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.
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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
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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 H2O (20
min) and 3% lead citrate (5min) and examined by transmission electron microscopy (Tecnai G2 20
TWIN, FEI Company, Oregon, USA) using an accelerating voltage of 80 kV or Tecnai G2 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 metaperiodate, 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 NaIO4 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
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the other Streptomyces strains that exhibited biofilm detachment, this was also delayed by coculturing 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.
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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).
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Fig 1. Winn et al.
15
Fig 2. Winn et al.
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Fig 3. Winn et al.
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Fig 4 Winn et al.
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