Biochemical Engineering Journal 121 (2017) 2537

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Biochemical Engineering Journal

journal homepage: www.elsevier.com/locate/bej

Biocoatings: A new challenge for environmental biotechnology

Susana Cortez a, , Ana Nicolau a , Michael C. Flickinger b , Manuel Mota a
a
Centre of Biological Engineering (CEB), University of Minho, Braga 4710-057, Portugal
b
Golden LEAF Biomanufacturing Training and Education Center (BTEC), Department of Chemical and Biomolecular Engineering, North Carolina State
University, Centennial Campus, Campus Box 7928, Raleigh, NC 27695-7928, USA

a r t i c l e i n f o a b s t r a c t

Article history: Adhesive biocatalytic coatings (biocoatings) have a nanoporous microstructure generated by partially
Received 7 September 2016 coalesced waterborne polymer particles that entrap highly concentrated living cells in a dry state stabi-
Received in revised form 8 December 2016 lized by carbohydrate osmo-protectants. Biocoatings can be deposited by high speed coating technologies,
Accepted 16 January 2017
aerosol delivery or ink-jet printed in multilayered, patterned coatings on exible nonporous or nonwoven
Available online 19 January 2017
substrates, preserving 1010 1012 non-growing viable microorganisms per m2 in 250 m thick layers.
Cells are rehydrated to restore their metabolism. The layers reactive half-life following rehydration can be
Keywords:
1000 s of hours. The planar structure of biocoatings enable uniform illumination of a high concentration
Biocatalysis
Environmental biotechnology
of photo-reactive microorganisms or algae and contact these microbe with thin liquid lms for efcient
Nanoporous latex coating mass transfer. This review highlights recent advances in biocoating technology for pollutants degradation,
Non-growing cells photo-reactive coatings, stabilization of hyperthermophiles for biocatalysis, environmental biosensors,
and biocomposite fuel cells. Engineering cells for desiccation tolerance, unveiling the metabolism of non-
growing cells, and engineering the interaction between the cell surface and adhesive polymer binders
are fundamental challenges to open the door to vast future applications of biocoatings for environmental
sensing and remediation.
2017 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
2. Using immobilized microorganisms in environmental remediation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
3. Combining novel microorganisms and novel materials as environmental catalysts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
4. Fundamentals of biocatalytic latex nanocoatings as biocoatings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
4.1. Early biocoatings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
4.2. Advantages of the engineered biocomposite materials approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
4.2.1. Key-Concepts: dry stabilization and non-growing concentrated microbes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
4.2.2. Biocoating microstructure and polymer chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
5. Preparation of biocoatings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
6. Example of environmental applications of biocoatings intensifying reactivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
7. Biocoatings: future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

1. Introduction

Biotechnology will play a major role to signicantly reduce

Corresponding author. Postal address: Centre of Biological Engineering (CEB),
energy misuse, to stop pollution, recycle gaseous carbon emis-
University of Minho, Campus de Gualtar, Braga 4710-057, Portugal.
E-mail addresses: susana cortez@deb.uminho.pt (S. Cortez),
sions, and reduce global warming. This has led to a search for
protozoa@deb.uminho.pt (A. Nicolau), mcicki@ncsu.edu (M.C. Flickinger), new composite materials which concentrate (intensify) and stabi-
mmota@deb.uminho.pt (M. Mota). lize microbes as environmental biocatalysts for rapid degradation

http://dx.doi.org/10.1016/j.bej.2017.01.004
1369-703X/ 2017 Elsevier B.V. All rights reserved.
26 S. Cortez et al. / Biochemical Engineering Journal 121 (2017) 2537

