Straight, low-aspect ratio micro flow cells are used to support biofilm attachment and preferential accumulation at the short side-wall, which progressively reduces the effective channel width. The biofilm shifts downstream at measurable velocities under the imposed force from the constant laminar co-flowing nutrient stream. The dynamic behaviour of the biofilm viscosity is modeled semi-analytically, based on experimental measurements of biofilm dimensions and velocity as inputs. The technique advances the study of biofilm mechanical properties by strongly limiting biases related to non-Newtonian biofilm properties (e.g., shear dependent viscosity) with excellent time resolution. To demonstrate the proof of principle, young Pseudomonas sp. biofilms were analyzed under different nutrient concentrations and constant micro-flow conditions. The striking results show that large initial differences in biofilm viscosities grown under different nutrient concentrations become nearly identical in less than one day, followed by a continuous thickening process. The technique verifies that in 50 h from inoculation to early maturation stages, biofilm viscosity could grow by over 2 orders of magnitude. The approach opens the way for detailed studies of mechanical properties under a wide variety of physiochemical conditions, such as ionic strength, temperature, and shear stress.

1.
P.
Barai
,
A.
Kumar
, and
P. P.
Mukherjee
, “
Mesoscale elucidation of biofilm shear behavior
,” preprint arXiv:1509.03926 (
2015
).
2.
E.
Bester
,
G.
Wolfaardt
,
L.
Joubert
,
K.
Garny
, and
S.
Saftic
, “
Planktonic-cell yield of a pseudomonad biofilm
,”
Appl. Environ. Microbiol.
71
(
12
),
7792
7798
(
2005
).
3.
E.
Bester
,
G. M.
Wolfaardt
,
N. B.
Aznaveh
, and
J.
Greener
, “
Biofilms' role in planktonic cell proliferation
,”
Int. J. Mol. Sci.
14
(
11
),
21965
21982
(
2013
).
4.
N.
Billings
,
A.
Birjiniuk
,
T. S.
Samad
,
P. S.
Doyle
, and
K.
Ribbeck
, “
Material properties of biofilms—A review of methods for understanding permeability and mechanics
,”
Rep. Prog. Phys.
78
(
3
),
036601
(
2015
).
5.
M.
Cancela
,
E.
Alvarez
, and
R.
Maceiras
, “
Effects of temperature and concentration on carboxymethylcellulose with sucrose rheology
,”
J. Food Eng.
71
(
4
),
419
424
(
2005
).
6.
M.
Charles
and
L.
Lilleleht
, “
Co-current stratified laminar flow of two immiscible liquids in a rectangular conduit
,”
Can. J. Chem. Eng.
43
(
3
),
110
116
(
1965
).
7.
F. C.
Cheong
,
S.
Duarte
,
S.-H.
Lee
, and
D. G.
Grier
, “
Holographic microrheology of polysaccharides from Streptococcus mutans biofilms
,”
Rheol. Acta
48
(
1
),
109
115
(
2009
).
8.
S. C.
Chew
,
B.
Kundukad
,
T.
Seviour
,
J. R.
van der Maarel
,
L.
Yang
,
S. A.
Rice
,
P.
Doyle
, and
S.
Kjelleberg
, “
Dynamic remodeling of microbial biofilms by functionally distinct exopolysaccharides
,”
MBio
5
(
4
),
e01536
(
2014
).
9.
S.
Das
and
A.
Kumar
, “
Formation and post-formation dynamics of bacterial biofilm streamers as highly viscous liquid jets
,”
Sci. Rep.
4
,
7126
(
2014
).
10.
Y.
Ding
,
N.
Peng
,
Y.
Du
,
L.
Ji
, and
B.
Cao
, “
Disruption of putrescine biosynthesis in Shewanella oneidensis enhances biofilm cohesiveness and performance in cr (vi) immobilization
,”
Appl. Environ. Microbiol.
80
(
4
),
1498
1506
(
2014
).
11.
B.
Dunsmore
,
A.
Jacobsen
,
L.
Hall-Stoodley
,
C.
Bass
,
H.
Lappin-Scott
, and
P.
Stoodley
, “
The influence of fluid shear on the structure and material properties of sulphate-reducing bacterial biofilms
,”
J. Ind. Microbiol. Biotechnol.
29
(
6
),
347
353
(
2002
).
12.
H. J.
Eberl
and
R.
Sudarsan
, “
Exposure of biofilms to slow flow fields: The convective contribution to growth and disinfection
,”
J. Theor. Biol.
253
(
4
),
788
807
(
2008
).
