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Biofilms benefiting plants exposed to ZnO and CuO NPs studied with a root-mimetic hollow fiber membrane Michelle Bonebrake, Kaitlyn C Anderson, Jonathan Valiente, Astrid Rosa Jacobson, Joan E. McLean, Anne J. Anderson, and David W. Britt J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02524 • Publication Date (Web): 19 Sep 2017 Downloaded from http://pubs.acs.org on September 20, 2017
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Journal of Agricultural and Food Chemistry
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Biofilms benefiting plants exposed to ZnO and CuO NPs
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studied with a root-mimetic hollow fiber membrane
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Michelle Bonebrake1, Kaitlyn Anderson1, Jonathan Valiente1, Astrid Jacobson2, Joan McLean3,
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Anne Anderson4, David Britt1*
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Affiliations: 1. Department of Biological Engineering, Utah State University, USA 84322; 2.
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Plants Soils Climate, Utah State University, USA 84322; 3. Utah Water Research Laboratory,
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Utah State University, USA 84322; 4. Department of Biology, Utah State University, USA
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84322
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*corresponding author: tel. 435 797 2158.
[email protected] 12 13 14
Key words: nanoparticles, rhizosphere, microbiome, extracellular polymeric material, root
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mimetic
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ABSTRACT
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Plants exist with a consortium of microbes that influence plant health, including responses to
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biotic and abiotic stress. While nanoparticle (NP) – plant interactions are increasingly studied,
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the effect of NPs on the plant microbiome is less researched. Here a root-mimetic hollow fiber
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membrane (HFM) is presented for generating biofilms of plant-associated microbes nurtured by
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artificial root exudates (AREs) to correlate exudate composition with biofilm formation and
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response to NPs. Two microbial isolates from field-grown wheat, a bacillus endophyte and a
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pseudomonad root surface colonizer, were examined on HFMs fed with AREs varying in N and
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C composition. Bacterial morphology and biofilm architecture were characterized using SEM
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and AFM, and responses to CuO and ZnO NP challenges of 300 mg / L evaluated. The bacillus
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isolate sparsely colonized the HFM. In contrast, the pseudomonad formed robust biofilms within
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3 d. Dependent on nutrient sources, the biofilm cells produced extensive extracellular polymeric
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substances (EPS) and large intracellular granules. Pseudomonad biofilms were minimally
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affected by ZnO NPs. CuO NPs, when introduced before biofilm maturation, strongly reduced
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biofilm formation. The findings demonstrate the utility of the HFM root-mimetic to study
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rhizoexudate influence on biofilms of root-colonizing microbes, but without active plant
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metabolism. The results will allow better understanding of how microbe-rhizoexudate-NP
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interactions affect microbial and plant health.
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Journal of Agricultural and Food Chemistry
INTRODUCTION
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Plants have co-evolved with soil microbes, forming a complex inter-kingdom relationship
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that helps each survive in the face of numerous abiotic and biotic stressors.1, 2. Endophytic
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microbes, initially existing within the seeds, may provide seedlings with a microbiome that
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becomes complimented by exogenous microbes from the environment. Microbial populations are
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higher in the root zone through the secretions of metabolites by root cells, the process of
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rhizoexudation, and the release of border cells from the root cap.3 The resulting boundary layer,
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the rhizosphere, is richer in sugars, amino acids, organic acids, and other secondary metabolites
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than the soil pore water. 4,5 Depending on plant species, developmental stage, and environmental
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conditions, plants may release as high as 40 % of the total carbon fixed through photosynthesis
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in root exudates.6,7 The actual input of C into the soil as exudates is uncertain, but this
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enrichment of the organic content of the rhizosphere establishes a microcosm with composition
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and functions that are influenced by the plant.2,8 Bacterial colonization of roots, through biofilm
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formation, is one of the consequences.9,10
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This is not, however, a one-way relationship. The plant microbiome influences plant
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health through a variety of mechanisms that provide protection against biotic and abiotic stress.11,
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(PcO6), produce antimicrobials that aid in protection against pathogens.13 Other metabolites
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modulate plant functions, including closure of stomates by the volatile butanediol, a process
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contributing to drought protection.14 Also, iron in the rhizosphere is routed to the bacteria and
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plant, but can be restricted from pathogenic microbes, through microbial secretion of highly
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effective, metal-chelating siderophores.15,16 This feedback loop between bacteria and plant is
Beneficial microbes, such as the bacterial root colonizer, Pseudomonas chlororaphis O6
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mutually beneficial. Thus, the multitude of external factors that directly influence one will, likely
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through indirect pathways, influence the other.
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It is becoming increasingly recognized that maintaining, and even augmenting, the plant
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microbiome will be necessary for sustainable agricultural output to keep pace with growing
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demand.17,18 To further optimize agricultural output, next generation fertilizers and pesticides are
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being explored, with targeted and timed delivery through nanoformulations.19 Some of the
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fertilizer applications may supply the nutrients directly, whereas others may be indirect. For
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instance, applications of TiO2 or Fe3O4 NPs may enhance P supply through acidification of the
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rhizosphere caused by NP challenge.20 Another mechanism associated with ZnO NP treatments
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that increase plant P involves stimulation of phosphatase activity in microbes through Zn release
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from dissolution of the NPs, the resulting Zn phosphate acting as a source of P.21 As these
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formulations are developed, model systems for studying rhizosphere processes are required to
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assess the interplay between the root, beneficial microbes, and external agents such as NPs as
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illustrated in Figure 1.
