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Application and microbial ecology of psychrotrophs in domestic wastewater treatment at low temperature
Accepted Manuscript Application and microbial ecology of psychrotrophs in domestic wastewater treatment at low temperature Zhenzhen Xu, Yue Ben, Zhonglin Chen, Anxi Jiang, Jimin Shen, Xiaoyun Han PII:
S0045-6535(17)31706-X
DOI:
10.1016/j.chemosphere.2017.10.121
Reference:
CHEM 20143
To appear in:
ECSN
Received Date: 26 December 2016 Revised Date:
11 October 2017
Accepted Date: 23 October 2017
Please cite this article as: Xu, Z., Ben, Y., Chen, Z., Jiang, A., Shen, J., Han, X., Application and microbial ecology of psychrotrophs in domestic wastewater treatment at low temperature, Chemosphere (2017), doi: 10.1016/j.chemosphere.2017.10.121. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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ACCEPTED MANUSCRIPT 1
Application and microbial ecology of psychrotrophs in domestic wastewater treatment at low temperature
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Zhenzhen Xua, Yue Ben
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b,c,
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*, Zhonglin Chenc,1, Anxi Jiangc, Jimin Shenc, Xiaoyun
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a
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P R China
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College of Geography and Environment, Shandong Normal University, Jinan 250014,
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b
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Research Institute Co. LTD, Beijing 100032, P R China
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c
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Technology, Harbin 150090, P R China
Water Works Department, State Nuclear Electric Power Planning Design and
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State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of
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A manuscript submitted to Chemosphere
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(December 25th, 2016)
20 * Corresponding author. Tel.: +86 010 58342558; Fax: +86 010 58342558. E-mail address: [email protected] (Z. Xu), [email protected] (Y. Ben), [email protected] (Z. Chen). 1
Co-corresponding author. 1
ACCEPTED MANUSCRIPT Abstract
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The feasibility of a bunch of screened psychrotrophs being applied to low-temperature
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wastewater treatment was investigated. The screened psychrophillic strains are capable
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of growth at a broad temperature-range from 0 to 40 °C and exhibit a preferable
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TTC-dehydrogenase activity at low temperature (4~10 °C). Along the sharply fluctuant
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temperatures (25 to 4 to 25 °C), the screened psychrotrophs (compared with the
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indigenous mesophiles) demonstrate less fluctuations of COD removal and more rapid
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recovery after temperature shocks. COD removal of approximate 80% was recorded by
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single psychrotrophs (while only 10% by single mesophiles) at low temperature (4 °C).
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Soft polyurethane foam showed better performance for psychrotrophs immobilization,
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with the optimal filling rate of 30% (v/v) in the bioreactor. The observation shows that
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the immobilized psychrotrophs demonstrated a relatively high performance on both
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conventional and emerging organic contaminants removals at low temperature. In
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order to check the feasibility of the screened psychrotrophs in treating actual domestic
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wastewater, a pilot-scale ICABR bioreactor was operated firstly at low temperature (4
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o
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COD of 150–600 mg L-1 was efficiently reduced to 40 ± 18 mg L-1 under the
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conditions of an overall hydraulic retention time of 10 h. Furthermore, psychrotrophs
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performed stably as the predominant bacteria family during the whole operation. This
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study provides evidence that microbial intensification with psychrotrophs was a
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feasible strategy to improve the efficiency of conventional wastewater treatment
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process at low temperature.
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Keywords: psychrotrophs; low temperature; wastewater treatment; aerobic biocontact
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reactor; immobilization
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1. Introduction Cold environment exists continuously in some regions of the Earth, such as polar
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surface, worldwide deep oceans and some moderately cold areas at higher altitudes in
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mountainous regions, and also appears seasonally in areas far away from the equator.
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Psychrophilic and/or psychrotrophic bacteria, which were detected in these cold areas,
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have been proved to play an important role in the environment-ecosystem (Rashid et
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al., 1999). As two typical cold-adapted microorganism communities, psychrotrophs
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and psychrophiles have frequently been isolated from these cold environments, and
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found to grow normally at temperatures near freezing point. Psychrophiles are
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specifically adapted to living in extremely cold environment, but cannot survive at
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temperature above 20 °C (Morita, 1975). In comparison, psychrotrophs are notable for
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having a particularly broad growth temperature range. With the upper limit as high as
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40 °C, psychrotrophs grow optimally at temperatures around 20–25 °C (Berchet et al.,
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2000; Margesin et al., 2002). Psychrotrophs have been reported on their considerable
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biotechnological potential (Gianese et al., 2001; Cavicchioli et al., 2002; Margesin and
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Feller, 2010; Russell, 2000) and well-demonstrated efficiencies in some environmental
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bioremediation processes (Zekker et al., 2016; Gilbert et al., 2014; Gratia et al., 2009;
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Siggins et al., 2011).
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Domestic wastewater, as one of the greatest sources of aquatic pollution, is related
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directly to water safety and public health issues. Since the occurrence of emerging 3
ACCEPTED MANUSCRIPT organic contaminants (EOCs) at different concentrations in municipal wastewater
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systems and natural water bodies (Jiang et al., 2013; Ternes et al., 2004; Matamoros et
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al., 2016) in recent years, new challenges are put forward on how to enhance the
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efficiency of wastewater treatment technology, which is expected to treat both
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traditional and emerging contaminants. For centuries, biological technology is chosen
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as the most sustainable and optimal option for wastewater treatment. Temperature was
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important for biological wastewater treatment on account of its effect on biochemical
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reactions in many ways, such as reaction rates, reaction pathway, microorganism yields,
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and death rates (ADM1, 2002). Wastewater temperature is apt to fall below 10 °C
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(some even below 5 °C) in winter as a result of climate seasonal changes. Due to
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seasonal
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microorganisms are severely depressed, leading to a poor biotransformation of
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contaminants in wastewater (Motta et al., 2003; Hai et al., 2011). Many ways had been
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utilized to improve the efficiency of low temperature wastewater treatment: heat
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preservation (Li et al., 2011), enlargement of returned sludge volume (Metcalf et al.,
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2003), reduction of sludge load rates (Tian et al., 2012), improvement of hydraulic
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retention time (Viraraghavan & Varadarajan, 1996; Daija et al., 2016) or other
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additional measures (Zekker et al., 2017), but all these ways would bring much higher
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cost. Rich biomass is anticipated to guarantee remarkable efficiency of biological
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systems. Immobilization technique has proved to be an effective way to make free cells
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more concentrated, more active and further stable. Additionally, compared with free
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cell systems, the immobilized cell systems benefit the cells separation from water
regional
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microbial
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organic solvents and microbial attack, polyurethane foam (PUF) has been widely used
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as a carrier in the immobilization of various microorganisms for wastewater treatment
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(Guimaraes et al., 2005; Lim et al., 2011; Zheng et al., 2009).
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The development of direct treatment systems operated at ambient temperatures
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(average temperature in Northeast China from 1961 to 2000, 5 °C; He et al., 2013),
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especially in winter, without doubt will have a great ecological and economic impact.
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In this respect, bioaugmentation and inoculation using psychrotolerant bacteria appears
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to be an appropriate way as psychrotrophs can thrive efficiently at low/moderate
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temperatures. Psychrotolerant microorganisms had attracted increasing interest for
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their capability and high efficiency for wastewater treatment at low temperature in
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recent years (Connaughton et al., 2006; Gratia et al., 2009; Yao et al., 2013; Huang et
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al., 2015). However, researches on the long-term treatment of actual wastewater
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(containing emerging organic contaminants) at seasonally changeable temperature
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were rarely reported.
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In this study, a series of preponderant psychrotrophs with high activity at 4 °C
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were isolated and identified. The biological characteristics and microbial ecology of
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the screened psychrotrophs were investigated. The primary objectives of this study
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were (1) to examine the resistance of screened psychrotrophs to sharp temperature
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changes (25 to 4 to 25 °C) and the performance of immobilized psychrotrophs on PUF;
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(2) to investigate the efficiency of the screened psychrotrophs in removing both
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conventional (COD) and emerging (para-chloronitrobenzene, pCNB) organic
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emerging organic contaminants into wastewater on psychrotrophs activity; (3) to
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evaluate the feasibility and stability of psychrotrophs application in a pilot-scale
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bioreactor system (open to climate change, long-term operation) for treating actual
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domestic wastewater at low temperature/seasonal varying temperatures.