of pollutants, solar energy harvesting, carbon recycling or gas- ties of natural biolms or hydrogels. Nanostructured materials in
cleaning [1,2]. the form of nanoparticles, nanotubes, nanobers and nanoporous
This review discusses the recent advances in fabrication meth- matrixes are advanced materials [18] as they have unique physi-
ods of biocoatings nanoporous adhesive composite materials cal and chemical properties such as large surface area to volume
containing highly concentrated living cells and their potential ratios or high interfacial reactivity [19]. One of the major advan-
environmental use. Biocoatings intensify catalytic conversion rates tages of incorporating advanced nano-structured materials with
for sensing/degradation of pollutants or solar energy harvesting microorganisms in composites is that their properties can be tai-
by highly concentrated dry-stabilized whole cells immobilized in lored for controlling both the physical properties of the composite
robust adhesive latex polymer coatings or coated/entrapped within (microstructure, adhesion, porosity, mechanical strength) and the
exible nonwoven materials. chemical environment immediately surrounding living cells, and
thus stabilize the cells reactivity [20]. An ideal adhesive polymer
immobilization matrix should be non-toxic, non-polluting, inex-
2. Using immobilized microorganisms in environmental
pensive, functional at ambient temperature, adhesive when wet,
remediation
exible, lightweight, mechanically and chemically stabile. In addi-
tion, the choice of a proper composite support material must take
Attractive features of whole cells as environmental biocatalysts
into account the capacity for retention of highly concentrated cells
compared to inorganic catalysts include versatility, high selectiv-
of different shapes and volume, and the porosity to allow diffu-
ity and temperature and pressure requirements that are close to
sion of nutrients, oxygen, substrates and products. A simple cost
environmental conditions [3,4]. However, the handling of growing
effective scalable fabrication procedure preferably using mature
microbes in large volumes of liquid media to process polluted air
manufacturing technologies such as high speed latex binder paper
and wastewaters presents severe drawbacks, such as uncontrolled
coating and waterborne coating synthesis methods is an advantage
cell density, growth toxicity of pollutants and products, nutritional
[2123]. Other criteria such as hydrophobic/hydrophilic balance,
limitations and difculties in recovering products. A physical sep-
optical and/or electronic properties and chemical functionalities
aration from the liquid phase by immobilization of the microbes
(i.e. solvation, wettability, etc.) are largely dependent on applica-
is highly advantageous, preserving high cell densities (without
tions [24] (Table 2).
leaching) to intensify reactivity, signicantly improving pollutant
Traditional microbe immobilization supports used to entrap
absorption/reaction rates and increasing volumetric productivity
living cells include hydrogels, natural polysaccharides (e.g. agar,
[5]. Moreover, immobilized whole cells reduce environmental cat-
carrageenan, alginate and chitosan), proteins (e.g. gelatin, collagen,
alyst cost and simplify downstream processing of products such as
albumin) and synthetic polymers (e.g. polystyrene, polyacrylamide,
recycling of carbon containing pollutants as chemicals and fuels
polyethylene glycol, polyvinylchloride, polyurethane). Inorganic
[6,7].
supports used for cell adsorption may be porous glass, ceramics,
The use of whole living cells, either in suspension or
clay, activated charcoal, zeolite, kieselgur, diatomaceous earth, or
immobilized in hydrogel beads, is well-established in industrial
loofa sponges [21,46]. Polymer hydrogels are not adhesive, col-
biotechnology [8,9]. Whole cells act as microbioreactors with the
lapse on drying and are often hundreds of microns to millimeters
ability to synthesize all necessary enzymes and cofactors [6,10,11].
thick, and can have severe mass transfer limitations [13,47]. These
Some whole cell biocatalysts can be stabilized with minimum
matrixes cannot be stored dry or frozen for prolonged periods of
preparation, e.g., adsorption onto an insoluble support, hydrogel
time without loss of cell viability. Also, when hydrogel-entrapped
entrapment with cross linking, as natural biolms, simple dry-
microorganisms are incubated with nutrients to sustain cell viabil-
ing, or lyophilization. All these methods reduce expensive enzyme
ity and regenerate activity, signicant cell release and outgrowth
purication [6,7,12]. Immobilized cells may be used in a growing or
occur because the gel matrix is macroporous (pores larger than the
non-growing state [13] or even as metabolically inactive cells [14].
entrapped microorganisms), the weak thin gel pore walls can be
The support matrix of natural biolms and hydrogels often results
broken by cell growth or the cells die and are sloughed from the
in severe mass transfer limitations for diffusion of substrates to the
surface [48].
cells and of cellular products into the bulk liquid. Embedding liv-
The utilization of inorganic ceramic materials for cell immobi-
ing cells in thin (250 m thick) nanoporous adhesive coatings can
lization offer many advantages, but the harsh conditions associated
signicantly reduce diffusion limitations. Growing cells produce
with their processing are often not compatible with cell viability
unwanted cell biomass, leading to severe mass transfer limita-
[48]. Some inorganic sol-gels can be processed at room temperature
tions and reactor plugging, and might have limited stability with
under mild conditions [5,48,49]. Furthermore, the porous nature
organic solvents and high concentrations of pollutants or prod-
of the sol-gel network allows easy access to external substrates or
ucts [6,8,15,16]. In contrast, non-growing cells are biocatalytically
pollutants [50]. Other attractive characteristics include rigidity (sil-
active, can withstand higher concentrations of toxic pollutants (no
ica or oxide ceramics), chemical inertness, high photochemical and
growth inhibition), do not produce biomass as a side product and
thermal stability and high optical transparency. Moreover, negligi-
are genetically stable [13]. However, microbial physiology knowl-
ble swelling in both aqueous and organic solvents is an advantage.
edge to engineer slow growing or non-growing cells as biocatalysts
Premkumar et al. [51] successfully entrapped recombinant biolu-
for thousands of hours of reactivity in a large-scale process is scarce
minescent Escherichia coli reporter strains in sol-gel silicate and
[15,17]. Table 1 summarizes the main characteristics of microen-
maintained viability and luminescence activity for more than three
capsulation, biolm and biocoating processes.
months at 4 C. The main drawbacks of sol-gel matrixes include
low sensitivity and reproducibility, and unwanted changes in the
3. Combining novel microorganisms and novel materials as chemical and biological properties of the cells due to the interac-
environmental catalysts tions with the inner surface of the pores [52], mainly because of the
intrinsic brittleness of sol-gel materials. To overcome this, poly-
An important aspect to consider in engineering immobilized mers (e.g., chitosan, polyhydroxyl polymers), have been coupled
whole cell biocatalysis is the material properties of the immobiliza- to sol-gel materials in the form of organic-inorganic composites,
tion matrix [10]. Engineering polymeric immobilization matrixes thereby limiting shrinkage during the process of sol-gel formation.
will have superior wet adhesion, controlled porosity, and the Sol-gel/hydrogel hybrids combine the rigidity with the biocompat-
ability to stabilize living cells when dried, which are not proper- ibility of hydrogels, thus avoiding sol-gel cracking and hydrogel
 S. Cortez et al. / Biochemical Engineering Journal 121 (2017) 2537 27

Table 1
Summary of main characteristics of microencapsulation, biolm and biocoating processes.

Process Entrapment material Geometry Growth

Microencapsulation Exogenous 3-D Growing of cells

(non-growing)
Biolm Endogenously produced by the cells 3-D Biolm growth (both cells and material growth)
Biocoating Exogenous 2-D Neither cells nor material growth
(non-growing)

