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Differential Sensitivity of Wetland-Derived Nitrogen Cycling Microorganisms to Copper Nanoparticles Vincent C. Reyes, Phillip B Gedalanga, Nancy Merino, Joy D Van Nostrand, Scott P. Keely, Susan K. De Long, Jizhong Zhou, and Shaily Mahendra ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b01868 • Publication Date (Web): 07 Aug 2018 Downloaded from http://pubs.acs.org on August 15, 2018
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ACS Sustainable Chemistry & Engineering
Differential Sensitivity of Wetland-Derived Nitrogen Cycling Microorganisms to Copper Nanoparticles Vincent C. Reyes†, Phillip B. Gedalanga†$, Nancy Merino†¥, Joy D. Van Nostrand‡, Scott P. Keely§, Susan K. De Long⊥, Jizhong Zhou‡ ⊤,+, and Shaily Mahendra†∞#* †
Department of Civil and Environmental Engineering, University of California, Los Angeles, CA 90095;
$
Department of Health Science, California State University, Fullerton, CA 92834;
¥
Earth-Life Science Institute, Tokyo Institute of Technology, Ookayama, Meguro-ku, Tokyo, Japan;
‡
Institute for Environmental Genomics and Department of Microbiology and Plant Biology, University of
Oklahoma, Norman, OK 73072; §
National Exposure Research Laboratory, US Environmental Protection Agency, Cincinnati, OH 45268;
⊥Department
of Civil and Environmental Engineering, Colorado State University, Fort Collins, CO,
80523; ⊤Earth +
Science Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720;
State Key Joint Laboratory of Environment Simulation and Pollution Control, School of Environment,
Tsinghua University, Beijing 100084, China; #
Institute of the Environment and Sustainability, University of California, Los Angeles, CA 900095;
∞
California NanoSystems Institute, University of California, Los Angeles, CA 900095;
Running Head: Nanoparticles Alter Microbial Communities in Wetlands Address correspondence to
[email protected] ACS Paragon Plus Environment
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ABSTRACT
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Metallic nanoparticles (NPs), the most abundant nanomaterials in consumer and industrial
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products, are the most probable class to enter and potentially affect the environment. In this
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study, wetland-derived microcosms were incubated with copper nanoparticles (Cu-NP) and ionic
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CuCl2 to investigate acute (10 days) and chronic (100 days) exposures to nitrogen cycling
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microorganisms. Gene abundance and expression changes were monitored using the GeoChip
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5.0 high throughput functional gene microarray and metatranscriptomic sequencing (RNA-seq),
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respectively. After 10 days, the Cu-NP impacted microbial communities experienced structural
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shifts within microorganisms associated with dissimilatory nitrogen reduction accompanied by
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lower nitrate removal as compared to the unexposed controls. By day 100, these differences were
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largely resolved and nitrate removal was similar to the unexposed control. Furthermore, the Cu-
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NP exposed microcosms tolerated copper and were more resilient and adaptive than the
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unexposed controls based on the abundance of copper oxidase (cueO), copper efflux (cusC), and
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bacterial adaptive response (opuE, soxS, desR, baeS) genes. These findings suggest that sudden
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influxes of Cu-NPs into wetland systems may impair nitrogen removal initially, but long-term
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microbial shifts and functional redundancy would promote the net flux of total nitrogen out of
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the wetlands.
