Stable-Isotope Probing Reveals the Activity and Function of

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Stable-isotope probing reveals activity and function of autotrophic and heterotrophic denitrifiers in nitrate removal from organic-limited wastewater Wei Xing, Jin-Long Li, Desheng Li, Jincui Hu, Shi-Hai Deng, Yuwei Cui, and Hong Yao Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b01993 • Publication Date (Web): 14 Jun 2018 Downloaded from http://pubs.acs.org on June 15, 2018

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Environmental Science & Technology

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Stable-isotope probing reveals activity and function of

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autotrophic and heterotrophic denitrifiers in nitrate

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removal from organic-limited wastewater

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Wei Xing †,‡, 1, Jinlong Li †,1, Desheng Li †,‡,*, Jincui Hu †, Shihai Deng †, Yuwei Cui †,

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Hong Yao †,‡

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†

School of Civil Engineering, Beijing Jiaotong University, Beijing 100044, China

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‡

Beijing Key Laboratory of Aqueous Typical Pollutants Control and Water Quality

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Safeguard, Beijing 100044, China

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*

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University, No. 3 Shangyuancun, Haidian District, Beijing 100044, PR China.

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E-mail: [email protected]; Tel/Fax: 86-10-5168-4986

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1

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Jiaotong University, No. 3 Shangyuancun, Haidian District, Beijing 100044, PR China.

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E-mail: [email protected] (Wei Xing); [email protected] (Jinlong Li)

Corresponding author: Desheng Li, School of Civil Engineering, Beijing Jiaotong

These authors contributed equally to this work. School of Civil Engineering, Beijing

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ACS Paragon Plus Environment


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ABSTRACT

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Combined heterotrophic and autotrophic denitrification (HAD) is a sustainable and

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practical method for removing nitrate from organic-limited wastewater. However, the

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active microorganisms responsible for denitrification in wastewater treatment have not

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been clearly identified. In this study, a combined micro-electrolysis, heterotrophic, and

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autotrophic denitrification (CEHAD) process was established. DNA-based stable isotope

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probing was employed to identify the active denitrifiers in reactors fed either 13C-labeled

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inorganic or organic carbon source. The total nitrogen removal efficiencies reached 87.2–

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92.8% at a low organic carbon concentration (20 mg/L COD). Real-time PCR analysis of

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the nirS gene as a function of the DNA buoyant density following ultracentrifugation of

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the total DNA indicated marked 13C-labeling of active denitrifiers. High-throughput

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sequencing of the fractionated DNA in H13CO3−/12CH312COO−-fed and

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H12CO3−/13CH313COO−-fed reactors revealed that Thermomonas-like phylotypes were

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labeled by 13C-bicarbonate, while Thauera-like and Comamonas-like phylotypes were

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labeled by 13C-acetate. Meanwhile, Arenimonas-like and Rubellimicrobium-like

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phylotypes were recovered in the “heavy” DNA fraction from both reactors. These results

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suggest that nitrate removal in CEHAD is catalyzed by various active microorganisms,

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including autotrophs, heterotrophs, and mixotrophs. Our findings provide a better

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understanding of the mechanism of nitrogen removal from organic-limited water and

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wastewater and can be applied to further optimize such processes.

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TOC/Abstract art

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INTRODUCTION Nitrate removal is one of the central objectives in water supply and wastewater

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treatment.1, 2 Biological denitrification, including heterotrophic and autotrophic

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denitrification, is a common method for nitrate removal.3 In particular, heterotrophic

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denitrification has been widely reported for its highly efficient denitrification in case of

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sufficient organic carbon substrate. Conversely, in autotrophic denitrification, microbes

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reduce nitrate by utilizing inorganic elements or compounds as electron donors and

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consuming inorganic carbon (bicarbonate) as a carbon source. Notably, autotrophic

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denitrification is considered an economically affordable and environmentally friendly

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process for organic-limited water and wastewater, because the dosing of additional

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organics (e.g., methanol, acetate) can increase the cost and risk of organic residue

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contamination.4 In recent years, sulfur-, hydrogen-, and biocathode-based autotrophic

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denitrification processes have been rapidly developed and have attracted widespread

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attention.5-7 In a previous study, we established a novel combined micro-electrolysis and

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autotrophic denitrification (CEAD) process based on iron-carbon micro-electrolysis

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carriers (MECs), which produced H2 and Fe2+ for autotrophic denitrifying bacteria via

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numerous microscopic galvanic cells.8, 9 With this system, total nitrogen (TN) removal

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efﬁciencies of 65–76% were achieved under organic-free conditions, without the need for

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external H2 or electricity.

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Combined heterotrophic and autotrophic denitrification (HAD) is considered a

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practical method for nitrogen removal from nitrogen-contaminated groundwater and

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drinking water, because natural water is usually organic-limited, rather than organic-free

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or organic-abundant.10, 11 HAD is also a promising method for wastewater treatment. For 4


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instance, secondary effluents from municipal wastewater treatment plants (WWTPs)

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contain insufficient organic carbon for tertiary denitrification; the application of HAD in

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advanced treatment can reduce TN emission by utilizing both the small amount of

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organics in the secondary effluent and inorganic electron donors, without the need for

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organic dosing. In previous studies, higher denitrification rates were achieved when

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limited organic carbon sources (liquid carbon, woodchips, and sludge) were subjected to

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sulfur- and ferrous-based autotrophic denitrification.12, 13 Therefore, to resolve

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complications with TN removal from organic-limited water and wastewater, it is essential

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to study the effects of organic carbon on autotrophic denitrification processes and to

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improve TN removal performance via the HAD process.