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2. Materials and methods
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2.1 Isolation and identification of psychrotolerant strains
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Samples of activated sludge were collected from a municipal wastewater
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treatment plant (Harbin, China) in deep winter and normally cultivated at 4 °C for
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more than one year. Luria-Bertani (LB) liquid medium (0.5% NaCl, 0.5% yeast extract,
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and 1% tryptone) was used for the initial enrichment and the isolation of psychrotrophs.
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After purification by spread-plate technique and marking method, the screened strains
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were prepared for physiological and biochemical characteristics test and 16S rDNA
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analysis. And the purified psychrotrophs were acclimated and enriched at 4 °C after
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certain amount of domestic wastewater appended to the LB liquid media.
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Mesophilic activated sludge was obtained from the aeration tank of a mesophilic
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(37 °C), lab-scale bioreactor (Li et al., 2014) provided by School of Municipal and
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Environmental Engineering, Harbin Institute of Technology, China. Mesophiles were
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inoculated and cultured at 25 °C. The isolation method is operated the same as the case
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of psychrotrophs.
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2.2 Source and quality of domestic wastewater
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Actual domestic wastewater was utilized as feeding material in all experiments. 6
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chamber of sewer nearby the preceptorial apartment of Harbin Institute of Technology
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(China), and the general characteristics of the wastewater are as follows: COD of
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100–600 mg L-1, TN of 18.5–105.5 mg L-1, TP of 1.5–10 mg L-1, pH of 6.5–8.5.
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2.3 Experiments
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2.3.1 Immobilization on PUF
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Polyurethane foams (bulk density 50–60 kg m-3, porosity 90–98%) were
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purchased from local market in China. Before filling into the reactor, the PUF was
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washed with distilled water and then air-dried. Cubiform PUF particles (side length of
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4–6 mm) were added into the well-shaked mixture of polyvinyl alcohol solution (10%)
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and psychrotrophs (pre-cultured to the logarithmic growth phase). Then saturated boric
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acid was used for cross-linking. After fixation for 24 h, PUF particles were washed
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with normal saline. The immobilized PUF cubes were used in the experiments as
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packing.
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2.3.2 Batch tests
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Batch tests were carried out to investigate the performance of screened
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psychrotrophs (free or immobilized) on COD and para-chloronitrobenzene (pCNB)
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removal. The reactor (1 L) was located in a water-bath tank (with thermostatted water
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bath or water-ice bath) which worked automatically to keep the target temperature.
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Bacteria strains were cultured and acclimatized with sterilized domestic wastewater
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(sterilized through pasteurization in an autoclave, maintained at 121 °C for 15 min).
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Strains that pre-cultured to the logarithmic growth phase were centrifugalized and the
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precipitate (the weight of these precipitates was defined as wet cell weight in the paper)
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was used as seed biomass. The hydraulic retention time (HRT) was 10 h, and the
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dissolved oxygen (DO) was 3–4 mg L-1. Experiments of sharp temperature-changes were carried out in a simulated
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continuous-mode to evaluate the activity and stability of psychrotrophs and mesophiles.
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Prior to use, raw domestic wastewater was let stand for a while in order to remove
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floating particles and inorganic sediment. The influent (sterilized domestic wastewater)
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was introduced to the bottom of the reactor by a water distributor. The collected
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effluent containing suspended strains was centrifuged every 2 h, and the precipitate of
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centrifugation was returned to the reactor to maintain MLSS concentration of 3000±
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150 mg/L.
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Experiments of immobilized psychrotrophs on polyurethane foams (soft PUF and
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hard PUF) were performed in a simulated SBR-mode (cycle time: 24 h, reaction: 10 h)
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to determine the preferable carrier, the optimum loading amount of psychrotrophs on
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PUF, the optimal volume concentration of loaded PUF, and to investigate the microbial
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morphology on PUF during the operation at low temperature.
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2.3.3 Experimental set-up and operation
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An internal circulation aerobic bioreactor (ICABR) was designed to investigate
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the performance of the immobilized psychrotrophs for actual domestic wastewater
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treatment at different temperatures. As shown in Fig. 7a, the pilot-scale ICABR system
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comprised two main reactor tanks (each volume is 115 L). The ICABR system was
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operated in a daily continuous mode by feeding with an influent pump. The whole 8
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100 days and (2) the reactor was open to seasonal temperature-changes for 290 days.
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Prior to the experimental process, the reactor had been conducted at 4 °C for 15 days to
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reach a stable state. During the operation, the hydraulic retention time (HRT) was hold
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at 10 h, the dissolved oxygen (DO) was 3–4 mg L-1, and the volume concentration of
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immobilized PUF (loading 3.33 mg wet weight of psychrotrophs per mL of PUF) was
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30%. The ICABR performance was monitored by regularly determining COD of the
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influent and effluent. Duplicate PUF-biofilm samples were collected for microbial
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analysis at steady operation stage of three seasons at days 150, 300 and 380 (marked as
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S1, S2 and S3 respectively).
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2.3.4 Analytical methods
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Water samples were filtered through qualitative filter paper (intermediate speed),
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and stored at 4 °C before analysis. COD was measured by a 5B-1 type COD
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quick-analysis instrument (Lanzhou Environmental Technology Co., Ltd). pCNB was
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extracted from filtered samples (0.45µm) using hexane and then the hexane extracts
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were analyzed using a GC as described in reference (Xu et al., 2009).
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Carrier structures and the attached biofilm were observed by microscope (BX51,
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Olympus, Japan) and scanning electron microscope (SEM, S-520, Hitachi, Japan).
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Microorganism-immobilized PUF was fixed by 2.5% glutaraldehyde at 4 °C, dried and
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then sputtered before SEM analysis.
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Quantification of biofilm on the PUF was performed by determining the CFU/mL
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suspension of the immobilized bacteria. Pieces of PUF from the different period of 9
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described previously (Alessandrello et al, 2017). The CFU/ml of this suspension was
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determined by diluted plate-count method.
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2.4 DNA extraction and PCR amplifications
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Total genomic DNA was extracted in duplicate from screened psychrotrophs
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samples using DNA Isolation Kit for Soil (Watson Biotechnologies, Inc, China)
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following the procedure described in the manufacturer’s instructions. Primer pair 27F
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(5'-AGAGTTTGATCCTGGCTCAG-3') and 534R (5'-ATTACCGCGGCTGCTGGC-3')
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was used to amplify the hyper-variable V3–V4 region of the 16S rRNA gene. The PCR
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program were conducted as follows: 3 min at 95 oC for initial denaturation, 35 cycles
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of 30 s at 95 oC, 30 s at 55 oC, and 40 s at 72 oC, and ended with a final extension at 72
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2.5 Cell growth of psychrotolerant strains
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Optimum growth temperatures of the isolates were determined in shake flasks
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containing LB liquid media (with pre-inoculation of the isolates) at different
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temperatures (0–40 °C). The test was carried out in duplicate at each temperature.
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Glycerol (5%) was added to the medium to avoid freezing when the test was conducted
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at 0 oC. The optical density of each incubate at 585nm was monitored by a UV–visible
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spectrophotometer (T6, Beijing, China).
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The growth curve of each isolate was examined in a side-arm triangular flask
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containing LB medium. The flasks with incubate were shaken at 4 °C at 110 rpm, and
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OD585 were monitored every 2 h.
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2.6 TTC-Dehydrogenase activity 10
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evaluate their activity. 2 mL of Tris–HCl buffer solution (0.2 M, pH=8.0), 2 mL of
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TTC (0.5%) and 2 mL of glucose solution (0.1 M) were added to 2 mL of sludge
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sample; the mixture was shaken (140 rpm) at room temperature for 20 min and then
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incubated at required temperature (4 °C, 10 °C, 20 °C, 30 °C) for 12 h. 100 µL of
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concentrated sulfuric acid was added to stop the deoxidization reaction. With the
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addition of 5 mL of toluene as extraction agent, the sample was shaken for 10 min and
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kept still for 1 min. The reaction mixture was centrifuged at 4000 rpm for 10 min, and
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the supernatant was measured spectrophotometrically at 492nm. Each test was
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performed in duplicate. TTC-dehydrogenase activity (DHA) test for psychrotrophs and
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mesophiles has been performed in duplicate at each temperature. The enzyme activity
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was expressed as the TTC amount reduced (generating TF) per liter sludge sample per
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hour.