swelling, as well as cell leaching [53]. To combine the merits of sol- on a carbon electrode using a lm-forming emulsion of methyl-
gel and a variant of polyvinyl acetate hydrogel, Jia et al. blended methacrylate and butyl acrylate copolymer. The device showed cell
both materials with a mixed culture of Thrichosporon cutaneum viability of nearly 100% with photoreactivity after rehydration
and Bacillus subtilis, on the surface of an oxygen probe [54]. This and illumination. The major difculties of these early biocoating
biochemical oxygen demand sensor retained activity and sensitiv- investigations, however, were low coating porosity, weak mechan-
ity for 40 days and could be stored for three months at 4 C while ical stability (delaminating from the support particles, creep), poor
keeping 80% of its initial activity. control of coating thickness, lack of uniformity, loss of reactivity,
Immobilized whole cell biocatalysts mimic natural biolms lack of dened coating microstructure (porosity, pore structure)
which have been successfully applied in bioremediation in wastew- and lack of cell viability data [67].
ater and exhaust gases [55]. The disadvantages of natural biolms Swope and Flickinger [68] used Mayer rod drawdown coat-
are time-dependent biological activity, limited control of cell con- ing methods with nontoxic acrylate/vinyl acetate copolymer latex
centration, limited control of lm thickness, complex structure binders without biocides to immobilize E. coli cell pastes in bio-
(channels) and uncontrolled cell adhesion/release (sloughing). The coatings of <80 m thickness as a two-layered coating in which
microbes in biolms produce their own extracellular carbohydrate the bottom layer (cell coat) consisted of a mixture of cells, latex
polymer immobilization matrix and are adapted to grow within and glycerol and the top layer (topcoat) consisted of a mixture
the matrix [56]. Natural biolms are inexpensive and can have of latex and glycerol. The top nanoporous layer sealed the cells
long-term activity (months to years) and enhanced tolerance to in the bottom layer (a sterile barrier). Glycerol was added as an
toxic reactants. These characteristics can overcome limited struc- osmoprotectant and to retard particle deformation, compaction
tural stability (sloughing) and control of porosity for wastewater and coalescence [69], resulting in enhanced cell viability following
treatment applications [55]. rehydration. Differential viability staining and laser scanning con-
focal microscopy (LSCM) techniques adapted from methods used
to characterize natural biolms were used to determine the via-
4. Fundamentals of biocatalytic latex nanocoatings as bility of the entrapped E. coli, which was 95% for several weeks
biocoatings following rehydraton. However, LSCM to determine cell viability
has limited penetration into the coatings due to photobleaching
4.1. Early biocoatings [68]. Subsequent work by Lyngberg et al. [57] using E. coli coat-
ings showed that the addition of sucrose plus glycerol combined
Different terms are used in the literature to describe bio- with controlled drying and controlling the ratio of cell size to
coatings: biocatalytic latex nanocoatings, biocatalytic coatings, polymer particle size could increase coating porosity and elim-
microbial paints and inks, latex copolymer lms, synthetic biolms, inate coat blistering during drying and rehydration. In addition
biomimetic leaves, cellular composite coatings and nanobiocom- to acting as porogens, carbohydrates such as sucrose or trehalose
posites. A biocoating consists of one or more thin layers of added to the latex formulation impart desiccation tolerance to the
metabolically active microorganisms entrapped between partially entrapped cells. During ambient drying, the coatings dry due to a
coalesced adhesive insoluble latex or non-lm forming polymer glass transition resulting in vitrication of extra- and intracellular
particles [57]. According to Mota et al. [5860] and Lyngberg et al. carbohydrates and ions, which form stabilizing complexes around
[61], the ratio between the size of the partially coalesced polymer and within the cells. Subsequently, a simple pressure sensitive vinyl
particles and the size of the cells as well as the volume ratio of each mask method was developed which eliminated coating edge effects
particle type are critical to the biocoating microstructure, adhesion loss of cells from the coating edge following rehydration [70].
and porosity (Fig. 1). Indeed, the porosity rst decreases with the Multilayer patch coatings of 12.5 mm to 35 mm in diameter were
volume ratio and, at a certain point (around 70%) it reaches a mini- fabricated from 5 to 65 m thick using these drawdown methods
mum from where it starts increasing. Biocoatings can be deposited [70]. These studies established methods to generate thin adhesive
on a wide variety of inexpensive and exible materials such as latex patch coatings (2 to 75 m thick) two orders of magnitude
polyester sheets, metals or porous materials wood, paper, bers, thinner than cross-linked hydrogels and most natural biolms used
yarns and other nonwoven materials [34]. for bioremediation, and to control latex coating porosity formed
Lawton, Bunning and Flanagan [6264] were the rst to entrap during ambient drying. These investigations also established that
living cells within latex coatings describing the immobilization of latex coatings maintained their adhesive properties and integrity
fungi, yeast and bacteria in polydispersed acrylate/vinyl acetate when rehydrated for cell concentrations as high as 50% of coating
copolymers (particle size 260 nm, glass transition temperature volume.
(Tg ) 13 C). Coating porosity was generated by integrating calcium
carbonate that was later leached with acid to form large pores
for colonization by microbial growth. Cantwell et al. [65] used 4.2. Advantages of the engineered biocomposite materials
bimodal blends of hard and soft polymer particles (Tg range of approach
60 to 60 C) to immobilize microbial cells. However, these cells
were not layered in thin coatings, but only in the form of oc- Biocoatings entrapping highly concentrated cell pastes stabi-
culates, 12 mm aggregates, and 2 mm diameter brils. No data lize viable but non-growing microorganisms in thin (2<50 m),
was presented on cell viability following entrapment or aggre- adhesive, nanoporous, partially-coalesced, insoluble polymer coat-
gate permeability. Martens et al. [66] immobilized Synechococcus ings with minimal diffusion resistance [57,70]. Colloid-based latex
 28
Table 2
Application of recent immobilized whole cells in environmental biocatalysis.

Microorganism Host support Purpose of Comments Refs.

immobilization

Escherichia coli, Mixture of 0.5% Biosensor for copper -80% of cell viability after 1 month (storage at 4 C) [25]
Saccharomyces agarose, 1% ion detection
cerevisiae polyvinylpyrrolidone
and 0.05% collagen
Pseudomonas species Polyvinyl alcohol Degradation of phthalic -the degradation rate of immobilized cells was higher than free cells; [26]
acid esters -the immobilization of microbial cells did not change their original metabolic pathway
Chlorella sorokiniana Loofa sponge Nickel (II) -immobilized cells accumulated 25% more nickel than free cells after 20 min of exposition [27]
accumulation
Spirulina platensis -Alginate Cadmium -the maximum biosorption capacities for alginate immobilized cells and silica immobilized [28]
-Silica accumulation cells were 70.92 and 36.63 mg Cd g1 biomass, respectively (high cadmium sorption capacity);
-the immobilized cells could be repeatedly used in the sorption process up to ve times
Genetically modied Silica sol-gel Degradation of atrazine -at room temperature, the encapsulated non-viable cells maintained a specic activity [29]
Escherichia coli between 0.44 0.06 and 0.66 0.12 mol g1 min1 for up to 4 months, comparing with free,