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In the HAD process, heterotrophic and autotrophic denitrifiers are supposed to

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coexist under organic-limited conditions, but it has been difficult to reveal their actual

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functions in this process. Since denitrification performance is closely associated with the

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activity of denitrifiers, identification of the functional denitrifiers is essential to reveal the

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microbiological mechanisms and for denitrification process optimization. Multiple

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studies have demonstrated that strains in genera such as Pseudomonas, Thauera, and

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Thiobacillus reduce nitrate.14, 15 Advanced molecular techniques (e.g., high-throughput

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sequencing) that can provide comprehensive information without cultivation have been

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successfully applied for microbial community analysis of denitrification processes.16, 17

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However, the characterization of the autotrophic and heterotrophic lifestyles of

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denitrifiers on the basis of 16S rRNA gene analysis alone has been challenging. For

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instance, it is difficult to determine whether uncultured bacteria of the genus Thauera are

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autotrophic or heterotrophic denitrifiers, because Thauera can predominate in all of

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autotrophic, heterotrophic, and HAD systems.13, 15, 18 DNA-based stable-isotope probing (DNA-SIP) is a promising technique for the

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identification of metabolically active bacteria that relies on the cell division of active

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microorganisms assimilating isotope-labeled substrates.19 Combined with fingerprinting

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or high-throughput sequencing, DNA-SIP has been successfully applied to investigate the

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functions and phylogenetic relationships of bacteria in natural environments,20, 21 and has

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been recently used in studies on drinking water purification and wastewater treatment.22,

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dominant autotrophic denitrifier in the CEAD process under organic-free condition.9

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By using DNA-SIP, we previously identified a Thiobacillus-like bacterium as a

In the present study, to further enhance nitrate removal from wastewater with

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subsistent but insufficient organic carbon, a combined micro-electrolysis, heterotrophic,

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and autotrophic denitrification (CEHAD) process was established by using synthetic

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wastewater with a low total organic carbon to nitrogen ratio (TOC/N; 7.5 mgTOC/L, 40

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mgNO3−-N/L). The DNA-SIP-based approach was applied to identify active autotrophic,

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heterotrophic, and mixotrophic denitrifiers on the basis of their different carbon

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assimilation lifestyles. We expected autotrophic, heterotrophic, and mixotrophic

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denitrifying phylotypes to be labeled by H13CO3−, 13CH313COO−, and both, respectively.

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Combined results of taxonomic identification and trophic characterization of denitrifiers

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provided insights into the microbiological mechanisms involved in nitrogen removal

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from organic-limited water and wastewater.

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MATERIALS AND METHODS

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CEHAD principles and experimental operation

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MECs were prepared using powdered iron (17.5% volume), scrap iron (25.0%

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volume), powdered activated carbon (35.0% volume, passed through a 200-mesh screen),

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three types of catalysts (each of 2.5% volume), adhesive X (10.0% volume), and foaming

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agent Y (5.0% volume), and were roasted under an oxygen-free atmosphere at 900–

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1000°C for 3 h.24 Numerous galvanic cell reactions occur once zero-valent iron and

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activated carbon in carriers coexist in wastewater. Fe2+ and H2 generated from the anode

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and cathode, respectively, were utilized as electron donors for the autotrophic denitrifiers,

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which have been described in detail in our previous studies.8, 25 Both autotrophic and

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heterotrophic denitrifiers, which use Fe2+/H2 and acetate as electron donors, respectively,

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contribute to nitrogen removal in the CEHAD process under low TOC/N conditions

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(Figure 1). Details of these principles are described in the Supporting Information.

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Three paralleled batch reactors were operated using three 250-mL serum bottles with

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tight caps, each containing 250 g (wet weight) of MECs and 170 mL of synthetic

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wastewater. All three reactors were placed in a shaking incubator (HZQ-F100; Donglian

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Instrument Co. Ltd., Harbin, China) at constant parameters of 30 °C and 90 rpm.

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Inoculated activated sludge (IAS) was collected from the GBD WWTP (Beijing, China).

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After centrifugation (3000 rpm for 3 min), 3 g of IAS was weighed and suspended in

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synthetic substrate, and then incubated with MECs in each reactor for the first three days,

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without substrate replacement. Starting on the fourth day, all of the liquid and excess

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suspended sludge (170 mL) in the reactors was replaced with the same volume of

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synthetic wastewater every 24 h (HRT = 24 h) for supplementation. After each liquid 7



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replacement, N2-stripping was performed, after which the reactors were sealed to

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establish an anoxic condition and to avoid atmospheric CO2 existing in the headspace.

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Synthetic wastewater was distilled water containing 40 mg/L NO3−-N, 40 mg/L inorganic

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carbon (IC; equal to 280 mg/L NaHCO3), 7.5 mg/L total organic carbon (TOC;

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CH3COONa, equal to 20 mg/L COD), and 1 mL/L trace element solution, the latter of

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which was prepared as described by Till et al.26 HCl solution (10%) was added to the

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reactors thrice a day to maintain the initial pH of 7.0–7.3.