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3. Results and discussion
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3.1 Identification and biological characteristics of psychrotrophs
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The isolated psychrotrophs were identified by 16S rDNA gene sequence analysis
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as shown in Fig. 1, and assigned to genus Pseudomonas, Bacillus, Sphingobacterium,
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Shewanella, Serratia, and Arthrobacter. The cell growth tests (see details in Table S-1)
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showed that, all isolates had a wide growth temperature range, with optimal growth
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temperature between 20–30 oC. Broad ecological amplitude of the isolates guaranteed
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that they would not be sifted out during the system running at different temperatures.
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The growth curve of each isolate at 4 oC was examined as shown in Fig. S-1. Results
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h.
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Fig. 1. Phylogenetic tree of the screened psychrotrophs.
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As a valuable indicator to evaluate the microbial viability, dehydrogenase was
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largely reported on the important role in environmental remediation. (Weaver et al.,
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2012) The degradation of organic pollutants in bioreactor system was proved to be
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significantly affected by enzymatic activity (Seifert et al., 2001; Weaver et al., 2012;
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Zhou et al., 2010). TTC dehydrogenase activity of the screened psychrotrophs and
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mesophiles were observed at different temperatures as presented in Fig. 2. The
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dehydrogenase activity of both microbes showed an initial sharp increase from 4 oC to
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20 oC, followed by a gradual increase from 20 oC to 30 oC, indicating that low
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temperature greatly inhibited the dehydrogenase activity of the microorganisms.
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However, the screened psychrotrophs exhibited superior activity over the mesophiles at
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low temperature (below 10 oC). Generally, aerobic bioreactor with normal mesophiles
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ran badly when temperature falls below 10°C in winter, and water temperature should
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be controlled at 10–35 °C (optimum of 20–30 °C) so as to obtain favorable efficiency
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of biotreatment process (Li et al., 2011). The application of psychrotrophs in bioreactor
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would bring a breakthrough for wastewater treatment.
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Fig. 2. Dehydrogenase activity of the screened psychrotrophs and mesophiles.
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3.2 Biodegradation of COD by the screened psychrotrophs at different
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temperatures Strains of psychrotrophs and mesophiles were isolated, enriched and cultured at
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4 °C and 25 °C, respectively. Beaker experiments of COD removal were carried out in
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a simulated continuous-mode at alternate temperature of 25 °C and 4 °C, results are
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shown in Fig. 3.
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A short start-up period (at 25 °C) of 10h was recorded for both psychrotrophs and
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mesophiles, during which time COD removal efficiencies increased gradually up to
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76% and 85%, respectively. Stable COD removal by both strains was obtained during
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P1 which lasted 20 hours at an operational temperature of 25 °C. Ultimate difference
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of 9% in COD removal efficiency was recorded between mesophiles and
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psychrotrophs at 25 °C.
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The stability of COD removal efficiencies by psychrotrophs and mesophiles was
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affected by the decreases in temperature implemented throughout P2. The temperature
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drop from 25 °C to 4 °C brought a sharp decline of COD removal efficiency by
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mesophiles at the initial 6h of P2, resulting in only 8% COD removal and keeping at
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this level until the end of P2. Reduction on the biodegradation of COD (from 76% to
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58%) by psychrotrophs was also observed at the first 2 hours of P2. Although obvious
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decrease in COD removal was exhibited by both strains on the beginning of P2,
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psychrotrophs recovered quickly to 70% in 6 h, while mesophiles remained at low
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levels. Finally, the COD removal efficiency of psychrotrophs was 62% higher than that
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of mesophiles at 4 °C. With the operational temperature return back to 25 °C in P3, both strains began to
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resuscitate their abilities in COD degradation. Psychrotrophs recovered rapidly to the
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COD removal of 76% which was in accord with that in P1 (before temperature drop).
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A marked augmentation in performance of mesophiles was noted after the temperature
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was increased to 25 °C. COD removal efficiency of mesophiles kept increasing for the
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duration of P3, but was not recovered by the end of the trial, resulting in COD removal
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efficiency of 59%.
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The above results indicated that psychrotrophs exhibited high capability and
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stability at fluctuant temperature, and thus showed great potential for wastewater
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treatment in seasonal temperature variations.
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Fig. 3. COD removal of the screened psychrotrophs and mesophiles at continuous
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period of P1: 25 °C, P2: 4 °C and P3: 25 °C (influent COD: 118–413 mg L-1).
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3.3 Comparison of soft and hard polyurethane foams as carrier of psychrotrophs
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in COD removal
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In this experiment, two types of polyurethane foam (PUF), the hydrophilic soft
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PUF and the hydrophobic hard PUF, were served as the microbial supporter of the
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screened psychrotrophs, and the activity enhancement of the immobilized bacterial
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community was investigated.
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Vacant polyurethane foams have plentiful interstices and large specific surface 14
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scanning electron microscopy (SEM) micrographs (inset in Fig. 4a and Fig. 4b). From
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SEM images, the structure of the soft and hard PUF carriers appeared to be somewhat
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different. The hard PUF (inset in Fig. 4a) was tightly structured with compact and
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orderly pores, while the soft PUF (inset in Fig. 4b) was loosely constructed with a lot
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of opening or holes in its structure.
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From results of blank test in Fig. 4a and Fig. 4b, the physical adsorption of COD
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by the carrier media in the reactors was found to be limited at a stable level during the
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operational period. After 40 days’ operation, only 12% and 5% of COD was removed
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by the adsorption of soft and hard PUF carriers, respectively. The biomass of
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psychrotrophs is a critical factor for COD biodegradation in wastewater treatment. The
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effect of wet cell weight (in the range of 0.5–3 g) on COD removal using soft and hard
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PUF as the carriers at 4 °C was illustrated in Fig. 4a and Fig. 4b.
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As shown in Fig. 4a, the increase of biomass loading on hard PUF carriers
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promoted COD removal in the reactor. When the wet cell weight increased from 0.5 g
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to 3 g, the COD removal increased from 15% to 54% on 41st day of the operation.
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However, slight influence of wet cell weight carried by soft PUF was observed on
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COD removal (Fig. 4b). With the augment of wet cell weight (from 0.5 g to 2.5 g)
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immobilized on soft PUF carriers, COD removal efficiency first increased from 76% to
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80% (wet cell weight of 1 g) and then decreased to 71% on 44th day of the operation. It
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might be attributed to that large biomass has intensified the population competition and
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then declined the biological activity, resulting in the drop of COD removal efficiency.
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Comparing Fig. 4a and Fig. 4b, soft PUF was more suitable for further studies because
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it is more effective (probably due to its hydrophilic character and porous structure)
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than hard PUF as carriers for COD removal. The effect of volume ratio of soft PUF to total working volume on COD removal
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at 4 °C was investigated. The concentrations of COD were determined at suitable time
335
intervals for a period of 40 d. The time courses of COD removal efficiency during the
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40-d aerobic batch assay were shown in Fig. 4c. It was found that the COD removal
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rate fluctuated between 38% and 80% with carrier volume concentrations in the range
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of 10 to 40%. The maximum removal rate of 80% was observed at carrier volume
339
concentration of 30%. When the carrier concentrations increased from 30% to 40%,
340
slight decline of COD removal was observed, which can be assigned to that
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over-packed carrier media resulted in less sufficient fluidization leading to the poor
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transfer of nutrients and dissolved oxygen.
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Fig. 4. Effect of wet cell weight on COD removal with (a) hard and (b) soft PUF as
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carriers at 4 °C (carrier volume concentrations: 30%), and (c) effect of carrier volume
346
concentrations on COD removal with soft PUF as carriers at 4 °C (wet cell weight: 1g),
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influent COD: 106–517 mg L-1.
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3.4 Immobilization and morphology of the screened psychrotrophs on soft
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polyurethane foams for COD and pCNB removal
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The screened psychrotrophs were immobilized on soft polyurethane foams and 16
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preceding results, soft PUF with immobilized psychrotrophs (loading 3.33 mg wet
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weight of psychrotrophs per mL of PUF) was put into the beaker-reactor, and the
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carrier volume concentration was hold at 30%.