13.
M. P.
Gashti
,
J.
Bellavance
,
O.
Kroukamp
,
G.
Wolfaardt
,
S. M.
Taghavi
, and
J.
Greener
, “
Live-streaming: Time-lapse video evidence of novel streamer formation mechanism and varying viscosity
,”
Biomicrofluidics
9
(
4
),
041101
(
2015
).
14.
P.
Gondret
,
N.
Rakotomalala
,
M.
Rabaud
,
D.
Salin
, and
P.
Watzky
, “
Viscous parallel flows in finite aspect ratio Hele-Shaw cell: Analytical and numerical results
,”
Phys. Fluids
9
(
6
),
1841
1843
(
1997
).
15.
G. W.
Hanlon
,
S. P.
Denyer
,
C. J.
Olliff
, and
L. J.
Ibrahim
, “
Reduction in exopolysaccharide viscosity as an aid to bacteriophage penetration through Pseudomonas aeruginosa biofilms
,”
Appl. Environ. Microbiol.
67
(
6
),
2746
2753
(
2001
).
16.
A.
Karimi
,
D.
Karig
,
A.
Kumar
, and
A.
Ardekani
, “
Interplay of physical mechanisms and biofilm processes: Review of microfluidic methods
,”
Lab Chip
15
(
1
),
23
42
(
2015
).
17.
J.
Kim
,
H.-S.
Kim
,
S.
Han
,
J.-Y.
Lee
,
J.-E.
Oh
,
S.
Chung
, and
H.-D.
Park
, “
Hydrodynamic effects on bacterial biofilm development in a microfluidic environment
,”
Lab Chip
13
(
10
),
1846
1849
(
2013
).
18.
J.
Kim
,
H.-D.
Park
, and
S.
Chung
, “
Microfluidic approaches to bacterial biofilm formation
,”
Molecules
17
(
8
),
9818
9834
(
2012
).
19.
I.
Klapper
,
C.
Rupp
,
R.
Cargo
,
B.
Purvedorj
, and
P.
Stoodley
, “
Viscoelastic fluid description of bacterial biofilm material properties
,”
Biotechnol. Bioeng.
80
(
3
),
289
296
(
2002
).
20.
V.
Körstgens
,
H.-C.
Flemming
,
J.
Wingender
, and
W.
Borchard
, “
Influence of calcium ions on the mechanical properties of a model biofilm of mucoid Pseudomonas aeruginosa
,”
Water Sci. Tech.
43
(
6
),
49
57
(
2001
).
21.
O.
Kroukamp
,
R. G.
Dumitrache
, and
G. M.
Wolfaardt
, “
Pronounced effect of the nature of the inoculum on biofilm development in flow systems
,”
Appl. Environ. Microbiol.
76
(
18
),
6025
6031
(
2010
).
22.
P. C.
Lau
,
J. R.
Dutcher
,
T. J.
Beveridge
, and
J. S.
Lam
, “
Absolute quantitation of bacterial biofilm adhesion and viscoelasticity by microbead force spectroscopy
,”
Biophys. J.
96
(
7
),
2935
2948
(
2009
).
23.
O.
Lieleg
,
M.
Caldara
,
R.
Baumgärtel
, and
K.
Ribbeck
, “
Mechanical robustness of Pseudomonas aeruginosa biofilms
,”
Soft Matter
7
(
7
),
3307
3314
(
2011
).
24.
J.
Liu
,
M.
Gardel
,
K.
Kroy
,
E.
Frey
,
B. D.
Hoffman
,
J. C.
Crocker
,
A.
Bausch
, and
D.
Weitz
, “
Microrheology probes length scale dependent rheology
,”
Phys. Rev. Lett.
96
(
11
),
118104
(
2006
).
25.
J.
Martin
,
N.
Rakotomalala
,
L.
Talon
, and
D.
Salin
, “
Viscous lock-exchange in rectangular channels
,”
J. Fluid Mech.
673
,
132
146
(
2011
).
26.
J.
Mathias
and
P.
Stoodley
, “
Applying the digital image correlation method to estimate the mechanical properties of bacterial biofilms subjected to a wall shear stress
,”
Biofouling
25
(
8
),
695
703
(
2009
).
27.
M. G.
Mazza
, “
The physics of biofilms—An introduction
,”
J. Phys. D: Appl. Phys.
49
(
20
),
203001
203024
(
2016
).
28.