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DNA-SIP incubations

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To distinguish active autotrophic and heterotrophic denitrifiers, the three reactors

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were fed synthetic wastewater labeled with 13C-bicarbonate or 13C-acetate in different

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serum bottles (Figure 1). The three reactors were fed the following substrates: (1)

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unlabeled H12CO3− and 12CH312COO−, with NO3− (control reactor, RC); (2) 13C-labeled

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H13CO3− and 12CH312COO−, with NO3− (13C-labeled inorganic carbon reactor, RIC); and

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(3) 13C-labeled 13CH313COO− and H12CO3−, with NO3− (13C-labeled organic carbon

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reactor, ROC). 13C-labeled H13CO3− and 13CH313COO− (99.9%) were purchased from

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Cambridge Isotope Laboratories, Inc. (Tewksbury, MA, USA). The reactors were

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operated for 46 days. To reduce the effect of cross-feeding from decayed microbes, 13C-

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labeled substrates were employed for 14 days only. During the first 32 days, 12C-

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unlabeled carbon substrates were used in all three reactors; as of the 33rd day, when the

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nitrogen removal efficiencies were stabilized, 13C-labeled carbon substrates were

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employed in RIC and ROC. At the end of the 46-day operation, MECs (100 g, wet

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weight) with attached biofilms were collected from each reactor for molecular analysis.

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Because most nitrogen in the reactors would be converted to N2 rather than being 8


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assimilated by the microbes, 15N labeling is not practical for evaluating denitrification.

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However, bacteria that can utilize 13C-substrate but are not involved in denitrification

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should be excluded. Hence, two supplementary DNA-SIP treatments (T1 and T2) were

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established together with two supplementary controls (C1 and C2) using the following

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substrates: (1) only H13CO3−, without NO3− (treatment T1); (2) only 13CH313COO−,

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without NO3− (treatment T2); (3) unlabeled H12CO3− and NO3− (control C1); (4)

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unlabeled 13CH313COO− and NO3− (control C2). The concentration of each substrate (if

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applicable) was identical to that used for the synthetic wastewater treatment in the above-

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described three reactors. More details are described in the Supporting Information.

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Analytical methods

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The concentrations of nitrogen, including NO3−-N, NO2−-N, and NH4+-N, were

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measured using an ultraviolet spectrophotometer (2102C; UNICO Co. Ltd., Shanghai,

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China), according to the APHA standard methods.27 The sum of values for NO3−-N,

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NO2−-N, and NH4+-N was used to estimate TN. TOC and IC were determined in the

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stable phase using a TOC analyzer (TOC 5000A; Shimatzu, Kyoto, Japan). pH was

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measured using a pH electrode (pHG-7685A; Tian-yi Co. Ltd., Shanghai, China).

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DNA extraction, SIP gradient fractionation, and microbial community analysis

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At the end of operation, seven biofilm samples were collected from the surfaces of

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the MECs of each of the three reactors (RC, RIC, and ROC) and the four supplementary

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treatments (T1 and T2) and controls (C1 and C2). Genomic DNA was extracted from the

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samples using a FastDNA Spin Kit for Soil (MP Biomedicals, Irvine, CA, USA). SIP

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fractionation was then performed by buoyant density gradient centrifugation according to

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previously described methods. 9, 28 Buoyant densities of each DNA fraction from all 9



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samples are shown in Table S1. Genomic DNA from RC, RIC, and ROC was used for

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real-time quantitative PCR (qPCR) analysis of the nirS and nirK genes (both encoding

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nitrite reductase). For all seven samples, the nirS gene was further quantified in the

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different DNA fractions across the buoyant density gradient. Each analysis was

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performed in triplicate. qPCR revealed no detectable labeling in the “heavy” DNA

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fractions of T1 and T2; therefore, only fractionated DNA from the three reactors (RC,

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RIC, and ROC) was used for high-throughput sequencing and clone analysis. High-

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throughput sequencing was performed on the Illumina MiSeq platform (Majorbio Bio-

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pharm Technology Co. Ltd., Shanghai, China) using a set of barcode-containing primers

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338F/806R for 16S rRNA gene amplification. In total, 40 samples were sequenced, which

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included genomic DNA samples from the IAS, RC, RIC, and ROC, as well as 12

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fractionated DNA samples from each reactor. After pre-processing using Trimmomatic

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software and FLASH, 20,761–41,629 effective sequences were obtained for the different

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samples. With random and even subsampling, 20,761 sequences in each sample were

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analyzed, and the average sequence length was 444 bp. Sequences were divided and

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clustered into operational taxonomic units (OTUs) with a 97% similarity threshold by

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Uparse. A heatmap was generated using R (version 3.2.3). Bacterial nirS and nirK clone

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libraries were constructed using the “heavy” DNA fractions of RIC and ROC (the 11th

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DNA fractions with buoyant densities of 1.745 and 1.746 g/mL, respectively) and the

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“light” DNA fractions of RC, RIC, and ROC (the 7th DNA fractions with buoyant

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densities of 1.729, 1.730, and 1.730 g/mL, respectively). For each sample, 30 clone

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sequences were obtained and analyzed. Details of the PCR conditions and primers (Table

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S2) are described in the Supporting Information.

