Characteristics of Streptomyces griseus biofilms in continuous flow tubular reactors

Michael Winn1, Eoin Casey2, Olivier Habimana2, and Cormac D. Murphy1*

1 UCD School of Biomolecular and Biomedical Science,2 UCD School of Chemical and Bioprocess

Engineering, University College Dublin, Belfield, Dublin 4, Ireland

*Corresponding author, Cormac.d.murphy@ucd.ie

Keywords: Attached growth; extracellular polymeric substances; continuous bioprocess

Running title: Streptomyces griseus biofilm

1

Abstract

The purpose of this study was to investigate the feasibility of cultivating the biotechnologically

important bacterium Streptomyces griseus in single-species and mixed- species biofilms using a

Tubular Biofilm Reactor (TBR). Streptomyces griseus biofilm development was found to be cyclical,

starting with the initial adhesion and subsequent development of a visible biofilm after 24 hours

growth, followed by the complete detachment of the biofilm as a single mass, and ending with the

re-colonization of the tube. Fluorescence microscopy revealed that the filamentous structure of the

biofilm was lost upon treatment with protease, but not DNase or metaperiodate, indicating that the

extracellular polymeric substance is predominantly protein. When the biofilm was cultivated in

conjunction with Bacillus amyloliquefaciens, no detachment was observed after 96 h, although once

subjected to flow detachment occurred. Electron microscopy confirmed the presence of both

bacteria in the biofilm and revealed a network of fimbriae-like structures that were much less

apparent in single-species biofilm, and are likely to increase mechanical stability when developing in

a TBR. This study presents the very first attempt in engineering Streptomyces griseus biofilms for

continuous bioprocess applications.

2

Introduction

Biofilms are microorganisms that grow attached to a surface, and are characterised by the

production of an extracellular matrix, increased tolerance to drugs and other xenobiotics, and

improved inter-cellular communication. In clinical settings, biofilms are commonly found on medical

implants and catheters; in industry, biofilms are problematic in pipes resulting in blockages, and

cause fouling and corrosion of crucial surfaces (Flemming, 2002, Hall-Stoodley et al., 2004). Biofilms

play an important role in wastewater treatment and their potential for degrading specific pollutants

has been investigated (Misiak et al., 2011). In recent years there has been a surge of interest in the

application of single species biofilms for biotechnology (Winn et al., 2012). Increased environmental

robustness, self-immobilisation and their ease of integration into continuous flow bioreactors make

biofilms ideal for production of fine chemicals at high productivities. For example, continuous

production of (S)-styrene oxide from styrene was enabled by Pseudomonas sp. strain VLB120∆C

cultivated in a tubular bioreactor, which had a volumetric productivity that exceeded that in a stirred

tank reactor with the same strain and had a process duration of over 50 days (Gross et al., 2010).

Biofilm catalysts employed in continuous processes also have the potential advantages of slower

biomass turnover and consequently reduced biomass waste compared with equivalent batch

systems.

Streptomyces spp. have a plethora of useful enzymes that can be applied to a range of

potentially valuable biotransformations. One class of these is the cytochromes P450 that catalyse

regio- and stereo-selective hydroxylation on unactivated carbon centres (Schulz et al., 2012).

Streptomyces griseus is one of the best genetically characterised members of the genus and has

many attributes that can be exploited for biotechnological applications. It is known to produce a

number of bioactive molecules, including the antibiotic streptomycin, and analysis of the complete

genome of S. griseus suggested that this strain has the capacity to produce over 34 secondary

metabolites (Ohnishi et al., 2008). Furthermore, the potential of the intrinsic cytochrome P450

activity of S. griseus has been demonstrated to enable production of human-like drug metabolites of

3

pharmaceuticals for subsequent toxicological evaluation (Alexandre et al., 2004, Bright et al., 2011).

Despite the biotechnological importance of this bacterium, no studies have yet been conducted to

investigate the potential of S. griseus biofilms in continuous flow biocatalysis. In this paper we

describe the first investigations into the characteristics of S. griseus when cultured in a tubular

biofilm reactor, and assess how co-cultivation with another bacterium might improve biofilm

stability.

