Michael
Winn
a,
Joanne M.
Foulkes
a,
Stefano
Perni
b,
Mark J. H.
Simmons
*b,
Tim W.
Overton
*b and
Rebecca J. M.
Goss
*a
aSchool of Chemistry, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, UK. E-mail: r.goss@uea.ac.uk
bSchool of Chemical Engineering, University of Birmingham, Birmingham, B15 2TT, UK. E-mail: m.j.simmons@bham.ac.uk; t.w.overton@bham.ac.uk
First published on 14th May 2012
The robust nature of biofilms makes them medicinally difficult to treat, however this same property renders them an attractive method for protecting and immobilising enzymes for biotransformation. Although biofilms consisting of a consortium of different microbial species have been routinely used in water purification for many decades, there are few reported examples of single species biofilms being harnessed for industrial applications. The potential of using tailored single species biofilms in order to catalyse a biotransformation of choice is attractive; we reflect upon recent advances in the use and generation of such platforms, from both biological and process engineering viewpoints.
Enzyme stability can be improved by increasing the structural rigidity by directed evolution and/or multipoint immobilisation (see Mateo 2007 and Hernandez 2011 for recent reviews);4,5 immobilised enzyme biocatalysts can also be more easily removed from reaction vessels, recycled or used in continuous flow reactors. However, the consequences of this increased rigidity may be poorer catalytic performance as enzymes often go through rapid and precise changes in structure during catalysis. Too much conformational stability can restrict flexibility and therefore activity.6 In some cases immobilisation may actually be advantageous to catalytic activity or site-directed mutagenesis applied to combat any negative affects but the consequences of immobilising a specific enzyme are hard to predict and many passes of directed evolution may be required for any improvements to be seen.
In industry, whole cell biocatalysts predominate as they present a number of advantages for large scale processes.3,7 Cells can protect enzymes from harsh external reaction conditions, thus maintaining structure and activity without the need to explore and optimise an immobilisation strategy. Preparation of whole cell biocatalysts is simple, not requiring lengthy protein purification procedures, and more complicated, multi-step syntheses that require multiple enzymes can all be contained inside single cells and expensive cofactors can be recycled by internal cellular mechanisms.8 Potential challenges of whole cell biocatalysts when compared to purified enzymes include increased mass transfer limitations and unwanted side reactions. In addition, although cells are able to protect enzymes from harsh reaction conditions, these harsh conditions may be toxic to cells. In some cases, biphasic systems have been used to great effect and the development of ionic solvents has minimised the problem of organic solvent toxicity but with added expense (for recent reviews see Murphy 2012 and Zhao 2010).9,10
Whole cells can also be immobilised inside calcium alginate beads for continuous flow processes,11 this however reduces the mass to catalytic activity ratio and requires intricate preparation steps which add to the cost. As a result the vast majority of whole cell catalysed biotransformation reactions are still performed as batch processes.2 An alternative immobilisation method makes use of the natural phenomenon of biofilms. Because of their increased antibiotic resistance and mechanical toughness biofilms are generally perceived as being problematic, both medically and in industry, for example in reactor fouling.23,24 However, recent studies have shown that these characteristics make biofilms an attractive biocatalytic platform as they can potentially withstand much harsher conditions than planktonic cells.25
Fig. 1 Cartoon illustrating biofilm formation by motile bacteria. Motile bacteria actively travel to a surface and attach via adhesins. Once attached some motility is lost and further adhesins and EPS components are made, generating a matrix. Continued twitching motility can give rise to mushroom shaped colonies and water channels. Variation in local conditions also leads to phenotypic diversification. Entrapped cell dispersion can be triggered by the action of shearing forces and/or the availability of nutrients. |
Microbial species | Use/Function | Reference |
---|---|---|
Wild type biofilms | ||
Thiomonas arsenivorans | Bioremediation of arsenic contaiminated mining effluent | Michel 200712 |
Zymomonas mobilis | Production of ethanol | Weuster-Botz 199313 |
Saccharomyces cerevisiae | Kunduru 199614 | |
Caldicellulosiruptor saccharolyticus | Generation of hydrogen | van-Groenestijn 200915 |
Clostridium beijerinckii | Production of butanol | Lienhardt 200216 |
Lactobacillus casei | Production of lactic acid | Demirci 200317 |
Pseudomonas sp. VLB120ΔC | Transformation of (S)-styrene oxide from styrene | Gross 200718 |
Gluconobacter oxydans | Production of dihydroxyacetone from glycerol | Hekmat 200719 |
Recombinant biofilms | ||
E. coli KO11 | Production of ethanol | Zhou 200820 |
Acetobacter xylinum | D-amino acid oxidase | Setyawati 200921 |
E. coli PHL644 | Biotransformation of haloindole into L-halotryptophan | Tsoligkas 201122 |
The EPS consists of a mixture of different biopolymers, including nucleic acids, proteins, and polysaccharides, responsible for a range of diverse functions.27 It is thought that this matrix of EPS together with the lower metabolic rate generally seen within biofilms contributes to their robustness when faced with harsh environments.28 However, EPS can present a potential problem for biotransformations if over-produced. An excess of EPS takes up room in bioreactors, sloughing of this matrix can complicate product purification and EPS can limit mass transfer, decreasing product yield.2 In one study, in which the cellular component of an Acetobacter xylinum biofilm grown in a shaken culture was only 25% of the entire biofilm mass, the activity of D-amino acid oxidase (DAAO) was six times lower than that exhibited by self-immobilised cells grown in static culture.21
The generation of industrially useful chemicals has been demonstrated using a Pseudomonas sp. VLB120ΔC biofilm for the production of (S)-styrene oxide (2). with a yield of 16 g Laq−1 day−1,18 as a model reaction to demonstrate prolonged asymmetric epoxide activity (see Scheme 1). In addition to protecting the cells from the toxic product, the immobilised nature of the biofilm also allowed in situ removal of product throughout the reaction.