S. Cortez et al. / Biochemical Engineering Journal 121 (2017) 2537

viable cell-specic activities (0.61 0.04 mol g1 min1 );
-when encapsulated, non-viable cells were assayed at 4 C, the activity increased threefold
over free cells
Mucor circinelloides Polyurethane foam Recovery of omega-3 -the optimum hydrolysis products were obtained at pH 7 and 35 C; [30]
from sh oil waste -a 12.6% increase of omega-3 concentration in comparison to initial omega-3 concentration in
the crude sh oil waste was observed
Bacillus sphaericus Silica sol-gel Copper and uranium -the immobilized cells accumulated more pollutants than matrixes with spores or S-layers [31]
accumulation
Rhodococcus Alumina bers Degradation of phenol -the immobilized cells showed high biocatalytic activity, high compactness and less shrinkage [32]
rhodochrous reinforced sol-gel during the drying;
-after immobilization, phenol concentrations up to 3 g L1 were tolerated and degraded
Rhodopseudomonas Biocide-free Hydrogen production -the immobilized cells were more reactive than an equivalent number of suspended or settled [33]
palustris polyvinylacetate-co- cells and remained active after hydrated storage for greater than 3 months in the dark and
acrylate copolymer 1 year when stored at 80 C
latex
Synechococcus Solvent-free acrylate Photosynthesis -the immobilized cells intensied up to 10-fold the specic photoreactivity of suspended cells [34]
PCC7002, Synechocystis copolymer latex binder in non-growth media designed from photosynthetic quotient data
PCC6803, Synechocystis
PCC6803, and Anabaena
PCC7120
Rhodotorula Sugarcane bagasse Biodiesel production -the maximum biodiesel yield was obtained at 85.29% with the optimum conditions of 1.5 g [35]
mucilaginosa whole cell biocatalyst, 1:12 methyl acetate to oil ratio, 10% water content (w/w), temperature
of 40 C, 60 h of reaction time and agitation at 250 rpm;
-the stability of immobilized whole cell biocatalyst was studied with 10 cycles of repeated
usage and it was shown that there was no signicant loss of lipase activity in the presence of
methyl acetate.
Genetically modied Mussel adhesive Biosensor for -the whole cell array biosensor system, prepared using optimal MAP coating (50 mg cm2 ) and [36]
Escherichia coli protein (MAP) organophosphate cell loading (4 OD600 ), detected paraoxon levels as low as 5 mM with high reproducibility, and
compounds detection its quantitative detection range was 5320 mM;
-the tested biosensor showed a good long-term stability for 28 day with 80% retained activity
and a reusability of up to 20 times;
-paraoxon in tap water was also successfully detected without a reduction in sensitivity
Activated sludge Polyethyleneglycol Nitrication -more than 90% of ammonia was reduced and 75% of the total organic compound was [37]
removed after treating 40 and 10 mg L1 synthetic ammonia wastewater with different C/N
ratios in 5 h under aeration conditions
Rhizopus oryzae Rigid polyethylene Biodiesel production -supplementation of the medium with carbon sources led to higher lipase activity and [38]
biomass support increased the biomass immobilized on the support;
-a cultivation period of 72 h in a basal medium supplemented with both cottonseed oil and
glucose is optimal for biodiesel production by R. oryzae, resulting in a fatty acid methyl ester
yield of 27.9 wt% (228.2 g L1 )
Table 2 (Continued)

Microorganism Host support Purpose of Comments Refs.

immobilization

Genetically modied Calcium alginate Degradation of -optimum bead loadings for bioreactor operation were found to be 200 g beads L1 for [39]
Escherichia coli organophosphate chlorferon degradation and 300 g beads L1 for diethlythiophosphate degradation;
compounds -using waste cattle dip solution as substrate, the degradation rate for an immobilized

S. Cortez et al. / Biochemical Engineering Journal 121 (2017) 2537

consortium of chlorferon-degrading bacteria was ve times greater than that for freely
suspended cells, and hydrolysis of coumaphos by immobilized recombinant Escherichia coli
was 2.5 times greater.
Geobacter Pectin embedded on Electrical current -immediately after immobilizing G. sulfurreducens on electrodes, electrical current was [40]
sulfurreducens graphite electrodes production (microbial obtained without addition of exogenous electron shuttles or electroactive polymers;
fuel cell) -electrodes maintained at +0.25 V initially produced 0.52 A m2 in the presence of acetate as
the electron donor;
-when electrodes were maintained at an oxidizing potential for 24 h, electron transfer to
electrodes increased to 1.75 A m2
Genetically modied Sodium alginate, Preservation of cell -more than 90% of the cell viability was preserved during the encapsulation process [41]
Escherichia coli cellulose sulfate viability for long term -the initial cell activity was fully maintained within encapsulated cells while it halved in free
storage cells
Aspergillus oryzae Polyurethane foam Biodiesel production -immobilized A. niger whole cell lipase showed biodiesel conversion of 80.97% and yield of [42]
cuboids 90.82% at 35 C, methanol to oil ratio of 5:1 with 2.5% water content based on oil weight and 6
biomass support particles with step-wise methanol addition
Pseudomonas Fe3 O4 chitosan Biodiesel production -a maximum biodiesel yield of 87.32% was obtained under the optimum operating condition [43]
mendocina microspheres (magnetic whole-cell biocatalysts concentration of 10 wt.%, water content of 10 wt.%, 35 C,
methanol to oil molar ratio of 4:1 and a four-step addition of methanol) for 48 h;
-magnetic whole-cell biocatalysts still gave a biodiesel yield of 83.57% after 10 cycles, which
was higher than that of Fe3 O4 uncontained whole cell biocatalysts (74.06%)
Bacillus fusiformis Alginate-polyvinyl Degradation of -more than 99.7% of naphthalene was removed within 12 h, containing bentonite 2% (w/v), [44]
alcohol (PVA)-clays naphthalene PVA 12% (w/v), alginate 0.3% (w/v) and 10% (v/v) initial biomass loading;
-immobilized cells remained stable after storage at 4 C for 35 days and being reused 8 times
(12 days);
-the naphthalene degradation rate of immobilized cells was maintained (94.3%) at the eighth
cycle
Ganoderma lucidum Ca-alginate (Ca)/poly- Remediation of -removal of ANT reached 96.2% 2% from soil after 20 days of incubation at pH 5.0 and 45 C [45]
-caprolactone anthracene (ANT) with an optimum PCL concentration in CA/PCL/corn-cobs beads of 12%
(PCL)/corn-cobs beads

29
30 S. Cortez et al. / Biochemical Engineering Journal 121 (2017) 2537

Fig. 1. Nanoporosity is function of cell loading and particles size ratio. (A) Dependence of normalized porosity (/0 ) on volume fraction of large particles in the mixture
(xD ) for different particles size ratio d/D, adapted from Mota et al. [58,59]. Curves 13 correspond to the model of Mota et al. [60]. D is the largest particle diameter in the
mixture microorganisms; d is the small particle diameter in the mixture latex; is the overall porosity of a mixed bed; 0 is the porosity of a bed of monosized particles,
0 = 0.407. (B) Microorganisms with approximately the triple size of polymer particles roughly corresponding to curve 3.