Materials and methods

Media and strains

Streptomyces strains were obtained from the German Collection of Microorganisms and Cell

Cultures (DSMZ) or were environmental isolates obtained from the UCD School of Biomolecular and

Biomedical Science culture collection. Spore stocks were prepared from strains grown on solid ISP4

medium (BD Biosciences) and re-suspended in 25% (v/v) glycerol and stored at -20 °C. Strains were

grown at 28 °C in either soybean medium (5 g L -1 Bacto Soytone, 20 g L-1 glycerol, 5 g L-1 yeast extract,

5 g L-1 KH2PO4, pH 7.0), ISP2 (10 g L-1 malt extract, 4 g L-1 yeast extract, 4 g L-1 glucose, pH 7.2),

tryptone soya broth (BD biosciences) or NMMP medium (van Keulen et al., 2003) supplemented with

20% (w/v) of either glucose or mannitol as carbon source. Planktonic cultures were grown in 50 mL

screw capped vials fitted with a 2 cm spring and agitated at 180 rpm.

Bacillus amyloliquefaciens was obtained from the American Type Culture Collection (ATCC

23844) and grown in liquid LB broth (10 g L -1 tryptone, 10 g L-1 NaCl, 5 g L-1 yeast extract) or on solid

LB and incubated at 28°C. Planktonic cultures were agitated at 180 rpm.

Well plate assay

S. griseus ATCC 13273 was cultivated in polystyrene 12-well plates (Corning, Cellbind). Each well

contained 2 ml of medium (ISP2, TSB or soybean) and was inoculated with 20 µL started culture. The

well plates were incubated at 30 °C for up to 96 h, either statically or with gentle agitation (75 rpm).

4

Biofilm growth was assessed after removing the liquid, rinsing the wells with phosphate buffered

saline and staining with crystal violet; the absorbance was measured at 595 nm.

Tubular biofilm reactor (TBR)

The TBR comprised of an inoculum reservoir, a medium reservoir fitted with a glass flow break, a

peristaltic pump (Watson Marlow Sci Q400) connected to a 100 cm-long length of silicone tubing (8

mm O.D., 5 mm I.D., Fisher Scientific) and a spent medium reservoir (Fig. 1a). A planktonic starter

culture was grown in soybean medium for 24 h and diluted 100-fold into the inoculum reservoir

containing fresh medium. This inoculum was pumped through the reactor at 10 mL min -1 until the

whole reactor was filled. The flow was switched off for 30 minutes to allow cells to adhere to the

silicone tubing, and then flow was initiated at 1 mL min-1 from the fresh medium reservoir. To test

the effect of laminar flow on biofilm development other reactors were inoculated either in the

absence of laminar flow or the flow was initiated after 16 hours following the formation of a thick

biofilm. The TBR was operated for at least 26 hours at 28°C to observe the biofilm detachment cycle.

Miniature static TBR

To enable convenient screening of other Streptomyces spp. for biofilm production a 20 cm length of

sterile silicone tubing (8 mm O.D., 5 mm I.D. Fisher Scientific) was connected to a 6 mL Luer Lock

syringe (Fisher Scientific). The syringe was used to draw appropriately inoculated soybean medium

(prepared as for the TBR reactor) into the tubing, which was capped at the other end by an

additional syringe. Adjustment of the two syringes was used to ensure no air bubbles remained

inside the tubing. The tube was then incubated at 28°C for 26-120 hours (Fig. 1b).