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The influent and effluent COD, as well as the calculated COD removal during the
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operation period of 60 day was shown in Fig. 5a. In the first 15 days (regarded as
358
start-up period), the sharp enhancement of COD removal from 30% to 70% was
359
observed. Changes in the influent COD did not evidently affect COD removal
360
efficiency in the reactor during the subsequent 45 days (regarded as steady-going
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period), and the COD removal efficiency tardily increased to 80% and remained in a
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steady level.
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With the stable COD removal efficiency in the steady-going period, the system
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was examined on the simultaneous removal of the emerging organic contaminant. The
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introduction of pCNB to reactor at a trace concentration of 500µg/L on day 36 did not
366
impact the COD removal efficiency (Fig. 5b). Although a lengthy acclimation period
367
(at 4 °C) of a month was observed for the reactor, pCNB removal efficiency exceeded
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90% by day 66 and remained at this level for the remainder days. Additionally, result
369
of adsorption experiments under the same conditions (initial pCNB concentration:
370
500µg/L, PUF concentration: 30% (v/v), temperature: 4 °C) show that the adsorbed
371
pCNB removal by soft PUF was less than 3%, which was considered to be negligible.
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Fig. 5. (a) COD and (b) pCNB removal of the immobilized psychrotrophs on soft
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polyurethane foams at 4 °C.
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Plentiful clear pores inside the fresh soft polyurethane foams can provide
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interspaces for microorganisms to grow and multiply. The morphological structure of
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the microbial population and its distribution on the surface of the soft PUF was
379
observed by SEM, and typical representative images at the start-up period (the 6th day)
380
and at the steady-going period (the 50th day) were presented in Fig. 6. At the beginning,
381
low density bacterial population comprised predominately of coccal and rod bacterium
382
was observed and the cells were sparsely distributed on the surface (Fig. 6a). After
383
acclimatization, the cell concentration and bacterial species were greatly enhanced,
384
resulting in thick and dense biofilm composed of diverse microbe on the surface of the
385
carrier (Fig. 6b). The large population of filamentous bacteria was distinctly visible in
386
Fig. 6b. Bacterial cells entrapped in the interior void spaces of the carriers were also
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observed.
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Fig. 6. SEM images of the microbe on the surface of soft polyurethane foams at (a)
390
start-up period and (b) steady-going period.
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3.5 Application and microbial stability of the screened psychrotrophs in
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pilot-scale ICABR for domestic wastewater treatment
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Fig. 7 shows the ICABR and its performance of wastewater treatment at 4 °C for
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100 days and at seasonal temperatures for 290 days. As shown in Fig. 7b, the ICABR 18
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wastewater throughout the whole operational period. During the first-stage operation,
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the ICABR system was operated at constant temperature of 4 °C. The influent COD of
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domestic wastewater fluctuated between 300–520 mg L-1, and the ICABR effluent
400
COD was maintained stable among 31–58 mg L-1, resulting in 83%–90% COD
401
removal; In the second-stage operation, the indoor temperature was no longer
402
artificially controlled as it in the first stage. Different wastewater temperature
403
transitions, from 4–10 °C in the winter to 20–28 °C in the summer and declined back
404
to 4–10 °C in the fall-winter, were recorded. For COD, 150–600 mg L-1 of influent was
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removed to 20–59 mg L-1 of ICABR effluent.
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Microbial community dynamic in ICABR operated at different seasons was
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investigated. With stable existence and activity throughout the whole operational
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period, Comamonas, Micrococcus, Arthrobacter, Bacillus and Pseudomonas were
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identified as the predominant bacteria family in ICABR. From log(sum of microbes) in
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Fig. 7, no obvious difference of microbial biomass was found when the system running
411
in winter (S1), summer (S2) and late autumn (S3). Besides, high temperature can bring
412
out some new microbes, such as Salmonella and Lactobacillus, which were detected
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and regarded as members of predominant bacteria family in ICBAR of
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summer-running.
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Throughout the entire operation of ICABR, COD removal efficiencies remained
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high (generally more than 80%) and stable. An average concentration of 40 ± 18 mg
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COD L-1 in effluent was observed, which was completely below the discharge limit of 19
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standard for pollutants discharge of municipal wastewater treatment plant in China).
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Fig. 7. (a) Schematic diagram and (b) performance and microbial ecology of ICABR
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with the immobilized psychrotrophs for COD removal during the operation at
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variational temperature.
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4. Conclusions
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The screened psychrotolerant strains in this study demonstrate the superiority in
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the removal of COD and a typical emerging contaminant from actual domestic
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wastewater at low temperature. The screened psychrotrophs, with TTC-dehydrogenase
429
activity ten times higher than that of mesophiles at 4 oC, are capable of sufficient
430
adaptation to the sudden temperature decrease from 25 to 4 oC. Soft polyurethane foam
431
was proved to be preferable in performance compared to the hard one, and the best
432
result was observed at concentration of 30% (v/v). The immobilized psychrotrophs on
433
soft polyurethane foam demonstrated efficient pCNB removal of up to 90%, and stable
434
COD removal of 80%, from domestic wastewater at 4 °C. Thick and dense biofilm
435
composed of diverse microbe was observed on the surface and in the interior void
436
spaces of soft polyurethane foam. A pilot-scale internal circulation aerobic bioreactor
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with the application of the screened psychrotrophs in treating actual domestic
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wastewater, operating at 4 °C for 100 days and at seasonally variational temperatures
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from 0 to 30 °C for 290 days, performed excellent and steady organic matter removal
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of 85% COD. No obvious difference of microbial biomass was observed when the
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system running at different seasons. Psychrotrophs performed stably as the
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predominant bacteria family in ICABR during the whole operation.
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Acknowledges
The authors would like to acknowledge the support given by the Natural Science
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Foundation of Shandong Province (ZR2014EEQ004), the National Natural Science
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Foundation of China (51408349, 51508353).
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26, 273–279.
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ACCEPTED MANUSCRIPT Pseudomonas veronii strain CIP 104663 Pseudomonas marginalis strain ICMP 3553 Pseudomonas antarctica strain CMS 35 H2 Pseudomonas sp. DSM 29165 100 Pseudomonas marincola strain KMM 3042 H1 79 100 Pseudomonas caeni strain HY-14 Shewanella profunda strain LT13a H5 100 100 Shewanella putrefaciens strain Hammer 95 68 Shewanella putrefaciens strain ATCC 8071 Serratia liquefaciens strain ATCC 27592 H6 100 Serratia proteamaculans strain DSM 4543 62 53 Serratia grimesii strain DSM 30063 H3 100 Bacillus idriensis strain SMC 4352-2 100 Bacillus pumilus strain ATCC 7061 98 Bacillus safensis strain NBRC 100820 99 H7 Arthrobacter psychrochitiniphilus strain GP3/JCM 13874/IARI-R-114 100 Arthrobacter psychrolactophilus JCM 12399 93 Arthrobacter psychrolactophilus strain B7 Sphingobacterium shayense strain HS39 Sphingobacterium nematocida strain M-SX103 100 H4 92 Sphingobacterium psychroaquaticum strain MOL-1 46
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Fig. 4. Effect of wet cell weight on COD removal with (a) hard and (b) soft PUF as carriers at 4 °C (carrier volume concentrations: 30%), and (c) effect of carrier volume concentrations on COD removal with soft PUF as carriers at 4 °C (wet cell weight: 1g), influent COD: 106–517 mg L-1.
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Fig. 6. SEM images of the microbe on the surface of soft polyurethane foams at (a)
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Research Highlights
Identified as Pseudomonas, Bacillus, Sphingobacterium, Shewanella, Serratia,
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Arthrobacter. Broad growth-range from 0 to 40 °C and powerful TTC-dehydrogenase activity at low temperature.
Immobilization on soft polyurethane foam led to better performance and stable
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Efficient and stable degradation of COD and pCNB in actual domestic wastewater at 4 oC.
