1

10−pH)

where pK1 is the dissociation of sulfide in water/seawater dependent on salinity andtemperature calculated using the following empirical formula:

pK 1=−98.08+5765.4

T+15.04555 ln(T )−0.157 S0.5+0.0135 S

where T is in Kelvin and S is in per mille (‰). The calculations are required to be corrected forporosity of the sediment and abiotic surfaces, however it was not done for this project.

Using first and second Fick's law of diffusion one can evaluate the diffusive flux and the rate ofchange of concentration for oxygen and sulfide. Here, sulfide depth profile for ILS4 was fittedwith an exponential curve, to analyze the profile of flux and rate of change of concentrationover time. It is observed that flux and rate of change of sulfide concentration will peak withdepth purely based on basic calculations. This postulates that there is a flow of sulfidemolecules from bottom surface to top layer (Figure 3).

Figure 2: Microsensor profiles of a) oxygen and b) sulfide for sediment mat: In lab site 2 (ILS2), In lab site3 (ILS3) and in lab site 4 (ILS4) used for incubating the three abiotic surfaces: sponge, carbon cloth and glass slides.

The samples (sponge and carbon cloth) were observed under the table top scanning electronmicroscope and confocal microscope (DAPI) for attachment for microbes (Figure 4 and Figure5).

Figure 3: a) Sulfide concentration of ILS4 sediment, b) the exponentially fitted curve for a depth of 13 mm, c) Diffusive flux calculations and d) Rate of change of concentration with depth

Figure 4: Confocal microscope image of the carbon cloth showing the presence of cells through DAPI.

Figure 5: SEM image showing the presence of organisms including diatoms.

The 16S sequences from the 48 colonies from each S3 sponge, S3 carbon cloth, ILS3 spongeand ILS3 carbon. The 16S data received was of very low quality, hence no statistical analysiscould be performed. However, the following few 16S rRNA data closely mtached with thefollowing phylogenetic groups within the four samples – Gammaproteobacteria,Deltaproteobacteria, Alphaproteobacteria, Betaproteobactera, Ignavibacteria, Epsilonbacteria(with more than 90% confidence threshold). These sequences were classified using ribosomaldatabase project (RDP) classifier.

CARD-FISH analysis provided more insight to the presence of bacteria (EUB338) andmethanogenic archaea (F-420 fluorescence) in the sponge as well as carbon cloth. Cell densityin S3 sponge was greater than S3 carbon, however for in lab experiment the attachment wassimilar on the sponge and carbon (Figure 6).

Figure 6: DAPI cell count for various incubations performed during the experiment point out that attachment on the surface of sponge in situ was higher than the cell of the incubations. Each scale bar is 20 m.

S3 Sponge S3 Carbon

ILS3 Sponge ILS3 Carbon

(1.23 ± 0.17) x 107 cells/ml (7.00 ± 2.14) x 106 cells/ml

(6.89 ± 1.5) x 106 cells/ml (6.36 ± 1.19) x 106 cells/ml

Many of the CARD-FISH experiment failed due to improper washing step. However, EUB338probe hybridization was repeated on filter sections from each sample and it confirmed thepresence of bacteria in the surfaces. Proper counting of probe signal (other than EUB338) wasmade impossible due to high background fluorescence.

Here, the possible presence of each probe is tabulated (Table 3).

Probe ID Target Organisms PresenceNON338 Nonsense -

ARCH915 Archaea +EUB338 I-III Bacteria +

ALF968 Alphaproteobacteria -BET42a Betaproteobacteria +

DELTA495a-c Deltaproteobacteria -EPSY914 Epsilonproteobacteria -GAM42a Gammaproteobacteria -DSS658 Desulfosarcina -

JTB1275 c1-4 Various clades -

Table 3: The detection of various clades observed via CARD-FISH technique.

F-420 and EUB338 probe signal was quantified for each sample by taking the ratio of probesignal with DAPI signal. The figure 7 and the table 4 below provides the qualitative andquantitative view of the probe signal.

Sample F-420 coenzyme EUB338S3 sponge 0.26 ± 0.11 0.76 ± 0.19S3 carbon 0.34 ± 0.12 0.72 ± 0.20

ILS3 sponge 0.42 ± 0.19 0.45 ± 0.14ILS3 carbon 0.22 ± 0.14 0.70 ± 0.23

Table 4: The ratio of F-420 and EUB338 signal with DAPI.

ConclusionsThis experiment verified that attachment on polyurethane sponge was greater than carbon clothfor in situ incubation experiment along the trunk river at site 3. The attachment of cells in thesponge and carbon cloth for incubation in lab did not differ much, confirming that sponge withhigher porosity has greater ability to capture organism in flow conditions. None of the glassslide showed any attachment to the surface, hence were not pursued for further analysis.

Micro-sensor analysis pointed towards presence of sulfur oxidizing and sulfur reducing bacteria, whichwere seen in clone library analysis. However not many probes were seen using CARD-FISH analysisdue to large background fluorescence.

This experiment can be used to study biofilm formation on abiotic surfaces for future. However, it isimportant to note that incubation period should be kept longer which was not possible due to timeconstraint during this mini-project. For this project only single cells were observed under the scanningelectron microscope and formation of any biofilm is inconclusive. Also, parallel incubations shouldhave been started to take time series measurement of cell count on the surfaces for quantifying growthon these surfaces.

Overall project is only proof of concept and needs further studies. This project helped me learn varioustechniques like microelectrode profiling, building clone libraries, confocal microscopy and CARD-FISH for phylogenetic identification of microorganisms.

Figure 7: DAPI, F-420 and probe (EUB338) signal from the ILS3 sponge sample shows the presence of bacteria. Scale bar = 20 m.

ILS3 Sponge

EUB338

F420

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