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Hydroponic growth of plants affords a detailed focus on the NP-plant interactions,
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revealing the uptake and translocation of NPs; for instance, CuO NPs travel through roots, into
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foliar tissues, and return to the roots.22 Thus, hydroponic systems provide key insights in terms of
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NP fate and translocation and plant cell responses under model conditions. Studies of NP
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influence on root-colonizing microbes also often take a similar reductionist approach,
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investigating NP-microbe interactions with planktonic microbes.23,24 Findings relevant to toxicity
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of distinct classes of engineered NPs are gained. However, bacteria associated with the root
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surface exist not only as planktonic cells, but are embedded in a matrix termed the biofilm.
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Biofilm growth confers resistance to many environmental challenges such as antibiotics.25 It also
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provides the microbes direct access to the rhizoexudates and, in return, bacterial metabolites are
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concentrated in the root zone, intensifying their influence on the plant or other rhizosphere
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organisms. Thus, studies under conditions of hydroponic plant growth or with planktonic cells do
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not duplicate the environment of the rhizosphere as established in agricultural soils.
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To study the influence of root exudates on soil microbial communities, root-models have
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been developed to focus on the biofilm lifestyle of rhizosphere microbes. Root-colonizing
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bacteria such as PcO6 form biofilms on synthetic surfaces, including polystyrene well plates in
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several media.26 In contrast, a plant-beneficial Burkholderia isolate from sugar cane only forms
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biofilms in well plates containing growth medium amended with metabolites prepared from
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macerated sugarcane roots.27 An in-situ platform functioning as a root-mimetic was developed
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by Ziegler et al. (2013), in which thin agar films containing artificial root exudates (AREs) are
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deposited on glass slides, that when placed in soil allow bacteria that metabolize the AREs to be
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detected.28 A continuous ARE-delivery artificial root was developed in which a rolled filter
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paper wick delivers nutrients into soil samples to assess changes in soil microbial composition as
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a function of carbon loading and distance from the wick. This method has also been used to
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augment the rhizosphere with AREs to assess the role of specific metabolites, such as 8-
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hydroxyquinoline, a potential allelochemical from knapweed, on microbial community
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composition in native soil.29 The filter paper wicks are conduits to deliver AREs into the soil,
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and as such, are not actual root-mimetics for assessing biofilm formation and response to ARE
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changes and/or external challenges.
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Here, we employ hollow fiber membranes (HFM) as root-mimetics for assessing biofilm
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growth as a function of ARE composition and NP challenges. NP interactions with root-
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colonizing microbes on living roots are complex as the response of the root to the NP challenge
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(whether directly, or through NP-induced alteration in rhizosphere metabolite production)
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convolutes the situation as depicted in Figure 1. Surface-sterilization of seeds and planting into
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sterilized growth matrix such as sand are employed to study the NP-plant interactions without
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bacterial contributions. However, to study the root-colonizing microbe as a biofilm, without
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influence from active plant metabolism, a model system such as a hemodialysis hollow fiber
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membrane (HFM), can be used to deliver isolated root exudates, or ARE, either through the
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lumen, or externally. HFMs, developed for dialysis and ultrafiltration, can be applied as bio-inert
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surfaces for culturing biofilms. Microbial trapping within HFMs has been used to stimulate
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growth of “unculturable” bacteria by reintroduction into soils, where the soil pore waters
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carrying the nutrients flow across the fiber walls stimulate growth.30 This system can be placed
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in the rhizosphere where it functions as a permeable-selective cell encapsulation platform for cell
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culture as an in-situ HFM bioreactor.
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The high-flux PS/PVP HFM fiber is evaluated here as root-mimetic where microbes
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colonize the outer surface, allowing for facile analysis of soil microbe biofilm formation as a
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function of ARE composition and in response to external challenges of engineered NPs. We have
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previously established that P. chlororaphis O6 (PcO6) colonizes the root surfaces of seedlings at
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densities that are not impaired by growth in sand amended with CuO or ZnO NPs31,32 and
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attribute their survival to the formation of the protectant biofilms on the root surface. Isolates of
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Bacillus subtilis are strong biofilm formers and also have biocontrol activity for the plant.33 The
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B. subtilis endophyte used in these studies, designated as Bs309, was from surface-sterilized
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commercial wheat seeds. Distinct biofilm forming abilities of the two microbes on the HFMs are
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demonstrated and correlations between ARE composition, biofilm architecture, and cell
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morphologies are observed. Challenges of CuO or ZnO NPs delivered at different time points
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reveal differential impacts on bacterial cell morphology and biofilm formation.
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MATERIALS AND METHODS
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Hollow Fiber Membranes. HFMs of PS/PVP, PS, and cellulose diacetate (CD) were
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obtained from Fresenius Medical Care North America, Ogden, UT. Bleach-treated PS/PVP
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HFMs were prepared in 0.57 % sodium hypochlorite at 70 °C for 2 min and rinsed 3x in double
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distilled water. The bleach-treatment is a facile means to reduce the PVP content of the fiber,
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creating a membrane whose properties can be tuned between those of PS/PVP and pure PS.34
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The HFMs have lumen diameters < 200 µm and wall thickness < 50 µm, and molecular weight
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(MW) cutoff < 60 kD.
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Artificial Root Exudate (ARE) Formulations. All of the chemicals used were of
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analytical grade and were obtained from either Sigma-Aldrich or Fisher Scientific. Five different
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media recipes were utilized as AREs to deliver nutrients to the bacteria: Luria Broth, LB, (ARE
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A); minimal medium (ARE B); ARE B with glycine, ARE B(+gly); ARE B with additional
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citrate, ARE B(+cit); and ARE B with glycine and additional citrate ARE B(+gly,+cit). Specific
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ARE recipes can be found in the supporting information as Table S1. ARE A and ARE B both
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support PcO6 biofilm formation.26
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Nanoparticle Source. Commercially available CuO (nominal particle size