Scheme 1 Pseudomonas catalysed biotransformation of (S)-styrene oxide (2) from styrene (1).18 |
The process was stable for 55 days, using silicone tubing as the substratum in the bioreactor. Later experiments increased the yield to a maximum of 70 g Laq−1 day−1, with the process appearing to select for a biofilm-forming phenotype.39
The production of the fine chemical dihydroxyacetone (4) from glycerol (3) by Gluconobacter oxydans in a fed-batch process was increased when using self-immobilised cells compared with planktonic cells (Scheme 2).19 However, only about 65% of the cells were immobilised, probably due to the strictly aerobic culture requirements of G. oxydans.
Scheme 2 Conversion of glycerol (3) into dihydroxyacetone (4) mediated by a Gluconobacter oxydans biofilm.19 |
The most recent study to use recombinant cells in a biofilm demonstrated the conversion of haloindoles and serine to L-halotryptophans (7) (see Scheme 3) using the biofilm-forming strain E. coli PHL644.22 The strain was transformed with the tryptophan synthase genes from Salmonella enterica sv Typhimurium, and artificially engineered biofilms were produced by spin-coating E. coli cultures onto glass slides which were then left to mature for 7 days in minimal media. The engineered biofilms matured more rapidly than naturally-formed biofilms, adhering more firmly to the glass substrate, and had formed mushroom structures, pores and channels by day 6 (see Fig. 2).40 Higher conversions were obtained than with cell-free-lysate, immobilised enzyme, or planktonic cells at twice the biomass, and in three sequential biotransformations for the production of 5-chloroindole, no loss of activity was observed. Although these initial investigations were conducted in batch the authors note that the immobilised nature of the biofilm and the lack of observed drop in yield after recycling the biofilm opens up the possibility for conducting these types of biotransformation reaction as a component of flow chemistry.
Scheme 3 Tryptophan synthase mediated biotransformation of L-halotryptophan (7) from haloindole (5) and L-serine (6) catalysed by recombinant E. coli PHL644 biofilms. X = F, Cl or Br.22 |
Fig. 2 Environmental scanning electron micrograph of an engineered E. coli biofilm on day 6 after attachment, showing the presence of EPS (white fibrous material) and the development of mushroom structures, pores and channels.22 |
In these last two cases the stated goal was to produce a general, easily formed and adaptable biofilm platform, composed of a recombinant organism, designed to be able to perform a range of required biotransformations.
The possibility of engineering biofilms from recombinant single species provides an attractive platform for plug-and-play biocatalysis, despite the mass transfer problems encountered by some workers. It could be envisioned that metabolic engineering and synthetic biology tools currently used to develop novel enzymatic reactions and metabolic routes within cells could be expanded to encompass biofilm catalysts, allowing development of multiple platform technologies to respond to challenging requirements in biocatalysis. In particular, biofilm catalysts could be envisioned to assist in the development of novel biotransformations involving highly toxic solvents, substrates or products using well-studied and genetically tractable organisms such as E. coli without the time-consuming necessity of isolating novel organisms that have resistance to the reaction conditions. In the last few years there has been a surge of interest in the potential of biofilms to perform biotransformation reactions,22,41 and engineered biofilms in particular appear to offer a low cost, high efficiency alternative to traditional fed-batch processes, and should be sufficiently flexible regarding the generation of a range of fine chemicals to warrant further study in this area.
This journal is © The Royal Society of Chemistry 2012 |