biocomposite adhesives can stabilize desiccated vegetative cells must be very low (0.1) for large volume catalysis [77,78]. New
without loss of reactivity, concentrate cells (5001000-fold) to a cell biomass is a waste product that signicantly increases E.
very high volume fraction (>50% by weight depending on cell size), For example, non-growing cells can be used as whole-cell
and incorporate carbohydrate osmoprotectants to maintain the biocatalysts for microbial production of hydrogen gas (H2 ). Photo-
integrity of microbial membranes during controlled ambient dry- heterotrophic bacteria usually exhibit high H2 yields when utilizing
ing [47,67,71]. After rehydration, these cellular composites were organic substrates, although their H2 production rate is low [79].
shown to be capable of new protein synthesis without outgrowth In contrast, fermentative H2 production by strict and/or faculta-
for thousands of hours [13]. tive anaerobes proceeds at a fast rate but with a low H2 yield.
This technology is based on biocide-free waterborne latex Gosse et al. [33] and Ishikawa et al. [80] studied immobilized
binder emulsions. Latex paint binder emulsions are low cost mate- Rhodopseudomonas palustris and immobilized E. coli, respectively
rials ($1 m2 ) [72] and can be formulated to protect the embedded (facultative bacteria); and reported continuous H2 production was
cells from UV damage [13]. Latexes can be applied using high speed possible as long as acetate or glucose was replenished periodi-
industrial coating and printing methods and offer great exibility in cally. Seol et al. [81] evaluated the H2 production activity of four
the design of polymers for engineered porosity, adhesion (particu- Enterobacteriaceae strains, under growing and non-growing condi-
larly wet adhesion) and lack of toxicity [73]. Polymer heterogeneity tions, using glucose or formate as the carbon source and concluded
that contributes to lm morphology and adhesion after drying in that formate was much better substrate than glucose for H2 pro-
latex coatings is well studied by the waterborne coating indus- duction. Gosse et al. [13] reported hydrogen gas evolution at a
try [74,75]. However, the complex molecular interactions between constant rate for over 4000 h using a submerged biocomposite
heterogeneous polymer particle surface chemistry, water, surfac- latex coating of R. palustris on polyester sheet without outgrowth.
tants, viscosity modiers, osmoprotectant carbohydrate, and the In order to better understand metabolic activities of non-growing
complex surface of living cells during lm formation and drying cells, and improve engineered biocatalysts, McKinlay et al. [15]
is only beginning to be explored by polymer emulsion chemists in tracked changes in biomass composition and global transcript
order to alter coating functionality using biotechnology [76]. Fur- levels of Rhodopseudomonas palustris when starved for nitrogen.
thermore, since carbohydrates such as sorbitol protect biological They observed that during starvation R. palustris diverted their
material, it is anticipated their application on coating mixture in metabolism from biosynthesis to mobilize electrons for H2 produc-
order to increase long-term stabilization and preservation of many tion, resulting in a 3.5-fold enhancement of hydrogen gas evolution
types of cells and biological materials in biocoatings. without cell division. This emphasizes that several approaches
may achieve non-growth, e.g., nutritional conditions, temperature,
4.2.1. Key-Concepts: dry stabilization and non-growing process conditions, or genetic methodologies, to optimize the per-
concentrated microbes formance of biocoatings.
The real advantage of combining advanced colloid and adhe- Biocoatings also depend on cell desiccation tolerance. Changes
sive polymer materials with microbes lies in the dry-stabilization of in the mobility of water and solvents may be the mechanism of
concentrated microbes that do not grow out from the microstruc- stabilization of living cells in nanoporous coatings resulting from
ture nano-pores (pores smaller than the microbes). This enables increasing uid viscosity and entropic connement during lm
the cells to remain catalytically active for thousands of hours with formation and simultaneous pore formation by arrested polymer
comparable half-lives to chemical catalysts. particle coalescence [67]. Common methods of dry-preservation
Most bioprocesses catalyzed by microbes continuously generate of live cultures include freezing in glycerol and freeze-drying.
biomass as the bacteria grow and multiply by consuming reactants Numerous microorganisms, insect larvae and insects known as
meant for conversion to a desired product. This wasteful use of reac- anhydrobiotes are able to survive with almost complete loss of
tants can be avoided by keeping the bacteria in a non-growing but free body water to levels of 0.50.01 g water/gram cell dry
active state. This concept is particularly important for large-scale weight. The dry organisms may remain in a state of anhy-
environmental processes such as anaerobic digestion where the drobiosis, for decades without apparent damage. When water
production of excessive biomass is not desirable. It is also impor- again becomes available, they rapidly rehydrate and resume their
tant for overall process efciency as dened by the efciency factor normal metabolic functions [82]. Anhydrobiosis depends on a
E the ratio of waste products produced divided by products. E series of complex physiological adaptations which are important
 S. Cortez et al. / Biochemical Engineering Journal 121 (2017) 2537 31