Chemical degradation of the biofilm

Biofilms for chemical degradation experiments were grown in three-channel biofilm flowcells

(BioCentrum-DTU, www.csm.bio.dtu.dk/Instrument%20Center/Resources/Biofilm%20Setup.aspx,

5

see Fig. 1c). Flowcell channels were sealed by fixing a microscope coverslip (24mm x 60mm

thickness range No.1.5, Thermo Scientific) onto the flowcell with silicone rubber compound (RS

components). The flowcell was connected to the flow system with silicone tubing (3 mm O.D., 1 mm

I.D., Saint-Gobain Performance Plastics) and a peristaltic pump with a flow rate of 10 mL h -1 and

sterilised by passing a 1% solution of Virkon (Antec International) through the channels for 15

minutes. The channels were then rinsed with PBS for an additional 15 minutes. To grow the biofilms

the channels were inoculated with an identical inoculum culture as prepared for the TBR reactor.

The biofilms were allowed to develop under static conditions for 2-3 days before the growth

medium was replaced with either H2O, DNAse I (Sigma, 500 µg mL-1 in 5 mM MgCl2), NaIO4 (Sigma, 40

mM in H2O) or Pronase (Roche, 10 mg mL-1 in Tris-HCl, pH 7.5). Solutions of the chemicals were

pumped through the channel for 10 minutes before being returned to static incubation at 30°C.

Streptomyces-Bacillus amyloliquefaciens co-culture biofilm

Various Streptomyces cultures were prepared as described. B. amyloliquefaciens was grown in a 50

mL screw cap vial in LB medium for 16 h at 28 °C. The starter cultures were diluted 100 fold into

fresh soybean medium and this co-culture was immediately used as the inoculum for either the

continuous flow or miniature static TBR reactors. The biofilm was allowed to develop statically for

16-92 hours.

Fluorescence microscopy

For fluorescence microscopy the detached biofilms were removed from the TBR and thin slices made

using a scalpel and placed onto a microscope slide. The slices were stained for 15 minutes with

acridine orange (0.1 %) and visualised with an Olympus BX51 microscope fitted with a BP460-490

excitation filter and a BA515-550 barrier filter.

Transmission electron microscopy

6

Samples for transmission electron microscopy (TEM) were first fixed in 2.5 % glutaraldehyde in 0.1 M

Sørensen’s phosphate buffer (40.5 mL 0.1M Na2HPO4 (Riedel-deHaën AG, Seelze, Germany) and 9.5

mL 0.1M NaH2PO4 (Riedel-deHaën AG, Seelze, Germany), pH 7.4) for a minimum of 2 hours at room

temperature and post-fixed in 1 % osmium tetroxide in Sørensen’s phosphate buffer for 1 hour at

room temperature. Subsequently, the specimens were dehydrated in a graded ethanol series (30,

50, 70, 90, 100 %). When dehydration was complete samples were transferred from 100 % ethanol

to acetone, from acetone to a mixture of 1 part of acetone and 1 part of epoxy resin (24 g agar 100

resin), 9.5 g DDSA (Dodecenyl Succinic Anhydride), 16.5 g MNA (Methyl Nadic Anhydride) and 1 g

DMP-30 (2,4,6- tri(dimethylaminoethyl)phenol) for 1 hour. To complete the resin infiltration the

samples were placed in 100% resin at 37 °C for 2 hours. Finally samples were embedded in resin,

placed at 60 °C for 24 hours until polymerisation was complete.

For orientation purposes, sections from each sample were cut at 1 m, stained with

toluidine blue (consisted of toluidine blue (Agar Scientific, Essex, UK) and 1 % sodium tetraborate

powder, Na2B4O7.10H2O (May & Baker Ltd., Degenham, England) in dH2O, and examined by light

microscopy (Leica DMLB, Leica Microsystems, Germany).

From these survey sections areas of interest were identified and ultrathin (80 nm) sections

were obtained using a Leica EM UC6 ultramicrotome (Leica Microsystems, Wetzlar, Germany). These

sections were collected on 200 mesh thin bar copper grids, stained with 2% uranyl acetate in H 2O (20

min) and 3% lead citrate (5min) and examined by transmission electron microscopy (Tecnai G 2 20

TWIN, FEI Company, Oregon, USA) using an accelerating voltage of 80 kV or Tecnai G 2 12 BioTWIN

using an accelerating voltage of 120kV).

