Fishing bacteria with a nanonet

*This image is a runner up in the Materials Today cover competition.

There has been an increasing interest in recent years on improving crop productivity to fulfil the food needs of an ever-increasing world population. The damage and risk to the environment (croplands, forests and water sources) and human health through impacts on agricultural practices (tillage, excessive fertilisation and pesticide treatments, and deforestation) to satisfy these needs, however, have enhanced the belief that such practices should be urgently replaced by more environmentally friendly processes and compounds [1].

In recent decades, conservation agriculture practices like the Integrated Crop and Pest Management (ICM & IPM) and organic farming have been aimed at preserving natural resources (soil, water, fuels) and limiting the use of agrochemicals [2]. However, the short-term reduction of crop yields, the low effectiveness and high cost of commercially available low-impact fertilisers (Slow-Release Fertilisers - SRF) and the time consuming research necessary to develop effective biopesticides (pesticides employing organisms and their products) have so far limited the application of these practices by farmers.
New strategies and tools are thus necessary to fill the gap between intensive and conservation agriculture.

Nanomaterials have recently been proposed in a multitude of contexts as groundbreaking materials to deal with a broad range of problems and surpass the limits posed by traditional approaches [3]. In agriculture, novel and groundbreaking tools have been developed employing nanomaterials to deliver agrochemicals to plants for both improving nutrition (nanofertilisers) and protecting plants (nanopesticides), but reducing the impact of these compounds on the environment and human health by reducing the global amount provided and improving the efficiency of their actions [4].

Similar results can be obtained, however, following a more ‘green’ and sustainable approach based on microorganisms. Microbes preferentially live in complex structures, adhering to surfaces, that are termed biofilms [5]. Such a particular lifestyle is extremely advantageous for these organisms to resist to harsh environmental conditions, toxic substances (pollutants), biocidal agents (antimicrobials, immune systems) and predators [6,7]. Such resistance, however, poses serious problems in contexts where the presence of harmful and detrimental microorganisms can cause damage (infection and disease, corrosion and fouling of metal substrates) [6]. Conversely, the same properties can be extremely advantageous to generate tools for specific applications, where the resistance of biofilms to adverse conditions can be a prerequisite for the success of the tool [8].

In the portion of soil surrounding plant roots, named rhizosphere, microorganisms mostly live in biofilms attached to both soil particles and root surfaces. In the rhizosphere, microbes actively and mutually interact with plants through their roots, mainly supporting their growth (beneficial effects), but also competing with them for nutrients and even causing diseases (pathogens) (negative effects). Benefits include nutrient supply and reduced damages from toxic substances, pollutants, pathogens (biopesticides and Induced Systemic Resistance – ISR) and environmental fluctuations (water stress and pH) [9].

We have proposed employing microbial biofilms grown onto nanomaterials. Some nanomaterials have been proven to support and stimulate cell growth, as demonstrated in nanomedicine for tissue engineering and in the creation of microbial fuel cells [10,11]: but to our knowledge, however, the latter is the only application where both microbial biofilms and nanomaterials are coupled to improving process performance together. Another possible application where such bio-nano-frameworks could provide extremely useful advancements is agriculture.

Recently we developed an electrospun nanofibrous scaffold as the support for microbial cell growth. Specifically, we grew a bacterial species with the ability to colonise the rhizosphere of several crop plants (e.g. maize, wheat, rice, oat, coffee) on electrospun 3D polycaprolactone nanofibres [12]. Since electrospinning conditions (polymer solution, potential, rate of deposition, collecting tool, rotation speed and distance from the spinneret) strongly affect the morphology of the materials deposited [13], several trials were performed aimed at generating an artificial 3D scaffold mimicking the micromorphology of the soil structure, i.e. resembling the features of surface distribution in soils. Microbial inoculum composition and environmental conditions for microbial growth were set according to those suitable for biofilm formation and development in conventional microbial media. The electrospun nanoscaffold was capable of providing a suitable surface for bacterial attachment, colonisation of the framework and development of a proper biofilm, and finally, of preserving its viability and vitality for months.