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Keywords: nanoscale, lagoon, sewage, anaerobic, microbiome, stress response
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INTRODUCTION
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Copper nanoparticles (Cu-NPs) and other metallic nanoparticles (NPs) are the most utilized engineered
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nanoparticles in consumer and industrial products.1 Due to their growing applications in anti-microbial
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coatings, electronics, textiles, cosmetics, wood additives, and ceramics, NPs will enter the environment
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through their intended uses and subsequent waste disposal.2 NPs influence the microbial composition in
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natural environments, implying that NPs have the potential to alter microbially driven processes like
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carbon, phosphorous, sulfur, and nitrogen cycling.3-5
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Managing the nitrogen cycle is recognized by the National Academy of Engineering as one of the Grand
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Challenges of the 21st century due to the staggering nitrogen cycle imbalances caused by human activity.6-
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denitrifying microbial communities, while fertility of agricultural soils is controlled by nitrogen-fixing
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bacteria. Cu-NPs have the potential to exacerbate existing nitrogen cycle imbalances by differentially
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impacting microbial populations involved in these processes. For example, greater inhibition of
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denitrifying microorganisms compared to nitrifying microorganisms could lead to elevated levels of
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nitrate in surface waters. Previous research has shown that denitrifying microorganisms in wastewater-
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derived sludge were more sensitive (IC50 0.95 mg/L) than nitrifying microorganisms (IC50 26.5 mg/L) to
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copper salts.8 Cu-NPs may have greater impacts than copper salts due to inherently higher catalytic
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activity resulting from increased surface to volume ratios.9-10 Denitrifying bacteria impacted by Cu-NP
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may contribute to climate change given that nitrous oxide, a potent greenhouse gas, is an intermediate of
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denitrification.11 Thus, branches of the nitrogen cycle that are sensitive to NPs need to be identified to
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facilitate proactive management strategies.
Nitrogen transformations in wetlands and in wastewater treatment plants are shaped by nitrifying and
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Previous research into NP impacts on nitrogen cycling microorganisms has mainly focused on pure
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cultures, while NP impacts on environmental microbial communities have yet to be adequately
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explored.12-16 Within mixed microbial communities, microorganisms function differently because of
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synergistic growth, functional redundancy, exchange of nutrients, and fortuitous novel gene variants.17
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Further, the few studies examining NP interactions with mixed microbial communities have focused on
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engineered wastewater systems and wastewater-derived microcosms.18-20 Thus, limited information exists
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regarding the influence of NPs on the health of other important ecosystems. One study has assessed the
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effects of Ag- and Cu-NPs on freshwater wetland mesocosms, providing insight into the microbial
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community structure and composition over time.21 However, NP impacts on the abundance and activity of
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specific microbial populations were not examined for wetlands.21
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Wetlands play an essential role in balancing nitrogen. In rural and less developed areas, natural or
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constructed wetlands act as cost-effective alternatives to advanced wastewater treatment systems by
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removing excess nitrogen.22-23 Biological nitrification and denitrification rates in wetland sediments have
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been recorded as high as 56.1 and 21.6 mg-N·kg-1 h-1, respectively.24 Further, wetlands play an important
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role in atmospheric nitrogen fixation. Estimates of nitrogen fixation rates for wetlands in the Florida
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Everglades are greater than 100 mg-N·m-2 d-1.25 Therefore, understanding how NPs may affect the growth
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and activity of specific wetland nitrogen cycling microorganisms is critical to understanding NP impacts
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on the local, as well as the global, nitrogen cycle.