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Data deposition Sequences obtained by high-throughput sequencing of genomic DNA (four samples)

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have been deposited in the NCBI short-read archive under accession numbers

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SRR4457989–4457992; sequences obtained from fractionated DNA (36 samples) were

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archived under accession numbers SRR4457945–4458007. All non-identical sequences

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from clone libraries also have been deposited in the NCBI archive under accession

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numbers MH432369–432507 (139 sequences for nirS) and MH432265–432359 (95

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sequences for nirK).

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RESULTS AND DISCUSSION

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Nitrogen removal performance in the CEHAD process

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As shown in Figure 2, nitrogen removal showed a similar trend in each reactor.

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Nitrate was rapidly reduced initially, likely because the decay of the seed sludge provided

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electron donors for denitrification. The average concentration of nitrate in the effluent

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was 9.8 mg/L between days 8 and 17, and decreased to less than 2.7 mg/L after day 21,

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indicating the establishment of biofilm culture. The average nitrate removal efficiencies

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during the stable phase in the three reactors ranged from 98.7% to 100%. Notably, NO3−-

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N can be converted to NH4+-N by chemical reactions (Eq. 6 in Supporting Information)

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or via the dissimilatory nitrate reduction to ammonium (DNRA) pathway in the presence

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of iron.29 The concentration of NH4+-N decreased gradually from 20.5–23.8 mg/L (in the

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first 17 days) to 0.6–1.5 mg/L (in the last three days), demonstrating that biological

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denitrification dominated over ammonium generation. At the end of operation, TN

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removal efficiencies of 87.2–92.8% were noted, indicating that highly efficient

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denitrification was achieved in all three reactors. Although small amounts of NO2−-N (1.9–4.4 mg/L) and NH4+-N (0.6–1.5 mg/L)

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existed in the effluent, CEHAD achieved higher TN removal efficiencies than those

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reported for Fe(0)-supported autotrophic denitrification, sulfur autotrophic denitrification,

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and electrochemical denitrification.12, 30, 31 Notably, the CEHAD process yielded lower

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levels of NH4+-N than the CEAD process under organic-free conditions, despite the use

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of the same carriers. As shown in Table S3, at the end of operation, the concentrations of

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NH4+-N in the effluents of the CEAD reactors were 5.6 ± 2.1 mg/L and 10.5 ± 2.2 mg/L

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(in duplicate) under similar conditions, without the addition of organic carbon.9

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Comparison of the CEHAD and CEAD performances indicated that a small amount of

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organic carbon, which was insufficient for complete heterotrophic denitrification,8 could

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enhance biological denitrification and improve TN removal efficiency through combined

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heterotrophic and autotrophic denitrification processes. These results were in accordance

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with those of other studies.12, 13 Thus, the CEHAD process is an efficient and promising

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method for organic-limited wastewater treatment, such as advanced nitrogen removal

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from secondary effluent in WWTPs.

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Because of precipitation and continuous consumption, Fe2+ and H2 supplied to

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autotrophic denitrifiers could not be accurately quantified. Instead, IC was determined to

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assess autotrophic denitrification activity from the process perspective. The IC and TOC

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concentrations in the influent and effluent during the last three days (stable phase) of

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activity indicated that 6.6 ± 3.6 mg IC/L and 6.3 ± 0.6 mg TOC/L were consumed in the

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reactors, respectively. These data suggested the existence of autotrophic and

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heterotrophic denitrification in the CEHAD reactors. However, part of the IC in the

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reactors was consumed by the autotrophic ammonia-oxidizing bacteria (AOB, with

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Nitrosomonas-like bacteria being dominant) and nitrite-oxidizing bacteria (NOB, with

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Nitrotoga-like bacteria being dominant), as evidenced by the high-throughput sequencing

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results which are discussed in the next subsection. The quantitative contributions of IC

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and TOC to autotrophic and heterotrophic denitrifiers were difficult to determine and

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require further study.

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Characterization of the bacterial communities present in the CEHAD reactor

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Bacterial community composition was analyzed by high-throughput sequencing of

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the 16S rRNA gene. Diversity indexes are listed in Table S4. The high coverage index

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values indicated that the sequence data were sufficient to effectively characterize

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bacterial communities. Figure S1 shows that the bacterial communities obtained from all

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three reactors were different from those observed in the IAS. Although they were not

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identical in specific OTUs (e.g., OTU181) due to random errors, the bacterial

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communities in the three reactors were generally similar. In the CEHAD reactors, the

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phylum Proteobacteria accounted for >80% of total bacteria. In total, 355 OTUs were

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classified at 97% similarity. As shown in Figure 3, OTU143, OTU1092, OTU181,

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OTU352, OTU170, and OTU174, which accounted for 21.7% ± 3.4%, 11.1% ± 2.7%, 8.1%

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± 6.7%, 7.7% ± 4.7%, 6.8% ± 2.7%, and 5.7% ± 2.8% of total bacteria, respectively,

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were the most predominant, and were affiliated with the genera Thauera, Arenimonas,

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Comamonas, Rubellimicrobium, Nitrotoga, and Thermomonas, respectively.