Predominant and changeless family in a pilot-scale ICABR bioreactor at seasonal
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S0045-6535(17)31706-X
DOI:
10.1016/j.chemosphere.2017.10.121
Reference:
CHEM 20143
To appear in:
ECSN
Received Date: 26 December 2016 Revised Date:
11 October 2017
Accepted Date: 23 October 2017
Please cite this article as: Xu, Z., Ben, Y., Chen, Z., Jiang, A., Shen, J., Han, X., Application and microbial ecology of psychrotrophs in domestic wastewater treatment at low temperature, Chemosphere (2017), doi: 10.1016/j.chemosphere.2017.10.121. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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S1 S2 S3
Salmonella Lactobacillus
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Diverse shapes of psychrotrophs grow vigorously at low temperature
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Actual domestic wastewater is utilized as feeding material
Log(number of each microbe)
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Psychrotrophs eat COD, ECs and …
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Application and microbial ecology of psychrotrophs in domestic wastewater treatment at low temperature
2 3
5
Zhenzhen Xua, Yue Ben
6
Hanc
b,c,
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*, Zhonglin Chenc,1, Anxi Jiangc, Jimin Shenc, Xiaoyun
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a
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P R China
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College of Geography and Environment, Shandong Normal University, Jinan 250014,
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b
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Research Institute Co. LTD, Beijing 100032, P R China
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c
13
Technology, Harbin 150090, P R China
Water Works Department, State Nuclear Electric Power Planning Design and
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State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of
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A manuscript submitted to Chemosphere
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(December 25th, 2016)
20 * Corresponding author. Tel.: +86 010 58342558; Fax: +86 010 58342558. E-mail address: [email protected] (Z. Xu), [email protected] (Y. Ben), [email protected] (Z. Chen). 1
Co-corresponding author. 1
ACCEPTED MANUSCRIPT Abstract
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The feasibility of a bunch of screened psychrotrophs being applied to low-temperature
23
wastewater treatment was investigated. The screened psychrophillic strains are capable
24
of growth at a broad temperature-range from 0 to 40 °C and exhibit a preferable
25
TTC-dehydrogenase activity at low temperature (4~10 °C). Along the sharply fluctuant
26
temperatures (25 to 4 to 25 °C), the screened psychrotrophs (compared with the
27
indigenous mesophiles) demonstrate less fluctuations of COD removal and more rapid
28
recovery after temperature shocks. COD removal of approximate 80% was recorded by
29
single psychrotrophs (while only 10% by single mesophiles) at low temperature (4 °C).
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Soft polyurethane foam showed better performance for psychrotrophs immobilization,
31
with the optimal filling rate of 30% (v/v) in the bioreactor. The observation shows that
32
the immobilized psychrotrophs demonstrated a relatively high performance on both
33
conventional and emerging organic contaminants removals at low temperature. In
34
order to check the feasibility of the screened psychrotrophs in treating actual domestic
35
wastewater, a pilot-scale ICABR bioreactor was operated firstly at low temperature (4
36
o
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COD of 150–600 mg L-1 was efficiently reduced to 40 ± 18 mg L-1 under the
38
conditions of an overall hydraulic retention time of 10 h. Furthermore, psychrotrophs
39
performed stably as the predominant bacteria family during the whole operation. This
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study provides evidence that microbial intensification with psychrotrophs was a
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feasible strategy to improve the efficiency of conventional wastewater treatment
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process at low temperature.
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Keywords: psychrotrophs; low temperature; wastewater treatment; aerobic biocontact
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reactor; immobilization
45
1. Introduction Cold environment exists continuously in some regions of the Earth, such as polar
47
surface, worldwide deep oceans and some moderately cold areas at higher altitudes in
48
mountainous regions, and also appears seasonally in areas far away from the equator.
49
Psychrophilic and/or psychrotrophic bacteria, which were detected in these cold areas,
50
have been proved to play an important role in the environment-ecosystem (Rashid et
51
al., 1999). As two typical cold-adapted microorganism communities, psychrotrophs
52
and psychrophiles have frequently been isolated from these cold environments, and
53
found to grow normally at temperatures near freezing point. Psychrophiles are
54
specifically adapted to living in extremely cold environment, but cannot survive at
55
temperature above 20 °C (Morita, 1975). In comparison, psychrotrophs are notable for
56
having a particularly broad growth temperature range. With the upper limit as high as
57
40 °C, psychrotrophs grow optimally at temperatures around 20–25 °C (Berchet et al.,
58
2000; Margesin et al., 2002). Psychrotrophs have been reported on their considerable
59
biotechnological potential (Gianese et al., 2001; Cavicchioli et al., 2002; Margesin and
60
Feller, 2010; Russell, 2000) and well-demonstrated efficiencies in some environmental
61
bioremediation processes (Zekker et al., 2016; Gilbert et al., 2014; Gratia et al., 2009;
62
Siggins et al., 2011).
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Domestic wastewater, as one of the greatest sources of aquatic pollution, is related
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directly to water safety and public health issues. Since the occurrence of emerging 3
ACCEPTED MANUSCRIPT organic contaminants (EOCs) at different concentrations in municipal wastewater
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systems and natural water bodies (Jiang et al., 2013; Ternes et al., 2004; Matamoros et
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al., 2016) in recent years, new challenges are put forward on how to enhance the
68
efficiency of wastewater treatment technology, which is expected to treat both
69
traditional and emerging contaminants. For centuries, biological technology is chosen
70
as the most sustainable and optimal option for wastewater treatment. Temperature was
71
important for biological wastewater treatment on account of its effect on biochemical
72
reactions in many ways, such as reaction rates, reaction pathway, microorganism yields,
73
and death rates (ADM1, 2002). Wastewater temperature is apt to fall below 10 °C
74
(some even below 5 °C) in winter as a result of climate seasonal changes. Due to
75
seasonal
76
microorganisms are severely depressed, leading to a poor biotransformation of
77
contaminants in wastewater (Motta et al., 2003; Hai et al., 2011). Many ways had been
78
utilized to improve the efficiency of low temperature wastewater treatment: heat
79
preservation (Li et al., 2011), enlargement of returned sludge volume (Metcalf et al.,
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2003), reduction of sludge load rates (Tian et al., 2012), improvement of hydraulic
81
retention time (Viraraghavan & Varadarajan, 1996; Daija et al., 2016) or other
82
additional measures (Zekker et al., 2017), but all these ways would bring much higher
83
cost. Rich biomass is anticipated to guarantee remarkable efficiency of biological
84
systems. Immobilization technique has proved to be an effective way to make free cells
85
more concentrated, more active and further stable. Additionally, compared with free
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cell systems, the immobilized cell systems benefit the cells separation from water
regional
low
temperatures,
microbial
activity
of
indigenous
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organic solvents and microbial attack, polyurethane foam (PUF) has been widely used
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as a carrier in the immobilization of various microorganisms for wastewater treatment
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(Guimaraes et al., 2005; Lim et al., 2011; Zheng et al., 2009).
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The development of direct treatment systems operated at ambient temperatures
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(average temperature in Northeast China from 1961 to 2000, 5 °C; He et al., 2013),
93
especially in winter, without doubt will have a great ecological and economic impact.
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In this respect, bioaugmentation and inoculation using psychrotolerant bacteria appears
95
to be an appropriate way as psychrotrophs can thrive efficiently at low/moderate
96
temperatures. Psychrotolerant microorganisms had attracted increasing interest for
97
their capability and high efficiency for wastewater treatment at low temperature in
98
recent years (Connaughton et al., 2006; Gratia et al., 2009; Yao et al., 2013; Huang et
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al., 2015). However, researches on the long-term treatment of actual wastewater
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(containing emerging organic contaminants) at seasonally changeable temperature
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were rarely reported.
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In this study, a series of preponderant psychrotrophs with high activity at 4 °C
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were isolated and identified. The biological characteristics and microbial ecology of
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the screened psychrotrophs were investigated. The primary objectives of this study
105
were (1) to examine the resistance of screened psychrotrophs to sharp temperature
106
changes (25 to 4 to 25 °C) and the performance of immobilized psychrotrophs on PUF;
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(2) to investigate the efficiency of the screened psychrotrophs in removing both
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conventional (COD) and emerging (para-chloronitrobenzene, pCNB) organic
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emerging organic contaminants into wastewater on psychrotrophs activity; (3) to
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evaluate the feasibility and stability of psychrotrophs application in a pilot-scale
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bioreactor system (open to climate change, long-term operation) for treating actual
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domestic wastewater at low temperature/seasonal varying temperatures.