E.
Meijering
,
O.
Dzyubachyk
, and
I. T.
Smal
, “
Methods for cell and particle tracking
,”
Methods Enzymol.
504
(
9
),
183
200
(
2012
).
29.
T.
Merkel
,
V.
Bondar
,
K.
Nagai
,
B.
Freeman
, and
I.
Pinnau
, “
Gas sorption, diffusion, and permeation in poly (dimethylsiloxane)
,”
J. Polym. Sci. Part B Polym. Phys.
38
(
3
),
415
434
(
2000
).
30.
S.
Neethirajan
,
D.
Karig
,
A.
Kumar
,
P. P.
Mukherjee
,
S. T.
Retterer
, and
M. J.
Doktycz
, “
Biofilms in microfluidic devices
,” in
Encyclopedia of Nanotechnology
(
Springer
,
2012
), pp.
213
219
.
31.
In this paper, we adopt the convention of denoting dimensional quantities with the ̂ symbol and dimensionless quantities without.
32.
Note that the parameters with a single overbar sign are averaged over ẑ and the parameters with two overbar sings are averaged over x̂ and ẑ.
33.
J.-C.
Ochoa
,
C.
Coufort
,
R.
Escudié
,
A.
Liné
, and
E.
Paul
, “
Influence of non-uniform distribution of shear stress on aerobic biofilms
,”
Chem. Eng. Sci.
62
(
14
),
3672
3684
(
2007
).
34.
R.
Pamies
,
R. R.
Schmidt
,
M.
d.
,
C. L.
Martínez
, and
J. G.
de la Torre
, “
The influence of mono and divalent cations on dilute and non-dilute aqueous solutions of sodium alginates
,”
Carbohydr. Polym.
80
(
1
),
248
253
(
2010
).
35.
F.
Paquet-Mercier
,
M. P.
Gashti
,
J.
Belllavance
,
S. M.
Taghavi
, and
J.
Greener
, “
Through thick and thin: A microfluidic approach for continuous measurements of biofilm viscosity
,”
Lab Chip
(published online,
2016
).
36.
L.
Pavlovsky
,
J. G.
Younger
, and
M. J.
Solomon
, “
In situ rheology of staphylococcus epidermidis bacterial biofilms
,”
Soft Matter
9
(
1
),
122
131
(
2013
).
37.
M. O.
Pereira
,
M.
Kuehn
,
S.
Wuertz
,
T.
Neu
, and
L. F.
Melo
, “
Effect of flow regime on the architecture of a Pseudomonas fluorescens biofilm
,”
Biotechnol. Bioeng.
78
(
2
),
164
171
(
2002
).
38.
C.
Picioreanu
,
M. C.
Van Loosdrecht
, and
J. J. T.
Heijnen
, “
Mathematical modeling of biofilm structure with a hybrid differential-discrete cellular automaton approach
,”
Biotechnol. Bioeng.
58
(
1
),
101
116
(
1998
).
39.
C.
Picioreanu
,
M. C.
Van Loosdrecht
, and
J. J. T.
Heijnen
, “
Effect of diffusive and convective substrate transport on biofilm structure formation: A two-dimensional modeling study
,”
Biotechnol. Bioeng.
69
(
5
),
504
515
(
2000
).
40.
C.
Picioreanu
,
M. C.
Van Loosdrecht
, and
J. J. t.
Heijnen
, “
Two-dimensional model of biofilm detachment caused by internal stress from liquid flow
,”
Biotechnol. Bioeng.
72
(
2
),
205
218
(
2001
).
41.
L.
Ptitsyn
,
G.
Horneck
,
O.
Komova
,
S.
Kozubek
,
E.
Krasavin
,
M.
Bonev
, and
P.
Rettberg
, “
A biosensor for environmental genotoxin screening based on an sos lux assay in recombinant Escherichia coli cells
,”
Appl. Environ. Microbiol.
63
(
11
),
4377
4384
(
1997
).
42.
L. D.
Renner
and
D. B.
Weibel
, “
Physicochemical regulation of biofilm formation
,”
MRS Bull.
36
(
05
),
347
355
(
2011
).
43.
B. E.
Rittman
, “
The effect of shear stress on biofilm loss rate
,”
Biotechnol. Bioeng.
24
(
2
),
501
506
(
1982
).
44.
S.
Rogers
,
C.
Van Der Walle
, and
T.