in protecting anhydrobiotes from desiccation damage, including Lyngberg et al. [57] studied the diffusive permeability of rehy-
up-regulation of highly hydrophilic proteins and production of drated wet latex polymer only and of E. coli-containing biocoatings
intracellular non-reducing disaccharides such as trehalose. Despite (delaminated from a metal substrate) as the effective diffusion coef-
the discovery of various mechanisms involved in desiccation and cient (Deff in cm2 s1 ) corrected for coating thickness expressed
water stress, knowledge of the regulatory network governing the as a dimensionless ratio (between 0 and 1) of the effective diffusiv-
stability of the cellular structures and the metabolic machinery ity to the diffusivity of tracers such as KNO3 or riboavin in pure
active during dehydration by ambient drying is almost unknown water (D). This ratio is the diffusive permeability of the coating irre-
[8284]. spective of the diffusing species. The decreasing effective diffusion
coefcient as a function of increasing E. coli cell dry weight indicates
that for a specied coating thickness, there is an optimum cell con-
4.2.2. Biocoating microstructure and polymer chemistry tent at which the biocoating has an effective diffusion coefcient
Creation of thin biocoating microstructure that allows diffusion both for reactant species and nutrients high enough to sustain cell
of reactants and products through the polymer matrix requires that viability at the innermost cell layers [67].
latex particle deformation and coalescence be carefully controlled
during the drying process. Choice of latex polymer, polymer par-
ticle size and distribution, control and manipulation of drying and 5. Preparation of biocoatings
hydration conditions, and use of additives are among the means
available to achieve this goal [73]. Biocoatings generated from a Some common biocoating deposition techniques include extru-
mixture of cell paste and an emulsion of pH adjusted non-toxic sion [34,90,91], spray drying [92], the wire wound rod drawdown
adhesive (deformable) latex binder emulsions, bimodal particle Mayer rod coating method [13,67,93], 3-D ink-jet deposition
blends or core shell lattices can have engineered adhesion to a wide [94] and the convective assembly [12,47,95]. Recently, dielec-
variety of surfaces [76]. Adhesion (to substrate, layer to layer) can trophoresis (DEP) has also been used to generate photoreactive
be altered by polymer particle glass transition temperature (Tg ), cyanobacteria cell monolayers on top of polyelectrolyte adhesives
latex particle surface chemistry (charge, charge density, surface [96].
grafting of crosslinking agents), and the ratio of particle diame- The wire wound or Mayer rod drawdown coating method (Fig. 2)
ter to cell size and polymer particle to cell concentration in the was used in early biocoatings to deposit biocomposites of randomly
coating emulsion (Fig. 1) [47,67]. If these porous composite coat- oriented microbial cells and 150 to 300 nm latex particles in
ings are strong enough, they can be stripped (delaminated) from 5100 cm2 -scale patches, strips, or sheets on exible polyester sub-
the substrate and mounted as stand-alone lms and their diffusion strates with a range of thicknesses from 10 to 250 m, in either
properties as a function of microstructure directly measured in a monolayer or multilayer biocoatings [13,33,57,87]. This method is
diffusion apparatus [57,67]. capable of fabricating biocoatings of <10 m thick [67]. Coating
Thin latex biocoatings containing carbohydrates stabilize, and preparation involves a controlled coalescence/drying step and, if
preserve the integrity of microbial membranes during drying, needed, a subsequent short-term storage period, both affected by
thereby preserving cell viability [67]. Coating thickness is con- temperature and usually carried out at 50%60% relative humidity
trolled by the deposition method, emulsion formulation (percent [33]. Coatings can be made in ltered air, in an anaerobic hood, or
solids), and by the number of layers applied. Polymer parti- in a contained coating enclosure with both humidity and air-borne
cle coalescence and therefore the permeability is controlled by particulate control. Coating thickness can be altered by rod wire
polymer-water interfacial tension, capillary pressure, layer com- diameter, emulsion formulation (percent solids), rod pressure and
pression, and interparticle adhesion [85]. Even after dried and mask thickness [67]. Biocoatings can be cast on non-sterile sub-
rehydrated, reduction in the coating permeability can occur by wet strates (cleaned with 70% ethanol) using non-sterile biocide-free
coalescence depending on time, relative humidity and tempera- latex emulsions (adjusted to neutral pH) under non-aseptic con-
ture for some low Tg polymer particle emulsions [67,85]. ditions [67]. The Mayer rod method relies on self-leveling low Tg
For optimal reactivity of entrapped cells, pores should be large acrylate co-polymer or non-lm forming polymer particle blends
enough to allow unrestricted transport of molecules including plus carbohydrates to alter compaction and arrest coalescence dur-
buffer ions, gases, substrates, and products but be nanopores ing evaporation to generate nanoporosity, which can result in a
(<1 m) to prevent leakage or outgrowth of microorganisms [86]. mixture of particle packing congurations [13,33,47,65,87].
Nanoporosity is generated by the addition of glycerol and carbohy- A continuous convective colloid assembly method (CCSA) has
drates to arrest polymer particle coalescence during lm formation recently been reported to deposit whole cells and waterborne
of low Tg emulsions. These additives also protect the entrapped polymer particles. These particles are ordered by evaporation of
cells from osmotic stress during coating formation prior to depo- the meniscus into thin (<10 m thick), organized lms with engi-
sition [12]. However, when rehydrated, the entrapped porogens neered adhesion, composition, thickness, and particle packing, over
are leached and porosity can slowly be lost as a function of time larger surface areas (Fig. 2) [12]. Jenkins et al. [47] found that net
and temperature as a result of wet coalescence [67]. Bimodal charge leading to repulsion between particles or between particles
polymer blend [87] and core-shell approaches can minimize wet and S. cerevisiae is important in coating assembly. CCSA was used
coalescence following rehydration [67]. Latexes with Tg above the to precisely order yeast or polymer particles into closely packed
temperature at which biocatalysis is to be performed afford greater monolayer coatings (one cell thick) with minimal void space. Kum-
mechanical strength and shear resistance [73]. norkaew et al. [97] demonstrated that when the meniscus height
Biocoatings must contain a density of microorganisms below the is larger than the particle diameter, multilayer coatings will form
critical concentration that would disrupt polymer particle coales- [97]. The number and type of layers is easily adjusted by altering the
cence. This is analogous to the maximum or critical pigment volume suspension volume fraction and coating knife speed, allowing for
concentration (PVC) in latex paints [88]. However, the presence of precise control in particle packing and coating thickness [95,97,98]
high concentrations of cells can decrease porosity and block poly- (Fig. 2B). Convective assembly deposition methods could lead to
mer particle coalescence depending on cell size and the interaction rapid fabrication of well-ordered arrays of cells for multi-layer
between the cell surface and the surface of the polymer particles systems with hybrid functionality [95]. Methods to generate cell
[12]. Biocoatings with E. coli densities as high as 50% by volume, monolayers (one cell thick adhesive biocoatings) with engineered
2 1011 cells cm3 of coating volume, have been reported [89]. adhesion and reactivity are the starting point for the construc-
32 S. Cortez et al. / Biochemical Engineering Journal 121 (2017) 2537

Fig. 2. Schematic of the coating apparatus used to produce a randomly orientated thick coating produced by the wire-wound rod drawdown deposition method (A) and a
thin ordered coating via batch convective assembly (B).