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Many Proteobacteria, particularly those of the α, β, and γ subclasses, are involved in

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global nitrogen cycling and pollutant removal.32, 33 Many strains in the most predominant

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genus, Thauera, have been reported to mediate nitrate or nitrite reduction under either 13



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autotrophic or heterotrophic conditions using hydrogen or organic carbon as the electron

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donor.13, 15 However, some strains of Thauera can only reduce nitrate to nitrite, leading to

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nitrite accumulation, which can be resolved by maintaining a pH close to neutral.34 In

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addition, Arenimonas, Comamonas, Thermomonas, and Rubellimicrobium are typical

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denitrifiers that are capable of reducing nitrate to nitrogen gas.17, 35 In addition to

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denitrifiers, one of the dominant genera, Nitrotoga, reportedly belongs to NOB.36 The

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genus Nitrosomonas, typically comprising AOB, was also detected in the reactors. This

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might be because NO3−-N can be converted to NH4+-N by chemical reduction or via the

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DNRA pathway, thereby providing substrates for AOB and NOB. Furthermore, although

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N2-stripping was performed to establish an anoxic condition, a strict oxygen-free

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condition is hard to maintain, and both Nitrotoga and Nitrosomonas can reportedly grow

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under low-oxygen conditions.37, 38

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Notably, Thiobacillus, which was identified as the most dominant autotrophic

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denitrifier in the CEAD process in our previous study,9 was not detected as a dominant

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genus in the current study. This discrepancy can be explained by the presence of organic

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carbon in the CEHAD process, which might reduce the competitiveness of obligate

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autotrophic denitrifiers. Moreover, the biofilms in the CEHAD reactors were cultivated

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directly from activated sludge of the WWTP, rather than from previously operating

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reactors; thus, other random factors might be responsible for the absence of Thiobacillus

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from the reactors used in this study.

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Identification of autotrophic and heterotrophic denitrifiers by DNA-SIP

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qPCR of the nirS and nirK genes from genomic DNA and fractionated DNA

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Both nirS and nirK genes were quantified from genomic DNA of RC, RIC, and ROC 14


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by qPCR (Figure S2). The results showed that nirK copy numbers in the reactors were

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much lower than those of nirS. After incubation for 14 days in the presence of 13C-

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bicarbonate or 13C-acetate as carbon substrates, respectively, autotrophic and

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heterotrophic denitrifiers were identified by DNA-SIP.

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Figure 4 shows the variation in nirS gene copy numbers in different fractionated

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DNA of the seven biofilm samples. In the unlabeled treatment (RC), all nirS gene copies

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were concentrated in the 5th–7th fractions (“light” fractions with buoyant densities of

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1.720–1.728 g/mL). As 13C-DNA is heavier than 12C-DNA, the large peak of nirS copies

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in the ROC shifted to the 11th fraction (“heavy” DNA fraction with a buoyant density of

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1.746 g/mL), indicating that 13C-acetate was strongly assimilated by heterotrophic

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denitrifiers. In the RIC, peak nirS copy number was observed in the 7th (“light”) DNA

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fraction; however, labeled 13C-DNA was also observed in the 9th–11th fractions (“heavy”

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fractions with buoyant densities of 1.737–1.745 g/mL), demonstrating that autotrophic

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denitrifiers that assimilated 13C-bicarbonate coexisted with heterotrophic denitrifiers. The

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nirS copy number in the “heavy” fractions of the ROC was higher than that in the “heavy”

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fractions of the RIC, indicating that the denitrifiers in this reactor were inclined to utilize

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acetate as a carbon source.

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As shown in Figure 4a, in the absence of nitrate, treatment T1, employing only

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H13CO3−, resulted in no detectable labeling in the 9th–11th (“heavy”) DNA fractions. In

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contrast, RIC fed H13CO3−, 12CH312COO−, and NO3− showed labeling in the “heavy”

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fractions. In addition, remarkable labeling in the “heavy” fractions was detected in the

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previously reported CEAD reactor fed H13CO3− and NO3−.9 Similar results were observed

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when comparing ROC and T2 (Figure 4b). These results suggest that the 13C-labeled

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DNA shown in the “heavy” fractions of RIC and ROC was mainly derived from bacteria

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actively involved in N-cycling. Before inoculation of the 13C-substrate, the biofilms in the

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CEHAD and CEAD reactors were enriched from specific organic-limited wastewater

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containing nitrate; thus, denitrifiers are expected to have been dominant. Therefore, in

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this study, bacteria that can utilize 13C-substrate but are not involved in N-cycling were

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not abundant and could not have greatly affected the identification of denitrifiers.

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However, for environmental samples in which denitrifiers are not highly enriched, 13C-

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labeled DNA in the “heavy” fraction in treatments T1 and T2 is supposed to originate

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from bacteria utilizing 13C-substrate without NO3−. In such case, microbial community

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analysis in the “heavy” DNA fractions of T1 and T2 should be performed to exclude

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potentially interfering communities.

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High-throughput sequencing of the fractionated DNA

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To analyze the carbon utilization patterns of the predominant OTUs, each DNA

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gradient fraction in RC, RIC, and ROC was subjected to high-throughput 16S rRNA gene

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sequencing. Based on differences in the relative abundances of OTUs, three typical

330

patterns were observed (Figure 5). The first pattern consisted a predominant peak in the

331

“heavy” fraction of the ROC (demonstrating that 13C-acetate was assimilated) and in the

332

“light” fraction of the RC and RIC (demonstrating that 13C-bicarbonate was not utilized).