Waigh
, “
Microrheology of bacterial biofilms in vitro: Staphylococcus aureus and Pseudomonas aeruginosa
,”
Langmuir
24
(
23
),
13549
13555
(
2008
).
45.
C. J.
Rupp
,
C. A.
Fux
, and
P.
Stoodley
, “
Viscoelasticity of Staphylococcus aureus biofilms in response to fluid shear allows resistance to detachment and facilitates rolling migration
,”
Appl. Environ. Microbiol.
71
(
4
),
2175
2178
(
2005
).
46.
R.
Rusconi
,
S.
Lecuyer
,
N.
Autrusson
,
L.
Guglielmini
, and
H. A.
Stone
, “
Secondary flow as a mechanism for the formation of biofilm streamers
,”
Biophys. J.
100
(
6
),
1392
1399
(
2011
).
47.
C. A.
Schneider
,
W. S.
Rasband
, and
K. W. T.
Eliceiri
, “
NIH image to ImageJ: 25 years of image analysis
,”
Nat. Methods
9
(
7
),
671
675
(
2012
).
48.
T.
Shaw
,
M.
Winston
,
C.
Rupp
,
I.
Klapper
, and
P.
Stoodley
, “
Commonality of elastic relaxation times in biofilms
,”
Phys. Rev. Lett.
93
(
9
),
098102
(
2004
).
49.
D. E.
Solomon
and
S. A.
Vanapalli
, “
Multiplexed microfluidic viscometer for high-throughput complex fluid rheology
,”
Microfluid. Nanofluid.
16
(
4
),
677
690
(
2014
).
50.
K. J.
Sreeram
,
H. Y.
Shrivastava
, and
B. U.
Nair
, “
Studies on the nature of interaction of iron (iii) with alginates
,”
Biochim. Biophys. Acta
1670
(
2
),
121
125
(
2004
).
51.
P.
Stoodley
,
R.
Cargo
,
C.
Rupp
,
S.
Wilson
, and
I.
Klapper
, “
Biofilm material properties as related to shear-induced deformation and detachment phenomena
,”
J. Ind. Microbiol. Biotechnol.
29
(
6
),
361
367
(
2002
).
52.
P.
Stoodley
,
Z.
Lewandowski
,
J. D.
Boyle
, and
H. M. T.
Lappin-Scott
, “
Structural deformation of bacterial biofilms caused by short-term fluctuations in fluid shear: An in situ investigation of biofilm rheology
,”
Biotechnol. Bioeng.
65
(
1
),
83
92
(
1999
).
53.
T. K.
Van Dyk
,
W. R.
Majarian
,
K. B.
Konstantinov
,
R. M.
Young
,
P. S.
Dhurjati
, and
R. A.
Larossa
, “
Rapid and sensitive pollutant detection by induction of heat shock gene-bioluminescence gene fusions
,”
Appl. Environ. Microbiol.
60
(
5
),
1414
1420
(
1994
).
54.
A.
Vinogradov
,
M.
Winston
,
C.
Rupp
, and
P.
Stoodley
, “
Rheology of biofilms formed from the dental plaque pathogen Streptococcus mutans
,”
Biofilms
1
(
01
),
49
56
(
2004
).
55.
Q.
Wang
and
T.
Zhang
, “
Review of mathematical models for biofilms
,”
Solid State Commun.
150
(
21
),
1009
1022
(
2010
).
56.
J. P.
Ward
and
J. R.
King
, “
Thin-film modelling of biofilm growth and quorum sensing
,”
J. Eng. Math.
73
(
1
),
71
92
(
2012
).
57.
A.
Wileman
,
A.
Ozkan
, and
H.
Berberoglu
, “
Rheological properties of algae slurries for minimizing harvesting energy requirements in biofuel production
,”
Bioresour. Technol.
104
,
432
439
(
2012
).
58.
G.
Wolfaardt
,
M.
Hendry
,
T.
Birkham
,
A.
Bressel
,
M.
Gardner
,
A.
Sousa
,
D.
Korber
, and
M.
Pilaski
, “
Microbial response to environmental gradients in a ceramic-based diffusion system
,”
Biotechnol. Bioeng.
100
(
1
),
141
149
(
2008
).
59.
J.
Zilz
,
C.
Schäfer
,
C.
Wagner
,
R. J.
Poole
,
M. A.
Alves
, and
A.
Lindner
, “
Serpentine channels: Micro-rheometers for fluid relaxation times
,”
Lab Chip
14
(
2
),
351
358
(
2014
).

Supplementary Material

You do not currently have access to this content.