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

Liu, X.M., Sheng, G.P., Luo, H.W., Zhang, F., Yuan, S.J., Xu, J., Zeng, R.J., Wu, J.G., Yu,

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

Int. Licens. 1–10.

van Oss, C.J., 2008. Chapter Three The Extended DLVO Theory. Interface Sci. Technol. 16,

31–48. doi:10.1016/S1573-4285(08)00203-2

van Oss, C.J., 1995. Hydrophobicity of biosurfaces — Origin, quantitative determination and

interaction energies. Colloids Surfaces B 5, 91–110. doi:10.1016/0927-7765(95)01217-7

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

Zhang, L., Narita, Y., Gao, L., Ali, M., Oshiki, M., Okabe, S., 2017b. Maximum specific

growth rate of anammox bacteria revisited. Water Res 116, 296–303.

doi:10.1016/j.watres.2017.03.027

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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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