333

The bacteria affiliated with these OTUs, such as OTU143 (Figure 5a, affiliated with

334

Thauera) and OTU181 (Figure 5b, affiliated with Comamonas), were identified as

335

heterotrophic denitrifiers. Thauera and Comamonas have been shown to exhibit nitrate

336

removal capacity using organic carbon compounds as electron donors.34, 39 However,

337

Thauera reportedly also is dominant in hydrogen-based autotrophic denitrification.15

16


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338

Therefore, it is difficult to determine whether Thauera is a heterotrophic or autotrophic

339

denitrifier based solely on phylogenetic analysis of the 16S rRNA gene. In the current

340

study, DNA-SIP analysis provided clear molecular evidence that the OTU affiliated with

341

Thauera in the CEHAD reactors represented heterotrophic denitrifiers assimilating

342

acetate.

343

The second pattern consisted of a predominant peak within the “heavy” fraction of

344

the RIC (demonstrating that 13C-bicarbonate was assimilated) and in the “light” fraction

345

of the RC and ROC (demonstrating that 13C-acetate was not utilized). OTU174 (Figure

346

5c), which was affiliated with Thermomonas, displayed this second pattern and

347

represented an autotrophic denitrifier that uses inorganic carbon as the carbon source. In

348

our previous study, Thermomonas was identified as a dominant genus (>20%) in the

349

CEAD process under organic-free conditions.8

350

The third pattern revealed labeled 13C-DNA in the “heavy” fractions of both RIC and

351

ROC, demonstrating that both 13C-bicarbonate and 13C-acetate were assimilated.

352

OTU1092 (affiliated with Arenimonas) and OTU 352 (affiliated with Rubellimicrobium),

353

presented this mixotrophic pattern, evidenced by the shifts to heavier DNA fractions in

354

both RIC and ROC, as shown in Figure 5d and 5e. In previous studies, Arenimonas has

355

been observed in both heterotrophic and bioelectrochemical autotrophic denitrification

356

processes,35, 40 and Rubellimicrobium mesophilum was reported to have a denitrification

357

pathway based on genome sequence data.41 OTUs revealing a mixotrophic pattern likely

358

show partial labeling, since individual species affiliated with a certain OTU can

359

incorporate inorganic carbon while others representing the same OTU can incorporate

360

organic carbon. In this study, OTU1092 showed a more legible peak in the “heavy”

17



361

fractions of the RIC than that of the ROC, indicating that it was preferentially labeled by

362

13

363

C-bicarbonate. Although multiple studies have reported various genera as denitrifiers, their

364

heterotrophic or autotrophic lifestyle is primarily deduced on the basis of whether organic

365

carbons are employed in the process.5, 13, 15 It is difficult to distinguish heterotrophic and

366

autotrophic denitrifiers in HAD processes because organic and inorganic electron donors

367

coexist in such systems. By combining DNA-SIP with qPCR and high-throughput

368

sequencing, we identified heterotrophic, autotrophic, and mixotrophic denitrifiers, using

369

labeled organic and inorganic carbon compounds as substrates.

370

Clone libraries and phylogenetic analysis of the nirS and nirK genes

371

As denitrifiers are difficult to be identified solely by 16S rRNA gene analysis, clone

372

libraries of the nirS and nirK genes were constructed using the “heavy” and “light” DNA

373

fractions. In nirS clone libraries, 21 OTUs were obtained from 150 clone sequences by

374

clustering at 95% similarity (Table S5). A phylogenetic tree generated on the basis of the

375

nirS clones is shown in Figure S3. NirS-Clone-OTUs 1 to 4 represented four dominant

376

OTUs, which accounted for 42.0%, 9.3%, 10.7%, and 8.7% of the 150 clone sequences.

377

NirS-Clone-OTU3 and nirS-Clone-OTU4 were both highly similar to Thauera sp. 27 in

378

GenBank. They existed in the “heavy” fraction of the ROC and the “light” fraction of the

379

RIC, but were negligible in the “light” fraction of the ROC and the “heavy” fraction of

380

the RIC, which indicated that they could reduce nitrate using acetate rather than

381

bicarbonate. Thus, these two clone OTUs affiliated with Thauera related to heterotrophic

382

denitrifiers. These findings are in agreement with the above-mentioned high-throughput

383

sequencing results, which demonstrated that Thauera-like phylotypes were predominant 18


Page 18 of 33

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384

in the reactors and could be identified as heterotrophic denitrifiers. In contrast, nirS-

385

Clone-OTU1 was present in similar proportions (13/30, 16/30, 10/30, 12/30, 12/30,

386

respectively) in each DNA fraction, indicating that it represented a mixotrophic

387

denitrifier. Furthermore, it was found to be similar to Arenimonas donghaensis.

388

Consistent with this finding, the OTU affiliated with Arenimonas in the high-throughput

389

sequencing analysis also showed a mixotrophic lifestyle. Finally, nirS-Clone-OTU2

390

existed only in the “heavy” fraction of the RIC and the “light” fraction of the ROC and

391

thus, represented an autotrophic denitrifier. The nirS sequence in OTU2 was found to be

392

similar to that of an uncultured bacterium in GenBank, and in all nirS genes of known

393

species, the closest one was from Thiohalobacter thiocyanaticus.