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High-resolution analysis of microbial ecosystems, their community structures, and functional activities
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has been facilitated by advanced molecular techniques including functional gene arrays and next-
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generation sequencing. These platforms have significant advantages over traditional culture-based
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strategies because of their ability to genetically probe non-culturable microorganisms. The GeoChip
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functional gene microarray allows for simultaneous and repeatable analysis of environmentally relevant
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processes, such as carbon, phosphorous, sulfur, and nitrogen cycling.26-28 Metatranscriptomic sequencing
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offers the advantage of identifying expressed genes, providing a more direct predictor of metabolic
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activity.29 Applications of metatranscriptomic sequencing to environmentally relevant systems remain
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limited. However, this approach has been used to elucidate the metabolic and biogeochemical responses
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of marine microorganisms to day/night cycles30 and to investigate adaptation mechanisms and microbial
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stress responses in acid mine drainage.31
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This study describes the sensitivities to Cu-NPs among nitrogen cycling microorganisms derived from a
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wetland ecosystem. A primary objective included determining the most sensitive nitrogen cycling
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processes to Cu-NP stress. Further, changes in microbial community composition as well as functional
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activities of nitrogen cycling communities at the gene expression level were assessed after 10 and 100
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days of exposure to differentiate acute and chronic effects because previous studies report that NP toxicity
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changes over time.32-33
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EXPERIMENTAL
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Nanoparticles
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Copper nanoparticles (99.9 % pure) of 50 nm nominal size were obtained from M. K. Impex Corp
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(Mississauga, ON, Canada). CuCl2 (>99% purity) salt was used in parallel experiments to assess the
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contribution of Cu2+ ions to observed toxicity. Prior to microcosm amendments, fresh copper stock
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suspensions (1000 mg/L as Cu) were prepared by mixing Cu-NPs into deionized water, followed by
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sonication in an ultrasonic bath for 30 min at maximum power (FS30H, Fisher Scientific, 100 W, 42
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kHz). All stocks were sonicated for 1 min before use and diluted to their final concentration within 3 hr of
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preparation.
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The stability of Cu-NPs and CuCl2 was characterized in the exposure medium, which was a 1:1 blend of
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basal salt growth solution and environmental water derived from a slurry mix collected from the Malibu
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lagoon (see supporting information for more).34 Hydrodynamic diameters of the particles were measured
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and surface zeta-potentials were computed to assess colloid stability in the medium (see supporting
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information).
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Microcosm Conditions
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Microcosm seed water and sediment were obtained from the Malibu Lagoon (34.0333° N, 118.6792 °W;
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Malibu Lagoon State Beach in Malibu, CA). The Malibu Lagoon and the accompanying beach have a
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history of elevated nutrient levels and fecal indicator bacteria.35 As a result, indigenous microbial
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populations may have had previous exposure and resistance to metals and other antimicrobials from
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anthropogenic inputs. Samples were collected in August 2013 prior to sunrise to reduce UV induced
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mortality of endemic organisms. Water and sediment samples were extracted from the surface water
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column down to 0.3 m into the sediment. Water and sediment samples were placed immediately on ice
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before transport to the laboratory, where they were stored at 4°C, and used within 6 hr. Samples contained
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less than 2 mg/L of nitrogen as ammonium, nitrate, or nitrite which was near the limit of detection for the
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ammonium assay (Hach, Loveland, CO, USA).
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Microorganisms within the wetland slurry samples were enriched for a period of 10 days prior to the start
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of the microcosm study. This enrichment period was implemented to allow for growth of mesophilic
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microorganisms. For each microcosm, 25 mL of slurry was mixed with 25 ml of a basal salt solution (see
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supporting information for details) in butyl-rubber stopper sealed sterile 100-mL serum bottles.36
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Microcosms were flushed with N2 gas to promote anaerobic conditions and incubations were carried out
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in a stationary incubator for 10 days at 30°C in the dark to prevent photosynthesis from occurring in the
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microcosms prior to introduction of either Cu-NPs or CuCl2. Incubation at 30°C was chosen to maintain
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selection of mesophilic microorganisms because many of these bacteria are important contributors to
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nutrient removal processes in wetlands.37-39 Samples were collected after the 10th day of enrichment and
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analyzed for ammonium, nitrite, and nitrate concentrations resulting from the basal salt solution and any
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potential microbial transformations occurring in this period.
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Triplicate microcosms exposed to Cu-NPs or CuCl2 (100 mg·L-1 as Cu) were established for 0-, 10-, and
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100-day incubation periods to study acute (10 days) and chronic (100 days) effects of Cu-NPs and CuCl2.
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Every 10 days, microcosms were flushed with N2 to promote anaerobic conditions and amended with
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sucrose (100 mg·L-1) and nitrate (350 mg·L-1) to replenish carbon and nitrogen, respectively. These
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amendments changed microcosm volumes by