Scanning electron microscopy

Samples for scanning electron microscopy (SEM) were processed the same way as the TEM samples

until the last 100% ethanol step. From 100% ethanol the samples were transferred to a mixture of

30% hexamethyldisilazane (HMDS) in acetone, from that mixture to a mixture of 60% HMDS in

7

acetone and then finally the samples were placed in 100% HMDS. HMDS was allowed to evaporate

at room temperature and fully dried samples were then mounted onto metal stubs with double

sided carbon tape. Finally, a thin layer of gold was applied over the sample using an automated

sputter coater (Agar Scientific, Essex, UK). The samples were imaged by using scanning electron

microscope (Hitachi S-4300, Tokyo, Japan).

Results and discussion

Streptomyces griseus biofilm

Initial experiments to assess the biofilm-forming ability of S. griseus ATCC13273 were conducted

using polystyrene well plates; however, no attachment was observed and the bacterium grew at the

air-liquid interface. A tubular biofilm reactor (TBR) was constructed (Fig. 1a) and inoculated with a

24 h-old planktonic culture of S. griseus ATCC 13273, grown in soybean medium, which was allowed

a 30 minute attachment period before a flow of fresh medium was initiated at a rate of 1 mL min -1.

Following incubation at 30°C for 12 hours a thin layer of biomass was visible on the bottom of the

tube. By 16 hours this initial layer had thickened to cover the lower 50% of the tubing (Fig. 2a). The

non-motile nature of Streptomyces spp. makes it more likely that a biofilm will develop on the base

of the tube rather than over the entire surface as the bacteria are driven downwards by gravity.

The biofilm continued to grow and thicken for the next 10 hours; however, 24-26 hours

following initial inoculation the surface adhesion of the biofilm was lost. This was characterised by

the entire biofilm becoming detached from the surface and curling into a cylindrical structure within

the silicone tube (Fig. 2a). Under the laminar flow this biofilm is pushed out of the reactor and can

be collected in a long, continuous fragment (Fig. 2b). The detachment was found to be independent

of the flow, since the same effect was observed if the biofilm was allowed to form entirely in the

absence of flow or if the biofilm was allowed to develop for 16 hours before flow was initiated.

Increasing the flow rate to 2 mL min -1 also had no temporal effect on the biofilm detachment. The

8

detachment was also observed in other complex media such as ISP2 or TSB and in the defined

minimal NMMP medium with either glucose or mannitol as carbon source. Previous studies with S.

coelicolor had highlighted that surface attachment is mediated by amyloid fimbriae that are

activated in NMMP medium only when mannitol is used as a carbon source (de Jong et al., 2009).

Although all visible traces of the biofilm are removed at this point a fresh tube-attached

biofilm subsequently developed over the same time period as the initial biofilm, without further

inoculation, hence some S. griseus cells remain attached to the tube. This fresh biofilm also became

detached after 24 hours of growth. This cycle of growth and detachment was seen to repeat at least

3 times inside the same TBR without any further inoculations. A detailed study of the surface of the

biofilm by SEM confirmed that it consisted largely of interlocking hyphae forming a very thick mesh

of cells (Fig. 2c), which accounts for the apparently strong cohesive properties.

Employing the TBR configuration shown in Fig 1B, other Streptomyces spp. were investigated

for their biofilm-forming ability. All of the strains tested formed biofilm and most showed signs of

detachment within the timeframe of the experiment (Supplemental Information, Fig S1).

Biochemical analysis of the biofilm

In order to assess the chemical composition of the S. griseus ATCC 13273 biofilm and any

extracellular matrix present, the biofilm was subjected to the standard triad of sodium meta-

periodate, protease (Pronase) and DNAse treatment (Seidl et al., 2008) to determine whether

carbohydrate, protein or extra-cellular DNA are present (Fig. 3). To enable convenient microscopic

analysis of the degradation, biofilms were grown inside flow cells (Fig. 1c). Under these conditions

the biofilm grew much more slowly, possibly due to poor oxygenation within the channels;

nevertheless, after two days there was sufficient growth to allow treatment with the different

reagents.