tion of structured multi-layer systems [97]. Biocoatings formed energy applications); Rhodopseudomonas palustris CGA009 (anaer-
from CCSA monolayers can also stabilize living cells which can add obic bacterial phototroph), which produces H2 gas; Clostridium
self-cleaning capabilities for releasing or digesting surface contam- ljungdahlii OTA1 (anaerobic autotrophic acetogenic ethanologen),
inants [12]. Once CCSA monolayer fabrication methods are rened, for conversion of syngas to ethanol; and the Synechococcus sp.
layer-by-layer (LBL) methods can be used to generate perfusive PCC7002 (cyanobacterial aerobic phototroph). Investigation of C.
channeled systems with tailored uid distribution, light scattering, ljungdahlii biocoated papers, coated without adhesive latex, has
gas harvesting and gas absorbing reactivity [13]. recently be extended by Schulte et al. [91], who optimized sur-
Many classical whole cell immobilization techniques such as face area, with mass-transfer power input reduced by 3 orders of
hydrogels are reversible and cells can be released from their entrap- magnitude compared to traditional bioreactors and reduced liq-
ment, enabling the measurement of cell number, viability and uid volume. His approach intensied the rate of CO uptake by over
reactivity of the recovered cells. Cell recovery is not possible in 17 fold. These studies demonstrate new designs for engineering
most adhesive insoluble biocoatings. Nondestructive protocols to reactive biolters that absorb gases (CO, CO2 , CH4 , H2 , NOx , SOx )
monitor bacteria directly using uorescent reporters and confocal or produce useful gases (O2 , H2 , CH4 ) for low-power-input large-
microscopy are available [68] similar to methods to monitor natural scale gas processing [100102]. This is leading to investigation
biolm structure and cell viability (Fig. 3). of biocoatings in thin-lm, falling lm or spinning disk bioreac-
tors, and rotating biological contactors (RBCs) for large-scale gas
absorption with signicantly reduced power input for mass transfer
6. Example of environmental applications of biocoatings [12,13,100104].
intensifying reactivity The possibility is emerging to exploit high intensity biocoatings
for wastewater treatment involving complex microbial consortia.
Biocoatings are beginning to be investigated for a variety of One example is signicantly increasing the rate of the nitrication-
environmental applications (Fig. 4). denitrication [105]. The conversion of organic nitrogen to gaseous
Estrada et al. [99] entrapped Pseudomonas putida F1 in a nitrogen involves a strict and balanced cooperation of three
nanoporous latex coating for intensication of biolters for toluene microorganisms, two of which are nitriers (Nitrosomonas europaea
vapors degradation. These biocoatings showed volumetric toluene and Nitrobacter vulgaris) and the last one a denitrier (Paracoc-
mineralization rates and specic biodegradation rates 10 times cus denitricans). Nitrifying bacteria are slow-growing, oxygen
higher than when using agarose biolms. The high rates were dependents and very sensitive microorganisms when compared
attributed to the markedly decreased diffusional resistance in the with denitriers (oxygen sensitive microorganisms). The devel-
thin biocoatings, demonstrating the intensication concept for opment of an articial consortium using three adjacent adhesive
engineering advanced biolters or biotrickling lters. Gosse et al. nanoporous layers of highly concentrated bacteria containing in
[90] demonstrated intensied absorption or evolution of H2 , CO, each layer a single species providing the substrate for the adja-
CO2 or O2 from biocoatings on paper located above the liquid cent layer has all the characteristics of becoming a groundbreaking
phase. The microbes used were Chlamydomonas reinhardtii CC124 environmental remediation technology (Fig. 5). In this process, an
(eukaryotic microalgae phototroph being engineered for renewable
 S. Cortez et al. / Biochemical Engineering Journal 121 (2017) 2537 33

Fig. 3. Methods for biocoating characterization.

Fig. 4. Advantages and environmental applications of immobilized cells in biocoatings.

Fig. 5. Scheme of application of biocoatings in a rotating biological contactors (RBC) reactor for wastewater treatment using different microorganisms.
34 S. Cortez et al. / Biochemical Engineering Journal 121 (2017) 2537