394

In nirK clone libraries, nine OTUs were obtained from 150 clone sequences by

395

clustering at 95% similarity (Table S6). A phylogenetic tree constructed on basis of the

396

nirK clones is shown in Figure S4. More than 80% of clone sequences were clustered

397

with nirK-Clone-OTU1, which showed high sequence similarity with Rubellimicrobium

398

mesophilum, indicating that most denitrifiers in CEHAD reactors detected by the nirK

399

gene primers were affiliated with Rubellimicrobium.

400

Intriguingly, the genera Comamonas and Thermomonas, which were detected by

401

high-throughput sequencing, were not found in the clone libraries. In a primer coverage

402

assessment using NCBI Primer-BLAST, we found that Comamonas serinivorans strain

403

DSM 26136 (CP021455) cannot be detected with the nirS1F/nirS6R pair used in this

404

study, though it was reported to harbor a nirS gene. Meanwhile, both Thermomonas fusca

405

(WP_028838532) and Thermomonas hydrothermalis (WP_072755949) harbor nirK

406

genes, but cannot be detected with the primer pair F1a/R3Cu. Moreover, some strains in

19



407

the genus Thauera, such as Thauera sp. 28 (WP_037984032), cannot be detected with

408

nirS1F/nirS6R, while others, such as Thauera humireducens strain SgZ-1 (CP014646)

409

can, although both contain a nirS gene. Because bacterial nirS and nirK genes might be

410

more labile than the 16S rRNA gene, it might be difficult to find an appropriate primer

411

pair targeting the nirS or nirK genes in all denitrifiers. It should be emphasized that the

412

primers for nirS and nirK used in this study need to be optimized for more

413

comprehensive studies.

414

Implications and perspectives In this study, autotrophic, heterotrophic, and mixotrophic denitrifiers were identified

415 416

by DNA-SIP combined with 16S rRNA, nirS, and nirK gene analysis. However, our

417

study had some limitations. Technical errors, e.g., in centrifugation efficiency, and

418

fractionation operation are inherent to the SIP technology. Part of the 12C-unlabeled

419

genomic DNA, which is supposed to be distributed to the “light” fractions, may have

420

been spun down during the isopycnic ultracentrifugation of the total DNA extract and

421

therefore be detected in the “heavy” DNA fractions, especially for the bacteria with

422

relatively high GC content or highly abundant bacteria. Therefore, in general, the

423

difference in the microbial abundances in the same DNA fraction of different treatments

424

(RC, RIC, and ROC) was reliable for identifying whether the bacteria assimilated

425

carbonate or acetate, rather than the absolute abundances in a certain treatment.

426

Furthermore, cross-feeding may have occurred in the reactors. After assimilating 13C-

427

labeled bicarbonate, autotrophic denitrifiers would decay and release 13C-labeled cell

428

substances, which could be secondarily assimilated by heterotrophic bacteria. Meanwhile,

429

13

C-labeled acetate could be decomposed by heterotrophic denitrifiers and generate

20


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Page 21 of 33


430

inorganic carbon, which could subsequently be used as inorganic carbon in autotrophic

431

denitrification. To avoid erroneous assumptions regarding carbon utilization patterns, the

432

following three strategies were applied. (1) 13C-labeled substrates were employed only

433

for 14 days, after the reactors had achieved stable performance. Thus, the incubation time

434

for 13C-labeled substrates was shortened, and the possibility of cross-feeding by decayed

435

microbes could be reduced. (2) The liquid and excess suspended sludge was replaced

436

with fresh synthetic wastewater daily, so that the potential for recycling of cell substances

437

was minimized. (3) Because TOC and IC were maintained at 7.5 mg/L and 40 mg/L,

438

respectively, in the synthetic wastewater, inorganic carbon generated by acetate

439

decomposition (a small portion of the TOC, considering that most organic carbon could

440

be assimilated) was much less than the inorganic carbon content in the influent.

441

Therefore, the effects of inorganic carbon generated by acetate decomposition on

442

autotrophic denitrifiers would not be significant. However, time-course DNA-SIP

443

analysis should be carried out in future, to characterize the optimal duration for

444

incubation with the 13C-substrate to separate 13C-labeled DNA and distinguish

445

heterotrophic, autotrophic, and mixotrophic denitrifiers more precisely.

446

This study showed that nitrate removal in CEHAD was catalyzed by a variety of

447

active microorganisms, including autotrophs (Thermomonas-like phylotypes),

448

heterotrophs (Thauera-like and Comamonas-like phylotypes), and mixotrophs

449

(Arenimonas-like and Rubellimicrobium-like phylotypes). The results lend strong support

450

for the performance of denitrification activity in the reactors, which indicated that a small

451

amount of organic carbon enhanced biological denitrification and benefited higher TN

452

removal efficiency. However, real wastewater has a more complex and variable

21



453

components, and thus, different microbial communities might be existed in systems fed

454

with real wastewater. Therefore, further studies using real wastewater are desired to

455

analyze the microbial communities in situ, by using the real wastewater as the influent

456

and adding the target 13C-labeled substrates in it. Further, studies with different TOC/N

457

and TOC/IC ratios should be conducted to illuminate the effect of component variation

458

on microbial species composition. The microbiological mechanisms of denitrification in

459

real wastewater treatment could thus be profiled comprehensively.