Treatment of the biofilm with DNAse I for 48 hours resulted in no change to the structure of

the biofilm (Fig. 3b) suggesting that extracellular DNA is not a component of the EPS. The addition of

9

40 mM sodium meta-periodate had mixed results; a number of filamentous structures remained

visible even after two days of treatment but evidence of biofilm breakdown could also be seen as

truncated filaments and cell debris (Fig. 3c). The most marked change of biofilm structure occurred

following Pronase treatment (10 mg mL-1) as almost all the filamentous structure was lost and the

biofilm was left as a collection of truncated filaments (Fig. 3d). Work by de Jong et al. (2009) on S.

coelicolor inferred that adhesion of this bacterium was mediated by amyloid-like fimbriae that

assemble along cellulose fibrils emerging from the surface of the cells. If S. griseus uses similar

appendages then proteases and the cellulose degrading NaIO4 could be corroding these structures

and therefore dispersing the biofilm cells. Proteases are also implicated in Streptomyces sporulation

and may be breaking up the cells in addition to degrading putative fimbriae (Kim & Lee, 1995). The

thick layers of filaments within the biofilm may act as a diffusion barrier to the NaIO 4 which may

explain the lack of complete dissociation with this reagent.

Streptomyces-Bacillus mixed culture biofilm

It has been previously shown with other bacteria that mixed culture biofilms have greater

resistance to mechanical forces than single species (Simoes et al., 2009). Therefore, experiments

were undertaken to improve the stability of the S. griseus biofilm by cultivating the bacterium in

conjunction with another strain. Bacillus amyloliquefaciens was selected for the co-culture

experiments since this organism is also known to produce a lactonase that might potentially disrupt

sporulation of S. griseus by degrading the A-factor γ-butyrolactone signalling molecule (Yin et al.,

2010). When inoculated with a mixed culture of S. griseus and B. amyloliquefaciens it was observed

that no detachment of the biofilm occurred inside a static TBR even after 96 hours (SI, Fig. 2),

whereas S. griseus alone detached after 24 h when grown under the same conditions. A control

experiment in which only B. amyloliquefaciens was cultured in the TBR resulted in a very weak

biofilm coating the very bottom of the tube and a similar co-inoculation of S. griseus with another

bacterium (E. coli) showed no similar effects on Streptomyces biofilm morphology or detachment. In

10

the other Streptomyces strains that exhibited biofilm detachment, this was also delayed by co-

culturing with B. amyloliquefaciens.

Scanning electron microscopy of the dual culture biofilm revealed that both bacteria are

present and are closely associated with each other (Fig. 4a). TEM analysis of a cross-section of the

single species biofilm show short hair-like projections on the surfaces of the cells that appear to

make contact with adjacent cells (Fig 4c). Other than these structures, extracellular matrix, which is

characteristic of biofilms and can be observed in other bacterial biofilms with electron microscopy

(Kwiecinski et al., 2009; Sriramulu et al., 2005; Tsoligkas et al., 2011), was not observed here. In fact

the filamentous structure of the biofilm produced by S. griseus more closely resembles that of fungal

biofilms (Seidler et al., 2008; Amadio et al., 2013). In S. coelicolor, fimbriae composed of bundled

amyloid fibrils of chaplin protein are responsible for attachment of the bacterium to surfaces.

Similar fimbriae might be involved in the biofilm of S. griseus, which would be consistent with the

dispersal of biofilm in the presence of pronase as observed in the flowcell experiments.

Furthermore, TEM images of cross-sections of the dual species biofilm revealed a more extensive

network of the putative fimbriae than was observed in the single species S. griseus biofilm (Fig. 4b),

which would account for the improved stability. This more robust S. griseus/B. amyloliquefaciens

biofilm still detached when a flow of fresh medium was initiated (1 mL min -1) suggesting that the

adhesive property of the biofilm is still not strong enough to withstand moderate shear forces.