ammonium-oxidizing organism (Nitrosomonas europaea) converts Biocoating stability and storage are important design param-
ammonium (N-NH4 + ) into nitrite (N-NO2 ), then a nitrite dissim- eters [21]. Lyngberg et al. [87] noticed that thin bimodal blend
ilatory organism (Nitrobacter vulgaris) oxidizes nitrite into nitrate coatings of acrylate/vinyl acetate latex are useful to concentrate
(N-NO3 ) and nally a denitrier (Paracoccus denitricans) converts and stabilize metabolically active Thermotoga maritima, an hyper-
nitrate into nitrogen gas (N2 ). thermophile, in articial sea water (ASW). These coatings preserved
A recent approach to improve solar energy harvesting by com- high amylase activity for 100 s of hours at 80 C in ASW. Fidaleo et al.
bining complementary photosynthetic microbes is to fabricate [93] demonstrated that a strictly aerobic acetic acid bacterium
biocoatings that mimic the function and stability of natural plant Gluconobacter oxydans could be preserved at ambient temper-
leaves by ordering layers of closely packed photosynthetic cells on a ature, concentrated, entrapped, and retained in thin nanoporous
surface with non-toxic adhesive polymer binders. Bernal et al. [34] multi-layer coatings under nitrogen-limited conditions and follow-
investigated this concept with a porous paper nonwoven substrate ing rehydration, carry out an oxygen-requiring sugar bioconversion
for supporting latex coatings of cyanobacteria above a liquid phase at high efciency and intensity. Similarly, Piskorska et al. [71]
to function as biosolar absorbers (or biomimetic leaves), absorbing observed that when stored in argon at <5% humidity and room
CO2 and producing O2 . Four different cyanobacteria strains (Syne- temperature, R. palustris biocoatings retained full H2 production
chococcus PCC7002, Synechocystis PCC6803, Synechocystis PCC6308, activity for 3 months.
and Anabaena PCC7120) with diverse morphologies were used,
and it was demonstrated that this simple method of latex binder
coating by extrusion is capable of intensifying 510-fold the spe- 7. Biocoatings: future perspectives
cic photoreactivity of suspended cyanobacteria in non-growth
nitrogen-limited media [34]. Since neither the cyanobacteria The cooperation between nanoscience, nanomaterials, water-
strains nor the biocomposite paper microstructure were optimized, borne coating technology, synthetic biology/biotechnology and
this level of biocoating photoreactivity is remarkable. microalgae scientists and engineers is resulting in emerging tech-
Rapid and selective biocoating lm as biosensors able to detect nologies to fabricate innovative functional biological nanosystems
pollutants at very low levels could enhance our understanding and containing stabilized living cells. In particular, the controlled explo-
protect the environment [106]. Biocoating sensors are receiving ration of engineered whole cells in biocoatings seems to be very
growing attention for measuring toxic gases and toxic soluble com- promising for low cost biochemical transformation of waste waters
pounds in water and wastewater. Lyngberg et al. [70] developed and waste gases with enhanced intensity (small footprint) and ef-
a single-use luciferase-based mercury biosensor using Escherichia ciency.
coli HB101 in a latex copolymer lm. The immobilized E. coli The vast potential of using engineered immobilized pho-
HB101 cells harbored a mer-lux plasmid construct and produced tosynthetic cells, where better illumination efciencies, higher
a detectable light signal when exposed to Hg(II). This cell Hg(II) photoreactivity and long-term stability can be obtained when cells
biosensor had a sensitivity similar to that of suspended cells but a are coated into thin structured layers, has been demonstrated.
signicantly larger detection range. The levels of mercury detected Since algae, plant cells and cyanobacteria are highly promising
ranged from 0.1 nM to 10 000 nM HgCl2. as energy converters, biocoating research should focus on these
The fabrication of thin (<10 m thick), robust biocoatings for organisms. Indeed, photoreactivity superior to natural leaves may
biosensors may be an important approach to overcome the slow be possible if photosaturation can be minimized by spatially isolat-
reactivity of whole cell biosensors [106]. In order to generate these ing and optimizing cells with altered photopigment content, alter
thin coatings, biocoating formulations have been developed as fast microstructure to control illumination intensity, and engineer coat-
drying microbial inks for 3-D printing of uniform coatings, ink-jet ing microstructures with altered reectance and light scattering
microstructures or as sensing components of bioelectronics devices properties.
[67]. Fidaleo et al. [94] demonstrated that ink-jet droplet deposi- The multiple capabilities of immobilized whole cells in biocoat-
tion of Gluconobacter oxidans can be controlled to precisely deliver ings as environmental biosensors and large scale biocatalysts has
droplets onto exible polyester to create nanoporous microstruc- not been fully explored. By investigating a combination of how
tures with dened height, uniform surfaces and large surface area different biosensing cells, miniaturized transducers and detection
without cell death during ink deposition and controlled drying. G. techniques could be incorporated in advanced polymer materials
oxidans is a useful model biosensor organism for detecting alcohols, or microuidic devices revolutionary new biosensing composite
carbohydrates and polyols. Cell densities of up to 1014 m2 could materials could be fabricated. Indeed, the exploration of solar
be achieved with 3-D printed microstructures with 5:1 height to energy harvesting through the utilization of biocoatings of non-
diameter ratio. This ink-jet technique indicates the potential for growing cyanobacteria, algae and plant cells, coupled with suitable
rapid fabrication of high surface area 3-D printed whole cell biosen- electrodes, could provide an ideal way to produce clean energy and
sor arrays on exible microelectronic or microuidic devices using recycle CO2 into chemicals and liquid fuels with minimal water use.
adhesive latex binders. Unfortunately, the biology of non-growing microorganisms is
Biocoatings may also be applied in energy production and stor- poorly understood. By devising how to manipulate the regulation
age to transfer electrons to conductive surfaces in microbial fuel of gene expression without growth, the specic reactivity of bio-
cells [40]. Recently, Wagner et al. [92] proposed a proof-of-concept coatings could be signicantly increased. Furthermore, mastering
immobilization approach that allows exoelectrogenic activity of the regulation of in vivo message stability and proteolysis in non-
cells on an electrode consisting of a layer of latex holding bacteria growing microbes may lead to strategies to alter protein expression
on surfaces. A single topcoat layer of airbrushcoating did not reduce and protease activity in biocoatings and to design enzymes with
the voltage produced by a biolm in a microbial fuel cell (MFC), and very long in vivo functional half-lives.
the more easily applied dip-and-blot coating reduced voltage by Understanding the desiccation tolerance in anhydrobiotes
only 11% in a microbial electrolysis cell (MEC). Dry-stabilized bio- organisms will enable us to induce or engineer tolerance in desicca-
coating entrapped cells may be stabilized ready-to-use reagents tion sensitive microbes, and devise strategies to enhance long-term
that can be stored for prolonged time under controlled conditions stabilization and preservation of many types of cells and biological
of moisture and, once activated, (e.g., by hydration, a temperature materials in a dry state in biocoatings.
change or addition of nutrients), generate a reproducible number There is an increasing interest in the application of biocoating
of reactive cells for sensing [21]. processes industrially. However, the translation of this laboratory
 S. Cortez et al. / Biochemical Engineering Journal 121 (2017) 2537 35

concept into large-scale waste treatment processes is currently Biochem. Eng. J. 105 (Part B) (2016) 391405, http://dx.doi.org/10.1016/j.
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Acknowledgements for biosensing purposes, Anal. Bioanal. Chem. 402 (2012) 17851797, http://
dx.doi.org/10.1007/s00216-011-5364-x.
[22] O.M. Zacheus, E.K. Iivanainen, T.K. Nissinen, M.J. Lehtola, P.J. Martikainen,
This study was supported by the Portuguese Foundation Bacterial biolm formation on polyvinyl chloride, polyethylene and
for Science and Technology (FCT) under the scope of the stainless steel exposed to ozonated water, Water Res. 34 (2000) 6370,
http://dx.doi.org/10.1016/S0043-1354(99)00113-X.
strategic funding of UID/BIO/04469/2013 unit and COMPETE
[23] T. An, L. Zhou, G. Li, J. Fu, G. Sheng, Recent patents on immobilized
2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation microorganism technology and its engineering application in wastewater
(NORTE-01-0145-FEDER-000004) funded by European Regional treatment, Recent Pat. Eng. 2 (2008) 2835, http://dx.doi.org/10.2174/
Development Fund under the scope of Norte2020 Programa 187221208783478543.
[24] A. Sinsawat, K.L. Anderson, R.A. Vaia, B.L. Farmer, Inuence of polymer
Operacional Regional do Norte. Susana Cortez acknowledges the matrix composition and architecture on polymer nanocomposite formation:
FCT individual grant SFRH/BPD/73720/2010. coarse-grained molecular dynamics simulation, J. Polym. Sci. Part B Polym.
Phys. 41 (2003) 32723284, http://dx.doi.org/10.1002/polb.10696.
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