Supplementary Material
Implementation of aerobic retentostat cultivation for B. subtilis
Defining the experimental set-up for retentostat cultivation of B. subtilis required a
number of modifications relative to other zero-growth studies (Boender et al., 2009; Ercan et
al., 2013). Because sporulation will interfere with reaching a zero-growth state and therefore
is undesired, a sporulation-deficient sigF mutant was used in this study. This strain can
initiate sporulation, but is unable to express the genes necessary for continuation of the
process at stage II in the sporulation cascade (Piggot and Coote, 1976; Setlow et al., 1991;
Dworkin and Losick, 2005). The use of a knockout strain may impose a limitation in terms of
being able to interpret the natural response of the organism. However, this particular mutant
is still able to express genes for sporulation initiation (Fawcett et al., 2000; Steil et al., 2005;
Wang et al., 2006) and thereby still allows us to see if sporulation is one of the responses B.
subtilis applies. For retentostat cultivation, bioreactors were equipped with a cross-flow filter
(Fig. 1). These filters were chosen for their particular large filtration surface and tangential
flow, minimizing the risk of clogging during cultivation (Koros et al., 1996). Complete
biomass retention was confirmed by regular plating of effluent. Continuous filtration for up to
42 days was possible without noticeable clogging or observation of growth in the effluent.
Culture purity can be an issue with such long-term cultivations since the risk of
contamination by other bacteria is high. To easily identify contamination by microscopic
examination, a GFP fusion to the promoter of the constitutively expressed ribosomal RNA
operon rrnB (Krásný and Gourse, 2004) was introduced in the strain cultivated.
Consequently, cells not expressing GFP could be identified as contamination. The retentostat
cultures described here did not experience any contamination.
Growth of B. subtilis in medium-supply tubing was found to be a potential problem
during prolonged (> 7 days) cultivation. The slow feed rate of 35 ml h-1 allowed the bacteria
to grow against the current when aerosols or droplets from the culture came in contact with
the medium inlet on the fermentor. To prevent growth in the tubing, a dropper was installed
between the medium inlet and the medium-supply tubing, preventing direct contact. A sterile
airflow in the dropper pushed the droplets to the reactor and prevented cells from entering the
dropper.
Vigorous mixing and sparging with air to keep the culture oxygenated in a bioreactor
will lead to formation of foam, which can cause problems. Formation of foam needs to be
prevented and during trial experiments it became clear that not every anti-foaming strategy
was adequate. When anti-foam was premixed with the supplied medium it precipitated in the
silicone tubing, partly blocking the inflow of the medium. Not all anti-foam reached the
reactor and as a consequence foaming was not prevented properly. Foaming was prevented
most effectively by supplying anti-foam on regular intervals. In our setup this was achieved
by automatic addition of 5 ml of a 5% (wt.wt-1) antifoam solution every 13 minutes via a
computer-controlled pump.
Supplementary Figures and Tables
A
B
Figure S1. Distribution of glucose (%) between maintenance and growth. Black
bars represent energy directed towards maintenance (ms ∙ Cx, viable). White bars
represent energy directed towards growth (µ ∙ Cx, viable / Ysxmax). (A) Retentostat 1. (B)
Retentostat 2.
Table S1. Highly differentially expressed genes in response to near-zero growth
conditions.
Gene
glnH
glnQ
yusK
fadN
glnP
gmuA
yxlG
yxlE
sigY
yxlF
gmuC
yxlD
yxlC
glnM
acdA
gmuR
gmuD
yhzC
gmuE
etfA
Function
Gene
levD
mtlD
mtlF
levE
levF
mtlA
levG
ntdB
mtnA
mtnK
sacC
mtnB
mtnX
hag
ntdC
pheS
gapA
flgK
aroH
flgM
Function
glutamine uptake
glutamine uptake
fatty acid degradation
fatty acid degradation
glutamine uptake
glucomannan uptake and phosphorylation
unknown; ABC transporter
unknown
maintenance of the SPß prophage
unknown; ABC transporter
glucomannan uptake and phosphorylation
negative regulation of the sigY operon
control of SigY activity
glutamine uptake
fatty acid degradation
regulation of glucomannan utilization
glucomannan utilization
unknown
glucomannan utilization
fatty acid degradation
fructose uptake and phosphorylation
mannitol utilization
uptake of mannitol
fructose uptake and phosphorylation
fructose uptake and phosphorylation
mannitol uptake and phosphorylation
fructose uptake and phosphorylation
synthesis of the antibiotic kanosamine
methionine salvage
methionine salvage
degradation of levan to fructose
methionine salvage
methionine salvage
motility and chemotaxis
synthesis of the antibiotic kanosamine
translation
catabolic enzyme in glycolysis
motility and chemotaxis
biosynthesis of aromatic amino acids
control of SigD activity
Fold change
7
7
6
5
4
4
4
4
3
3
3
3
3
3
3
3
3
3
3
3
p-value
1.01E-08
6.33E-07
7.50E-03
6.83E-03
7.92E-08
1.32E-06
2.46E-04
1.26E-03
5.82E-04
7.02E-04
1.71E-06
1.76E-03
3.47E-03
5.13E-06
2.88E-04
8.09E-05
5.84E-06
1.33E-04
2.49E-05
9.36E-04
Fold change
-19
-18
-18
-12
-12
-11
-9
-6
-5
-5
-5
-5
-4
-4
-4
-4
-3
-3
-3
-3
p-value
1.22E-05
9.51E-07
7.97E-06
2.43E-05
8.01E-05
1.88E-05
1.25E-04
8.20E-03
3.84E-08
1.48E-08
6.23E-07
2.52E-15
2.75E-12
1.02E-05
1.01E-02
2.37E-12
2.57E-05
1.23E-06
3.64E-10
3.80E-06
Table S2. SNPs resulting in missense mutations.
Mutated locus
Gene product
Amino acid changes
yozW
hypothetical protein
T9N, M11N
yozX
hypothetical protein
I45R, H52Q
yoqL
endonuclease
G11R, S3I
yoqG
hypothetical protein
Y54F
yomI
lytic transglycosylase
I58V, T60A, I2082L
yomG
hypothetical protein
A763P
uvrX
lesion bypass phage DNA polymerase
S323N, E307D, R187H
yolD
hypothetical protein
K85R
yolC
SPbeta phage protein
L89F
hypothetical protein
2bp→AC
Retentostat 1
Retentostat 2
yotM