Conclusion

Although a small number of studies on streptomycete biofilms has been conducted (Kim & Kim,

2004; Morales et al., 2007; de Jong et al., 2009), none has been studied in a continuous flow biofilm

reactor. In this paper we have described for the first time the characteristics of the

biotechnologically important bacterium S. griseus when cultivated as a biofilm in a tubular reactor.

The biofilm that forms is relatively unstable and detaches after 24 h; however, without further

inoculation fresh biofilm regrows on the tube. The cohesive strength of the biofilm is evident since

11

the biofilm detaches as a single piece of biomass, and this is accounted for by the dense network of

filaments observed by SEM. Although no extracellular matrix was apparent in the electron

micrographs, pronase, and to a lesser degree metaperiodate, caused the biofilm to disintegrate,

suggesting that these polymers form the EPS. Co-cultivation of S. griseus with B. amyloliquefaciens

resulted in a more stable biofilm that could be at least partially explained by the observation by TEM

of a much more extensive network of putative fimbriae compared with single species biofilm. It is

likely that these fimbriae provide improved mechanical strength to the biofilm.

S. griseus produces important secondary metabolites and catalyses biotechnologically

relevant reactions, and these attributes might be further exploited by employing biofilms. The

findings reported here suggest that in a submerged environment S. griseus is capable of forming

thick surface attached biofilms. However, spontaneous surface detachment occurs and controlling

this detachment is the key to forming a stable biofilm, thus enabling continuous flow applications.

Acknowledgements

This work was by Science Foundation Ireland under Grant 11/TIDA/B2007. The authors thank Dimitri

Scholz and Tiina O’Neil, UCD Conway Institute, for assistance with the electron microscopy. The

authors confirm no financial interest or benefit arising from the research.

References

Alexandre V, Ladril S, Maurs M & Azerad R (2004) Microbial models of animal drug metabolism - Part 5. Microbial preparation of human hydroxylated metabolites of irbesartan. Journal of Molecular Catalysis B-Enzymatic 29: 173-179.Amadio J, Casey E & Murphy C (2013) Filamentous fungal biofilm for production of human drug metabolites. Applied Microbiology and Biotechnology 97: 5955-5963.Bright TV, Clark BR, O'Brien E & Murphy CD (2011) Bacterial production of hydroxylated and amidated metabolites of flurbiprofen. Journal of Molecular Catalysis B-Enzymatic 72: 116-121.de Jong W, Wosten HAB, Dijkhuizen L & Claessen D (2009) Attachment of Streptomyces coelicolor is mediated by amyloidal fimbriae that are anchored to the cell surface via cellulose. Molecular Microbiology 73: 1128-1140.Flemming HC (2002) Biofouling in water systems - cases, causes and countermeasures. Applied Microbiology and Biotechnology 59: 629-640.