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2. Materials and methods
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2.1 Isolation and identification of psychrotolerant strains
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Samples of activated sludge were collected from a municipal wastewater
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treatment plant (Harbin, China) in deep winter and normally cultivated at 4 °C for
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more than one year. Luria-Bertani (LB) liquid medium (0.5% NaCl, 0.5% yeast extract,
119
and 1% tryptone) was used for the initial enrichment and the isolation of psychrotrophs.
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After purification by spread-plate technique and marking method, the screened strains
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were prepared for physiological and biochemical characteristics test and 16S rDNA
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analysis. And the purified psychrotrophs were acclimated and enriched at 4 °C after
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certain amount of domestic wastewater appended to the LB liquid media.
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Mesophilic activated sludge was obtained from the aeration tank of a mesophilic
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(37 °C), lab-scale bioreactor (Li et al., 2014) provided by School of Municipal and
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Environmental Engineering, Harbin Institute of Technology, China. Mesophiles were
127
inoculated and cultured at 25 °C. The isolation method is operated the same as the case
128
of psychrotrophs.
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2.2 Source and quality of domestic wastewater
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chamber of sewer nearby the preceptorial apartment of Harbin Institute of Technology
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(China), and the general characteristics of the wastewater are as follows: COD of
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100–600 mg L-1, TN of 18.5–105.5 mg L-1, TP of 1.5–10 mg L-1, pH of 6.5–8.5.
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2.3 Experiments
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2.3.1 Immobilization on PUF
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Polyurethane foams (bulk density 50–60 kg m-3, porosity 90–98%) were
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purchased from local market in China. Before filling into the reactor, the PUF was
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washed with distilled water and then air-dried. Cubiform PUF particles (side length of
140
4–6 mm) were added into the well-shaked mixture of polyvinyl alcohol solution (10%)
141
and psychrotrophs (pre-cultured to the logarithmic growth phase). Then saturated boric
142
acid was used for cross-linking. After fixation for 24 h, PUF particles were washed
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with normal saline. The immobilized PUF cubes were used in the experiments as
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packing.
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2.3.2 Batch tests
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Batch tests were carried out to investigate the performance of screened
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psychrotrophs (free or immobilized) on COD and para-chloronitrobenzene (pCNB)
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removal. The reactor (1 L) was located in a water-bath tank (with thermostatted water
149
bath or water-ice bath) which worked automatically to keep the target temperature.
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Bacteria strains were cultured and acclimatized with sterilized domestic wastewater
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(sterilized through pasteurization in an autoclave, maintained at 121 °C for 15 min).
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Strains that pre-cultured to the logarithmic growth phase were centrifugalized and the
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precipitate (the weight of these precipitates was defined as wet cell weight in the paper)
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was used as seed biomass. The hydraulic retention time (HRT) was 10 h, and the
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dissolved oxygen (DO) was 3–4 mg L-1. Experiments of sharp temperature-changes were carried out in a simulated
157
continuous-mode to evaluate the activity and stability of psychrotrophs and mesophiles.
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Prior to use, raw domestic wastewater was let stand for a while in order to remove
159
floating particles and inorganic sediment. The influent (sterilized domestic wastewater)
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was introduced to the bottom of the reactor by a water distributor. The collected
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effluent containing suspended strains was centrifuged every 2 h, and the precipitate of
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centrifugation was returned to the reactor to maintain MLSS concentration of 3000±
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150 mg/L.
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Experiments of immobilized psychrotrophs on polyurethane foams (soft PUF and
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hard PUF) were performed in a simulated SBR-mode (cycle time: 24 h, reaction: 10 h)
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to determine the preferable carrier, the optimum loading amount of psychrotrophs on
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PUF, the optimal volume concentration of loaded PUF, and to investigate the microbial
168
morphology on PUF during the operation at low temperature.
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2.3.3 Experimental set-up and operation
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the performance of the immobilized psychrotrophs for actual domestic wastewater
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treatment at different temperatures. As shown in Fig. 7a, the pilot-scale ICABR system
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comprised two main reactor tanks (each volume is 115 L). The ICABR system was
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operated in a daily continuous mode by feeding with an influent pump. The whole 8
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100 days and (2) the reactor was open to seasonal temperature-changes for 290 days.
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Prior to the experimental process, the reactor had been conducted at 4 °C for 15 days to
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reach a stable state. During the operation, the hydraulic retention time (HRT) was hold
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at 10 h, the dissolved oxygen (DO) was 3–4 mg L-1, and the volume concentration of
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immobilized PUF (loading 3.33 mg wet weight of psychrotrophs per mL of PUF) was
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30%. The ICABR performance was monitored by regularly determining COD of the
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influent and effluent. Duplicate PUF-biofilm samples were collected for microbial
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analysis at steady operation stage of three seasons at days 150, 300 and 380 (marked as
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S1, S2 and S3 respectively).
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2.3.4 Analytical methods
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Water samples were filtered through qualitative filter paper (intermediate speed),
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and stored at 4 °C before analysis. COD was measured by a 5B-1 type COD
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quick-analysis instrument (Lanzhou Environmental Technology Co., Ltd). pCNB was
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extracted from filtered samples (0.45µm) using hexane and then the hexane extracts
190
were analyzed using a GC as described in reference (Xu et al., 2009).
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Carrier structures and the attached biofilm were observed by microscope (BX51,
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Olympus, Japan) and scanning electron microscope (SEM, S-520, Hitachi, Japan).
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Microorganism-immobilized PUF was fixed by 2.5% glutaraldehyde at 4 °C, dried and
194
then sputtered before SEM analysis.
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Quantification of biofilm on the PUF was performed by determining the CFU/mL
196
suspension of the immobilized bacteria. Pieces of PUF from the different period of 9
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198
described previously (Alessandrello et al, 2017). The CFU/ml of this suspension was
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determined by diluted plate-count method.
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2.4 DNA extraction and PCR amplifications
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Total genomic DNA was extracted in duplicate from screened psychrotrophs
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samples using DNA Isolation Kit for Soil (Watson Biotechnologies, Inc, China)
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following the procedure described in the manufacturer’s instructions. Primer pair 27F
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(5'-AGAGTTTGATCCTGGCTCAG-3') and 534R (5'-ATTACCGCGGCTGCTGGC-3')
205
was used to amplify the hyper-variable V3–V4 region of the 16S rRNA gene. The PCR
206
program were conducted as follows: 3 min at 95 oC for initial denaturation, 35 cycles
207
of 30 s at 95 oC, 30 s at 55 oC, and 40 s at 72 oC, and ended with a final extension at 72
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2.5 Cell growth of psychrotolerant strains
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Optimum growth temperatures of the isolates were determined in shake flasks
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containing LB liquid media (with pre-inoculation of the isolates) at different
212
temperatures (0–40 °C). The test was carried out in duplicate at each temperature.
213
Glycerol (5%) was added to the medium to avoid freezing when the test was conducted
214
at 0 oC. The optical density of each incubate at 585nm was monitored by a UV–visible
215
spectrophotometer (T6, Beijing, China).
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containing LB medium. The flasks with incubate were shaken at 4 °C at 110 rpm, and
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OD585 were monitored every 2 h.
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2.6 TTC-Dehydrogenase activity 10
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evaluate their activity. 2 mL of Tris–HCl buffer solution (0.2 M, pH=8.0), 2 mL of
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TTC (0.5%) and 2 mL of glucose solution (0.1 M) were added to 2 mL of sludge
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sample; the mixture was shaken (140 rpm) at room temperature for 20 min and then
224
incubated at required temperature (4 °C, 10 °C, 20 °C, 30 °C) for 12 h. 100 µL of
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concentrated sulfuric acid was added to stop the deoxidization reaction. With the
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addition of 5 mL of toluene as extraction agent, the sample was shaken for 10 min and
227
kept still for 1 min. The reaction mixture was centrifuged at 4000 rpm for 10 min, and
228
the supernatant was measured spectrophotometrically at 492nm. Each test was
229
performed in duplicate. TTC-dehydrogenase activity (DHA) test for psychrotrophs and
230
mesophiles has been performed in duplicate at each temperature. The enzyme activity
231
was expressed as the TTC amount reduced (generating TF) per liter sludge sample per
232
hour.