460

ACKNOWLEDGEMENTS

461

This study was financially supported by the National Natural Science Foundation of

462

China (grant nos. 51408028 and 51278034). We would like to express our deep gratitude

463

to Prof. Zhongjun Jia at the Institute of Soil Science, Chinese Academy of Sciences, for

464

his technical support with the DNA-SIP experiments.

465

ASSOCIATED CONTENT

466

Supporting Information

467

Detailed methods for the establishment of the CEHAD process, supplementary

468

experiments, and molecular biological methods. Additional Tables S1 to S6 showing

469

buoyant density of each DNA fraction, PCR primers, CEAD performance, diversity

470

indices, and the results of nirS and nirK clone libraries. Additional Figures S1 to S4

471

related to abundances of bacterial populations, qPCR results of genomic DNA, and

472

phylogenetic trees on the basis of nirS and nirK clones.

473

This information is available free of charge via the Internet at http://pubs.acs.org.

22


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474

AUTHOR INFORMATION

475

Corresponding Author:

476

Desheng Li

477

First Authors:

478

Wei Xing

Tel: +86-10-5168-5917; E-mail: [email protected]

479

Jinlong Li

Tel: +86-10-5168-5917; E-mail: [email protected]

480

Notes

481

The authors declare no competing financial interest.

482

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483

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613

28


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614 615

Figure 1. Schematic of the experimental procedures. Three paralleled reactors were

616

operated for 46 days, including 32-day unlabeled incubation and 14-day DNA-based

617

stable-isotope probing (DNA-SIP) incubation periods. Unlabeled 12C-substances, 13C-

618

labeled inorganic carbon, and 13C-labeled organic carbon were employed in the RC

619

(control), RIC (reactor with inorganic carbon), and ROC (reactor with organic carbon),

620

respectively.

621 622

29



623 624

Figure 2. Performance of the combined micro-electrolysis, heterotrophic, and

625

autotrophic denitrification (CEHAD) process in the presence of low total carbon

626

to nitrogen (TOC/N) ratios. (a–c) Results obtained from the (a) control reactor with

627

12

628

with 13C-labeled organic carbon (ROC). The dashed line indicates the time point at

629

which the labeled substrates were added; it divides the periods of unlabeled incubation

630

(first 32 days) and DNA-based stable-isotope probing (DNA-SIP) incubation

631

(subsequent 14 days). NRE represents total nitrogen (TN) removal efficiency.

C-substrate (RC), (b) reactor with 13C-labeled inorganic carbon (RIC), and (c) reactor

30


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632 633

Figure 3. Hierarchical heat map analysis of the genomic DNA obtained from the

634

IAS, RC, RIC, and ROC, according to high-throughput sequencing of 16S rRNA at

635

the OTU level. OTUs were clustered with 97% similarity. The relative abundance of

636

OTUs is indicated by color intensity, which represents the percentage of the targeted

637

OTU reads in the total bacterial 16S rRNA gene reads in each genomic DNA. All OTUs

638

accounting for >5% in the RC, RIC, and ROC are listed. OTU, operational taxonomic

639

unit; IAS, inoculated active sludge; RC, control reactor with 12C-substrate; RIC, reactor

640

with 13C-labeled inorganic carbon; ROC, reactor with 13C-labeled organic carbon.

641 642

31



643 644

Figure 4. Quantitative distribution of the nirS gene copies ratio in different

645

fractionated DNA in a) RC, RIC, C1, and T1; b) RC, ROC, C2, and T2. The nirS

646

copies ratios were defined by copy numbers in the present fraction to that in total

647

fractions. RC, control reactor with 12C-substrate (H12CO3− and 12CH312COO−, with NO3−);

648

RIC, reactor with 13C-labeled inorganic carbon (H13CO3− and 12CH312COO−, with NO3−);

649

ROC, reactor with 13C-labeled organic carbon (H12CO3− and 13CH313COO−, with NO3−);

650

supplementary T1 (only contains H13CO3−, without NO3−); control C1 (H12CO3− and

651

NO3−); supplementary T2 (only contains 13CH313COO−, without NO3−); control C2

652

(12CH312COO− and NO3−).

653

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Page 32 of 33

Page 33 of 33


654 655

Figure 5. Relative abundance of 16S rRNA gene sequences across the entire buoyant

656

gradient of the DNA fractions for three reactors, based on high-throughput

657

sequencing. (a–e) Results of (a) OTU143, (b) OTU181, (c) OTU174, (d) OTU1092, and

658

(e) OTU352, affiliated with the genera Thauera, Comamonas, Thermomonas,

659

Arenimonas, and Rubellimicrobium, respectively. The relative abundance is expressed as

660

the percentage of the targeted OTU reads in the total bacterial 16S rRNA gene reads in

661

each DNA gradient fraction. OTU, operational taxonomic unit; RC, control reactor with

662

12

663

labeled organic carbon.

C-substrate; RIC, reactor with 13C-labeled inorganic carbon; ROC, reactor with 13C-

33


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