Post on 16-Aug-2020
For submission to Water Research
Aggregation Ability of Three Phylogenetically Distant Anammox
Bacterial Species
Supplemental Information
By
Muhammad Alia,b, Dario Rangel Shawa, Lei Zhangb, Mohamed Fauzi Haroonc,
Yuko Naritab, Abdul-Hamid Emwasd, Pascal E. Saikalya,*, and Satoshi Okabeb,**
a King Abdullah University of Science and Technology, Biological and Environmental
Sciences and Engineering Division, Water Desalination and Reuse Center, Thuwal
23955-6900, Saudi Arabia. b Division of Environmental Engineering, Faculty of Engineering,
Hokkaido University, North 13, West-8, Sapporo, Hokkaido 060-8628, Japan. c Department
of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA 02138, USA. d
King Abdullah University of Science and Technology, Core Labs, Thuwal 23955-6900, Saudi
Arabia.
*Corresponding author: Pascal E. Saikaly, Al-Jazri Building, Office 4237, Thuwal 23955-
6900, Saudi Arabia, E-mail: pascal.saikaly@kaust.edu.sa
**Corresponding author: Satoshi Okabe, North 13, West-8, Sapporo, Hokkaido 060-8628,
Phone & Fax: +81-(0)11-706-6266, E-mail: sokabe@eng.hokudai.ac.jp
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Surface energy calculations. Surface energies were calculated from the contact angle
measurements as described elsewhere (Van Oss et al., 1988). In summary, the total surface
energy (γtotal) was the sum of the Lifshitz-van der Waals (γLW) and Lewis acid-base (γAB)
components. γAB comprises of the electron-acceptor γ+ and electron-donor γ- parameters:
γ AB=2√γ+¿γ −¿ ¿¿. So the total surface tension of bacterial cell (γS) and liquid (γL) in this work
can be respectively expressed as equation 1 and 2, respectively.
γS=γ SLW+γ S
AB=γ SLW+2√γS
+¿ γS−¿¿¿ (1)
γ L=γ LLW +γ L
AB=γ LLW+2√γ L
+¿γ L−¿ ¿¿ (2)
The Young-Dupré equation (Equation 3) describes the relationship between γLW, γ+, γ-
and contact angle () for bacterial cell surface (S) and a drop of liquid (L). Thus, the surface
tension component and parameters of bacterial cell were calculated with Equation 3 by
contact angle () measurement with three different liquids (water, glycerol and formamide) of
known γLW, γ+, and γ-.
¿¿ (3)
Then, the total interfacial tensions between the bacterial cell surface and water were
calculated with the following equation:
γSL=γ SLLW +γ SL
AB (4)
γSLLW=¿ (5)
γSLAB=2¿ (6)
With the equations above, the interfacial free energy (ΔGadh) between the bacterial particles
and water could be evaluated with the following equations:
ΔGadh=ΔGadhLW+ ΔGadh
AB =−2( γ BLLW +γ BL
AB ) (7)
with
ΔGadhLW=−2 γ BL
LW (8)
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ΔGadhAB =−2γ BL
AB (9)
Extended DLVO Theory. Decay of energy of interaction as a function of distance between
the bacterial cells was calculated using the extended DLVO theory. The total energy of
interaction (WT) was modeled by summation of the electric double layer (WR), van der Waals
energies (WA) and acid-base interaction (WAB) as described elsewhere (Hou et al., 2015; Liu
et al., 2010; van Oss, 2008).
❑❑❑❑❑❑❑❑ (10)
with
❑❑❑❑
❑ (11)
where, ABLB is the effective Hamaker constant, which is measured using contact angle
approach and can be determined using Eq. 12 as described elsewhere (van Oss, 1995):
❑❑❑❑❑❑❑
❑ (12)
❑❑❑❑
¿(❑❑
❑ )¿ (13)
The double layer interactions can be calculated from the following Eq. 14 as described in
(Trefalt and Borkovec, 2014)
❑❑❑❑❑❑❑ exp( ) (14)
H is the separation distance between the cells.
R is the sludge cell radius, which is assumed to be spherical.
l0 is the minimum equilibrium distance between the two surfaces (≈0.157nm) (van Oss,
1995).
ε is the dielectric constant of the liquid; for water, ε ≈ 80
❑❑ is the permittivity of vacuum, ❑❑❑❑❑❑❑❑❑❑
ΨS is the stern potential and could be replaced by zeta potential measurement.
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k is the inverse of thickness of the diffuse electrical (Debye) double layer.
λ is the correlation length of molecules in the liquid medium (0.6 ~ 13 nm) (van Oss, 1995).