12

Gross R, Lang K, Bühler K & Schmid A (2010) Characterization of a biofilm membrane reactor and its prospects for fine chemical synthesis. Biotechnology and Bioengineering 105: 705-717.Hall-Stoodley L, Costerton JW & Stoodley P (2004) Bacterial biofilms: From the natural environment to infectious diseases. Nature Reviews Microbiology 2: 95-108.Kim IS & Lee KJ (1995) Physiological roles of leupeptin and extracellular proteases in mycelium development of Streptomyces exfoliatus SMF13. Microbiology-Uk 141: 1017-1025.Kim YM & Kim JH (2004) Formation and dispersion of mycelial pellets of Streptomyces coelicolor A3(2). Journal of Microbiology 42: 64-67.Kwiecinski J, Eick S & Wojcik K (2009) Effects of tea tree (Melaleuca alternifolia) oil on Staphylococcus aureus in biofilms and stationary growth phase. International Journal of Antimicrobial Agents 33: 343-347.Misiak K, Casey E & Murphy CD (2011) Factors influencing 4-fluorobenzoate degradation in biofilm cultures of Pseudomonas knackmussii B13. Water Research 45: 3512-3520.Morales DK, Ocampo W & Zambrano MM (2007) Efficient removal of hexavalent chromium by a tolerant Streptomyces sp affected by the toxic effect of metal exposure. Journal of Applied Microbiology 103: 2704-2712.Ohnishi Y, Ishikawa J, Hara H, et al. (2008) Genome sequence of the streptomycin-producing microorganism Streptomyces griseus IFO 13350. Journal of Bacteriology 190: 4050-4060.Schulz S, Girhard M & Urlacher VB (2012) Biocatalysis: Key to Selective Oxidations. Chemcatchem 4: 1889-1895.Seidl K, Goerke C, Woiz C, Mack D, Berger-Baechi B & Bischoff M (2008) Staphylococcus aureus CcpA affects biofilm formation. Infection and Immunity 76: 2044-2050.Seidler MJ, Salvenmoser S & Muller FMC (2008) Aspergillus fumigatus forms biofilms with reduced antifungal drug susceptibility on bronchial epithelial cells. Antimicrobial Agents and Chemotherapy 52: 4130-4136.Simoes M, Simoes LC & Vieira MJ (2009) Species association increases biofilm resistance to chemical and mechanical treatments. Water Research 43: 229-237.Sriramulu DD, Lunsdorf H, Lam JS & Romling U (2005) Microcolony formation: a novel biofilm model of Pseudomonas aeruginosa for the cystic fibrosis lung. Journal of Medical Microbiology 54: 667-676. Tsoligkas AN, Winn M, Bowen J, Overton TW, Simmons MJH & Goss RJM (2011) Engineering biofilms for biocatalysis. Chembiochem 12: 1391-1395.van Keulen G, Jonkers HM, Claessen D, Dijkhuizen L & Wosten HAB (2003) Differentiation and anaerobiosis in standing liquid cultures of Streptomyces coelicolor. Journal of Bacteriology 185: 1455-1458.Winn M, Foulkes JM, Perni S, Simmons MJH, Overton TW & Goss RJM (2012) Biofilms and their engineered counterparts: A new generation of immobilised biocatalysts. Catalysis Science & Technology 2: 1544-1547.Yin XT, Xu L, Fan SS, Xu LN, Li DC & Liu ZY (2010) Isolation and characterization of an AHL lactonase gene from Bacillus amyloliquefaciens. World Journal of Microbiology & Biotechnology 26: 1361-1367.

13

Figure legends

Figure 1. Schematic of apparatus used to grow biofilms. (a) A continuous flow tubular biofilm reactor

with a total reactor length of 100 cm. (b) Short tubular biofilm section for screening of biofilm

formation with 20 cm total reactor length, secured at both ends with a syringe. (c) Biofilm flow cell.

Inoculum passes through narrow channels and biofilm grows on glass coverslide on base.

Figure 2. (a) 16 mm long section of biofilm tubing showing formed S. griseus biomass over 40 hours.

Initial surface coverage is observed by 16 hours. By 26 hours the biomass lifts off the surface of the

tube and curls into a tubular structure within the tubing. By 40 hours the tubular structure can still

be seen detached from the tubing. (b) If laminar flow is present the detached biomass at 26 hours is

removed from the tubing by the flow and can be collected as a single piece of biomass. (c) SEM

showing that the biofilm is composed of many interlocking filaments.

Figure 3. Fluorescence microscopy images following chemical treatment of S. griseus biofilm.

Pronase treatment (10 mg mL-1) leads to fragmentation of the filamentous structure of the biofilm.

NaIO4 treatment seems to lead to some fragmentation but some structures do remains. DNAse I

treatment (500 μg mL-1) had no effect. Scale bar represents 100 μm.

Figure 4. Scanning (a) transmission (b) electron micrographs of the dual S. griseus/B.

amyloliquefaciens biofilm. The SEM image demonstrates the presence of the two cell types (the

rods are the B. amyloliquefaciens); the TEM image shows that there is a more extensive network of

fimbriae-like structures compared with the single species biofilm (c).

14

Fig 1. Winn et al.

15

Fig 2. Winn et al.

16

Fig 3. Winn et al.

17

Fig 4 Winn et al.

18