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3. Results and discussion
234
3.1 Identification and biological characteristics of psychrotrophs
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The isolated psychrotrophs were identified by 16S rDNA gene sequence analysis
236
as shown in Fig. 1, and assigned to genus Pseudomonas, Bacillus, Sphingobacterium,
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Shewanella, Serratia, and Arthrobacter. The cell growth tests (see details in Table S-1)
238
showed that, all isolates had a wide growth temperature range, with optimal growth
239
temperature between 20–30 oC. Broad ecological amplitude of the isolates guaranteed
240
that they would not be sifted out during the system running at different temperatures.
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The growth curve of each isolate at 4 oC was examined as shown in Fig. S-1. Results
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243
h.
244
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Fig. 1. Phylogenetic tree of the screened psychrotrophs.
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As a valuable indicator to evaluate the microbial viability, dehydrogenase was
248
largely reported on the important role in environmental remediation. (Weaver et al.,
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2012) The degradation of organic pollutants in bioreactor system was proved to be
250
significantly affected by enzymatic activity (Seifert et al., 2001; Weaver et al., 2012;
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Zhou et al., 2010). TTC dehydrogenase activity of the screened psychrotrophs and
252
mesophiles were observed at different temperatures as presented in Fig. 2. The
253
dehydrogenase activity of both microbes showed an initial sharp increase from 4 oC to
254
20 oC, followed by a gradual increase from 20 oC to 30 oC, indicating that low
255
temperature greatly inhibited the dehydrogenase activity of the microorganisms.
256
However, the screened psychrotrophs exhibited superior activity over the mesophiles at
257
low temperature (below 10 oC). Generally, aerobic bioreactor with normal mesophiles
258
ran badly when temperature falls below 10°C in winter, and water temperature should
259
be controlled at 10–35 °C (optimum of 20–30 °C) so as to obtain favorable efficiency
260
of biotreatment process (Li et al., 2011). The application of psychrotrophs in bioreactor
261
would bring a breakthrough for wastewater treatment.
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Fig. 2. Dehydrogenase activity of the screened psychrotrophs and mesophiles.
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3.2 Biodegradation of COD by the screened psychrotrophs at different
266
temperatures Strains of psychrotrophs and mesophiles were isolated, enriched and cultured at
268
4 °C and 25 °C, respectively. Beaker experiments of COD removal were carried out in
269
a simulated continuous-mode at alternate temperature of 25 °C and 4 °C, results are
270
shown in Fig. 3.
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A short start-up period (at 25 °C) of 10h was recorded for both psychrotrophs and
272
mesophiles, during which time COD removal efficiencies increased gradually up to
273
76% and 85%, respectively. Stable COD removal by both strains was obtained during
274
P1 which lasted 20 hours at an operational temperature of 25 °C. Ultimate difference
275
of 9% in COD removal efficiency was recorded between mesophiles and
276
psychrotrophs at 25 °C.
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The stability of COD removal efficiencies by psychrotrophs and mesophiles was
278
affected by the decreases in temperature implemented throughout P2. The temperature
279
drop from 25 °C to 4 °C brought a sharp decline of COD removal efficiency by
280
mesophiles at the initial 6h of P2, resulting in only 8% COD removal and keeping at
281
this level until the end of P2. Reduction on the biodegradation of COD (from 76% to
282
58%) by psychrotrophs was also observed at the first 2 hours of P2. Although obvious
283
decrease in COD removal was exhibited by both strains on the beginning of P2,
284
psychrotrophs recovered quickly to 70% in 6 h, while mesophiles remained at low
285
levels. Finally, the COD removal efficiency of psychrotrophs was 62% higher than that
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of mesophiles at 4 °C. With the operational temperature return back to 25 °C in P3, both strains began to
288
resuscitate their abilities in COD degradation. Psychrotrophs recovered rapidly to the
289
COD removal of 76% which was in accord with that in P1 (before temperature drop).
290
A marked augmentation in performance of mesophiles was noted after the temperature
291
was increased to 25 °C. COD removal efficiency of mesophiles kept increasing for the
292
duration of P3, but was not recovered by the end of the trial, resulting in COD removal
293
efficiency of 59%.
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The above results indicated that psychrotrophs exhibited high capability and
295
stability at fluctuant temperature, and thus showed great potential for wastewater
296
treatment in seasonal temperature variations.
297
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Fig. 3. COD removal of the screened psychrotrophs and mesophiles at continuous
299
period of P1: 25 °C, P2: 4 °C and P3: 25 °C (influent COD: 118–413 mg L-1).
300
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3.3 Comparison of soft and hard polyurethane foams as carrier of psychrotrophs
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in COD removal
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In this experiment, two types of polyurethane foam (PUF), the hydrophilic soft
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PUF and the hydrophobic hard PUF, were served as the microbial supporter of the
305
screened psychrotrophs, and the activity enhancement of the immobilized bacterial
306
community was investigated.
307
Vacant polyurethane foams have plentiful interstices and large specific surface 14
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309
scanning electron microscopy (SEM) micrographs (inset in Fig. 4a and Fig. 4b). From
310
SEM images, the structure of the soft and hard PUF carriers appeared to be somewhat
311
different. The hard PUF (inset in Fig. 4a) was tightly structured with compact and
312
orderly pores, while the soft PUF (inset in Fig. 4b) was loosely constructed with a lot
313
of opening or holes in its structure.
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From results of blank test in Fig. 4a and Fig. 4b, the physical adsorption of COD
315
by the carrier media in the reactors was found to be limited at a stable level during the
316
operational period. After 40 days’ operation, only 12% and 5% of COD was removed
317
by the adsorption of soft and hard PUF carriers, respectively. The biomass of
318
psychrotrophs is a critical factor for COD biodegradation in wastewater treatment. The
319
effect of wet cell weight (in the range of 0.5–3 g) on COD removal using soft and hard
320
PUF as the carriers at 4 °C was illustrated in Fig. 4a and Fig. 4b.
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As shown in Fig. 4a, the increase of biomass loading on hard PUF carriers
322
promoted COD removal in the reactor. When the wet cell weight increased from 0.5 g
323
to 3 g, the COD removal increased from 15% to 54% on 41st day of the operation.
324
However, slight influence of wet cell weight carried by soft PUF was observed on
325
COD removal (Fig. 4b). With the augment of wet cell weight (from 0.5 g to 2.5 g)
326
immobilized on soft PUF carriers, COD removal efficiency first increased from 76% to
327
80% (wet cell weight of 1 g) and then decreased to 71% on 44th day of the operation. It
328
might be attributed to that large biomass has intensified the population competition and
329
then declined the biological activity, resulting in the drop of COD removal efficiency.
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Comparing Fig. 4a and Fig. 4b, soft PUF was more suitable for further studies because
331
it is more effective (probably due to its hydrophilic character and porous structure)
332
than hard PUF as carriers for COD removal. The effect of volume ratio of soft PUF to total working volume on COD removal
334
at 4 °C was investigated. The concentrations of COD were determined at suitable time
335
intervals for a period of 40 d. The time courses of COD removal efficiency during the
336
40-d aerobic batch assay were shown in Fig. 4c. It was found that the COD removal
337
rate fluctuated between 38% and 80% with carrier volume concentrations in the range
338
of 10 to 40%. The maximum removal rate of 80% was observed at carrier volume
339
concentration of 30%. When the carrier concentrations increased from 30% to 40%,
340
slight decline of COD removal was observed, which can be assigned to that
341
over-packed carrier media resulted in less sufficient fluidization leading to the poor
342
transfer of nutrients and dissolved oxygen.
343
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Fig. 4. Effect of wet cell weight on COD removal with (a) hard and (b) soft PUF as
345
carriers at 4 °C (carrier volume concentrations: 30%), and (c) effect of carrier volume
346
concentrations on COD removal with soft PUF as carriers at 4 °C (wet cell weight: 1g),
347
influent COD: 106–517 mg L-1.