Reference:
Hou, X., Liu, S., Zhang, Z., 2015. Role of extracellular polymeric substance in determining
the high aggregation ability of anammox sludge. Water Res. 75, 51–62.
doi:10.1016/j.watres.2015.02.031
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H.Q., 2010. Contribution of extracellular polymeric substances (EPS) to the sludge
aggregation. Environ. Sci. Technol. 44, 4355–4360. doi:10.1021/es9016766
Narita, Y., Zhang, L., Kimura, Z. ichiro, Ali, M., Fujii, T., Okabe, S., 2017. Enrichment and
physiological characterization of an anaerobic ammonium-oxidizing bacterium
“Candidatus Brocadia sapporoensis.” Syst. Appl. Microbiol. 40, 448–457.
doi:10.1016/j.syapm.2017.07.004
Trefalt, G., Borkovec, M., 2014. Overview of DLVO Theory. Creat. Commons Attrib. 4.0
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Van Oss, C.J., Chaudhury, M.K., Good, R.J., 1988. Interfacial Lifshitz-van der Waals and
polar interactions in macroscopic systems. Chem. Rev. 88, 927–941.
doi:10.1021/cr00088a006
Zhang, L., Narita, Y., Gao, L., Ali, M., Oshiki, M., Ishii, S., Okabe, S., 2017a. Microbial
competition among anammox bacteria in nitrite-limited bioreactors. Water Res. 125,
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249–258. doi:10.1016/j.watres.2017.08.052
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growth rate of anammox bacteria revisited. Water Res 116, 296–303.
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TABLE CAPTIONS:
Table S1: 16S rRNA gene-targeted primers and probes for qPCR assay.
Primer/ probe name
Specificity Sequence (5'-3') Reference
BRS95F Ca. B. sinica GATGGGAACAACAACGTTCCA (Zhang et al., 2017a)BRS170R TTCTTTGACTGCCGACACCA
BRS130P FAM-CCGAAAGGGTTGCTAATTCTCA-MGB-NFQ
JEC447F Ca. J. caeni GTAAGGGGGTGAATAGCCCTC (Zhang et al., 2017a)JEC629R TCCAGCCCTATAGTATCAACT
JEC512P FAM-CAGCAGCCGCGGTAATACAGA-MGB-NFQ
BRSP454F Ca. B. sapporoensis
GCAAGGATGTTAATAGCGTTC (Narita et al., 2017)BRSP660
RTCAAGCCATGCAGTATCGGAT
JEC512P FAM-CAGCAGCCGCGGTAATACAGA-MGB-NFQ
Table S1S2: Contact angle and Zeta potential of the three enriched anammox cultures.
Description Water (°) Glycerol (°) Formamide (°) Zeta potential (mV)
Ca. B. sinica 79.5±2.5 54.5±2.3 76.0±2.0 -21.3±2.1
Ca. J. caeni 72.5±3.1 50.2±3.8 70.3±4.6 -20.5±2.0
Ca. B. sapporoensis 60.8±1.9 41.3±2.3 63.5±2.6 -19.2±2.2
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FIGURE CAPTIONS:
Figure S1. Up flow glass column reactor. Effective volume of the reactor was 40 mL.
Figure S2. The interaction energies profiles calculated according to the extended DLVO
theory as a function of distance between the bacterial enrichment cultures of “Ca. B. sinica”
(A), “Ca. J. caeni” (B) and “Ca. B. sapporoensis” (C).
Figure S2S3. Photos of the three reactors (Reactor 1, 2 and 3) taken at different time periods.
Figure S3S4. Aggregate size at the end of reactor operation (i.e. after 3 months). Photos in
panel A, B and C, represent anammox bacterial aggregates of Reactor 1, 2, and 3,
respectively. Scale bars represent 25mm.
Figure S4S5. Modeled growth curves of “Ca. B. sinica” (red), “Ca. J. caeni” (blue) and “Ca.
B. sapporoensis” (green). The specific growth rate (µ) was calculated as a function of nitrite
as limiting substrate concentration by using the Monod equation. The biokinetic parameters
(the maximum specific growth rate, µmax and the apparent half-saturation constant for nitrite,
KS) were adopted from previous studies (Narita et al., 2017; Zhang et al., 2017b). The Monod
growth curves were calculated using the upper and lower limit of KS values.
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Figure S1. Up flow glass column reactor. Effective volume of the reactor was 40 mL.
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Figure S2. The interaction energies profiles calculated according to the extended DLVO
theory as a function of distance between the bacterial enrichment cultures of “Ca. B. sinica”
(A), “Ca. J. caeni” (B) and “Ca. B. sapporoensis” (C).
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Figure S2S3. Photos of the three reactors (Reactor 1, 2 and 3) taken at different time periods.
.
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Figure S3S4. Aggregate size at the end of reactor operation (i.e. after 3 months). Photos in panel A, B and C, represent anammox bacterial
aggregates of Reactor 1, 2, and 3, respectively. Scale bars represent 25mm.
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Figure S4S5. Modeled growth curves of “Ca. B. sinica” (red), “Ca. J. caeni” (blue) and “Ca.
B. sapporoensis” (green). The specific growth rate (µ) was calculated as a function of nitrite
as limiting substrate concentration by using the Monod equation. The biokinetic parameters
(the maximum specific growth rate, µmax and the apparent half-saturation constant for nitrite,
KS) were adopted from previous studies (Narita et al., 2017; Zhang et al., 2017b). The Monod
growth curves were calculated using the upper and lower limit of KS values.
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