The Materials Today winning image* shows a Scanning Electron Microscopy (SEM) micrograph (JEOL JSM 6010LA) of the biofilmed nanoscaffold captured using the following steps: (1) pre-fixation in cold conditions in the presence of buffered glutaraldehyde at neutral pH 7.3 and ruthenium red; (2) fixation in similar conditions, after extensive washing, without ruthenium red; (3) post-fixation with cold buffered osmium tetroxide; (4) ethanol dehydration according to the critical point method, using CO2 in a Balzers Union CPD 020; 5) sputter-coating with gold in a Balzers MED 010 unit, after attachment to aluminium stubs. Steps 2 to 4 were performed after extensive washings in the buffered solution.
Specifically, the SEM micrograph shows the soil-like nanoscaffold, with beads roughly resembling the morphology of soil mineral particles and the electrospun fibrous network mimicking the 2D and 3D fibrous polymeric structure of the soil organic matter (SOM). The picture illustrates a crucial moment in the development of biofilms.

The deposition of an additional polymeric organic material (the so-called “conditioning film” generated by bacteria in the very initial step of biofilm formation to facilitate their attachment to surfaces) is visible as a rough coating on the beads and nanofibres (here appearing much thicker than soon after electrospinning deposition, fused together and mostly laid on the beads). Notwithstanding the humorous title of this article, bacteria are not truly fished by the nanonet structure (passive action), but they actively do demonstrate intracellular and extracellular actions to specifically adhere to surfaces. Bacteria cells appear at this stage as both isolated and in small colonies.

Bacteria in colonies here appear already embedded in a matrix of polymeric organic material, mainly made of polysaccharides (EPS = exopolysaccharides), which they secreted to protect them from harsh environmental conditions (e.g. pH, moisture, temperature, toxicants and pollutants). Interestingly, bacteria show a preferential attachment to fibres, both as single fibres and in networks. Upon the bacterial strain and the nanostructured framework employed, the biofilmed nanotools here developed exhibited plant promoting activities over both the short- and long-term and are demonstrated to be promising tools for creating artificial rhizosphere and improving crop yields.

Further reading

[1] H. Kirchmann, G. Thorvaldsson
Eur. J. Agron., 12 (2000), pp. 145-161

[2] National Research Council (U.S.)
Toward Sustainable Agricultural Systems in the 21st Century
National Academies Press, Washington, DC (2010)
ISBN: 978-0-309-14896-2

[3] B.S. Murty, et al.
Applications of nanomaterials
B.S. Murty, et al. (Eds.), Textbook in Nanoscience and Nanotechnology, Springer, Berlin Heidelberg (2012), pp. 107-148
ISBN: 978-3-642-28030-6

[4] S. Huang, et al.
Agron. Sustain. Dev., 35 (2015), pp. 369-400

[5] M.E. Davey, G.A. O’Toole
Microbiol. Mol. Biol. Rev., 60 (4) (2000), pp. 847-867

[6] T.-F.C. Mah, G.A. O’Toole
Trends Microbiol., 9 (1) (2001), pp. 34-39

[7] C. Matz, S. Kjelleberg
Int. J. Antimicrob. Agents, 35 (2010), pp. 322-332

[8] H.-C. Flemming, et al.
Nat. Rev. Microbiol., 14 (2016), pp. 563-575

[9] M.V.B. Figueiredo, et al.
Plant growth promoting rhizobacteria: fundamentals and applications
D.K. Maheshwari (Ed.), Plant Growth and Health Promoting Bacteria, Springer-Verlag, Berlin Heidelberg (2010), pp. 21-43
ISBN: 978-3-642-13612-2

[10] Mustakeem
Mater. Renew. Sustain. Energy, 4 (22) (2015), pp. 1-11

[11] J.-H. Jang, et al.
Adv. Drug Deliv. Rev., 61 (2009), pp. 1065-1083

[12] F. De Cesare, et al.
Growth of bacterial biofilm on electrospun polycaprolactone nanofibrous scaffold for agricultural uses Electrospinning, 2016 – 4th Int. Conf. on Electrospinning 28 June–1 July, Otranto (2016), p. 1216

[13] I. Greenfeld, E. Zussman
Controlling the nanostructure of electrospun polymeric fibers
A. Macagnano, et al. (Eds.), Electrospinning for High Performance Sensors, Springer International Publishing (2015), pp. 35-64

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DOI: 10.1016/j.mattod.2017.05.005