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3.4 Immobilization and morphology of the screened psychrotrophs on soft
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polyurethane foams for COD and pCNB removal
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The screened psychrotrophs were immobilized on soft polyurethane foams and 16
ACCEPTED MANUSCRIPT applied for COD and pCNB removal in domestic wastewater at 4 °C. According to the
353
preceding results, soft PUF with immobilized psychrotrophs (loading 3.33 mg wet
354
weight of psychrotrophs per mL of PUF) was put into the beaker-reactor, and the
355
carrier volume concentration was hold at 30%.
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The influent and effluent COD, as well as the calculated COD removal during the
357
operation period of 60 day was shown in Fig. 5a. In the first 15 days (regarded as
358
start-up period), the sharp enhancement of COD removal from 30% to 70% was
359
observed. Changes in the influent COD did not evidently affect COD removal
360
efficiency in the reactor during the subsequent 45 days (regarded as steady-going
361
period), and the COD removal efficiency tardily increased to 80% and remained in a
362
steady level.
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With the stable COD removal efficiency in the steady-going period, the system
364
was examined on the simultaneous removal of the emerging organic contaminant. The
365
introduction of pCNB to reactor at a trace concentration of 500µg/L on day 36 did not
366
impact the COD removal efficiency (Fig. 5b). Although a lengthy acclimation period
367
(at 4 °C) of a month was observed for the reactor, pCNB removal efficiency exceeded
368
90% by day 66 and remained at this level for the remainder days. Additionally, result
369
of adsorption experiments under the same conditions (initial pCNB concentration:
370
500µg/L, PUF concentration: 30% (v/v), temperature: 4 °C) show that the adsorbed
371
pCNB removal by soft PUF was less than 3%, which was considered to be negligible.
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Fig. 5. (a) COD and (b) pCNB removal of the immobilized psychrotrophs on soft
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polyurethane foams at 4 °C.
375
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Plentiful clear pores inside the fresh soft polyurethane foams can provide
377
interspaces for microorganisms to grow and multiply. The morphological structure of
378
the microbial population and its distribution on the surface of the soft PUF was
379
observed by SEM, and typical representative images at the start-up period (the 6th day)
380
and at the steady-going period (the 50th day) were presented in Fig. 6. At the beginning,
381
low density bacterial population comprised predominately of coccal and rod bacterium
382
was observed and the cells were sparsely distributed on the surface (Fig. 6a). After
383
acclimatization, the cell concentration and bacterial species were greatly enhanced,
384
resulting in thick and dense biofilm composed of diverse microbe on the surface of the
385
carrier (Fig. 6b). The large population of filamentous bacteria was distinctly visible in
386
Fig. 6b. Bacterial cells entrapped in the interior void spaces of the carriers were also
387
observed.
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Fig. 6. SEM images of the microbe on the surface of soft polyurethane foams at (a)
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start-up period and (b) steady-going period.
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3.5 Application and microbial stability of the screened psychrotrophs in
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pilot-scale ICABR for domestic wastewater treatment
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Fig. 7 shows the ICABR and its performance of wastewater treatment at 4 °C for
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100 days and at seasonal temperatures for 290 days. As shown in Fig. 7b, the ICABR 18
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wastewater throughout the whole operational period. During the first-stage operation,
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the ICABR system was operated at constant temperature of 4 °C. The influent COD of
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domestic wastewater fluctuated between 300–520 mg L-1, and the ICABR effluent
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COD was maintained stable among 31–58 mg L-1, resulting in 83%–90% COD
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removal; In the second-stage operation, the indoor temperature was no longer
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artificially controlled as it in the first stage. Different wastewater temperature
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transitions, from 4–10 °C in the winter to 20–28 °C in the summer and declined back
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to 4–10 °C in the fall-winter, were recorded. For COD, 150–600 mg L-1 of influent was
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removed to 20–59 mg L-1 of ICABR effluent.
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Microbial community dynamic in ICABR operated at different seasons was
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investigated. With stable existence and activity throughout the whole operational
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period, Comamonas, Micrococcus, Arthrobacter, Bacillus and Pseudomonas were
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identified as the predominant bacteria family in ICABR. From log(sum of microbes) in
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Fig. 7, no obvious difference of microbial biomass was found when the system running
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in winter (S1), summer (S2) and late autumn (S3). Besides, high temperature can bring
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out some new microbes, such as Salmonella and Lactobacillus, which were detected
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and regarded as members of predominant bacteria family in ICBAR of
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summer-running.
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Throughout the entire operation of ICABR, COD removal efficiencies remained
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high (generally more than 80%) and stable. An average concentration of 40 ± 18 mg
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COD L-1 in effluent was observed, which was completely below the discharge limit of 19
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standard for pollutants discharge of municipal wastewater treatment plant in China).
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Fig. 7. (a) Schematic diagram and (b) performance and microbial ecology of ICABR
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with the immobilized psychrotrophs for COD removal during the operation at
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variational temperature.
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4. Conclusions
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The screened psychrotolerant strains in this study demonstrate the superiority in
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the removal of COD and a typical emerging contaminant from actual domestic
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wastewater at low temperature. The screened psychrotrophs, with TTC-dehydrogenase
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activity ten times higher than that of mesophiles at 4 oC, are capable of sufficient
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adaptation to the sudden temperature decrease from 25 to 4 oC. Soft polyurethane foam
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was proved to be preferable in performance compared to the hard one, and the best
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result was observed at concentration of 30% (v/v). The immobilized psychrotrophs on
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soft polyurethane foam demonstrated efficient pCNB removal of up to 90%, and stable
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COD removal of 80%, from domestic wastewater at 4 °C. Thick and dense biofilm
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composed of diverse microbe was observed on the surface and in the interior void
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spaces of soft polyurethane foam. A pilot-scale internal circulation aerobic bioreactor
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with the application of the screened psychrotrophs in treating actual domestic
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wastewater, operating at 4 °C for 100 days and at seasonally variational temperatures
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from 0 to 30 °C for 290 days, performed excellent and steady organic matter removal
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of 85% COD. No obvious difference of microbial biomass was observed when the
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system running at different seasons. Psychrotrophs performed stably as the
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predominant bacteria family in ICABR during the whole operation.
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Acknowledges
The authors would like to acknowledge the support given by the Natural Science
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Foundation of Shandong Province (ZR2014EEQ004), the National Natural Science
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Foundation of China (51408349, 51508353).
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ACCEPTED MANUSCRIPT Pseudomonas veronii strain CIP 104663 Pseudomonas marginalis strain ICMP 3553 Pseudomonas antarctica strain CMS 35 H2 Pseudomonas sp. DSM 29165 100 Pseudomonas marincola strain KMM 3042 H1 79 100 Pseudomonas caeni strain HY-14 Shewanella profunda strain LT13a H5 100 100 Shewanella putrefaciens strain Hammer 95 68 Shewanella putrefaciens strain ATCC 8071 Serratia liquefaciens strain ATCC 27592 H6 100 Serratia proteamaculans strain DSM 4543 62 53 Serratia grimesii strain DSM 30063 H3 100 Bacillus idriensis strain SMC 4352-2 100 Bacillus pumilus strain ATCC 7061 98 Bacillus safensis strain NBRC 100820 99 H7 Arthrobacter psychrochitiniphilus strain GP3/JCM 13874/IARI-R-114 100 Arthrobacter psychrolactophilus JCM 12399 93 Arthrobacter psychrolactophilus strain B7 Sphingobacterium shayense strain HS39 Sphingobacterium nematocida strain M-SX103 100 H4 92 Sphingobacterium psychroaquaticum strain MOL-1 46
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Fig. 4. Effect of wet cell weight on COD removal with (a) hard and (b) soft PUF as carriers at 4 °C (carrier volume concentrations: 30%), and (c) effect of carrier volume concentrations on COD removal with soft PUF as carriers at 4 °C (wet cell weight: 1g), influent COD: 106–517 mg L-1.
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Fig. 6. SEM images of the microbe on the surface of soft polyurethane foams at (a)
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Research Highlights
Identified as Pseudomonas, Bacillus, Sphingobacterium, Shewanella, Serratia,
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Arthrobacter. Broad growth-range from 0 to 40 °C and powerful TTC-dehydrogenase activity at low temperature.
Immobilization on soft polyurethane foam led to better performance and stable
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Efficient and stable degradation of COD and pCNB in actual domestic wastewater at 4 oC.
Predominant and changeless family in a pilot-scale ICABR bioreactor at seasonal
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microbe population.