Permeabilization and immobilization of whole-cell Pseudomonas nitroreducens SP.001 to improve its l-glutaminase performance.

BACKGROUND: L-Glutaminase is considered to be an important industrial enzyme in both the pharmaceutical and food industries, especially for producing functional glutamyl compounds, such as l-theanine. Pseudomonas nitroreducens SP.001 with intracellular l-glutaminase activity has been screened previously. In the present study, three physical permeabilization methods were used to improve l-glutaminase activity. Then, the whole-cell immobilization conditions of permeabilized cells using sodium alginate as an embedding agent were optimized to enhance the enzyme's stability and reusability. The characteristics of the immobilized cells were investigated in comparison with those of permeabilized cells. RESULTS: The results obtained showed that cell permeabilization using osmotic shock with 155 g L-1 sucrose markedly improved enzyme activity. Then, an effective procedure for immobilization of permeabilized P. nitroreducens cells was established. The optimum conditions for cell immobilization were: sodium alginate 40 g L-1 , calcium chloride 30 g L-1 , cell mass 100 g L-1 and a curing time of 3 h. After successful immobilization, characterization studies revealed that the thermostability and pH resistance of l-glutaminase from immobilized cells were enhanced compared to those from permeabilized cells. Moreover, the immobilized biocatalyst could be reused up to 10 times and retained 80% of its activity. CONCLUSION: The stability and reusability of the permeabilized cells were improved through the immobilization. These findings indicated that immobilized whole-cell l-glutaminase from P. nitroreducens SP.001 possesses more potential for various industrial biotechnological applications than free cells. © 2020 Society of Chemical Industry.

Purification and Biochemical Characterization of a Tyrosine Phenol-lyase from Morganella morganii.

Tyrosine phenol-lyase (TPL) is a valuable and cost-effective biocatalyst for the biosynthesis of L-tyrosine and its derivatives, which are valuable intermediates in the pharmaceutical industry. A TPL from Morganella morganii (Mm-TPL) was overexpressed in Escherichia coli and characterized. Mm-TPL was determined as a homotetramer with molecular weight of 52 kDa per subunit. Its optimal temperature and pH for ß-elimination of L-tyrosine were 45 °C and pH 8.5, respectively. Mm-TPL manifested strict substrate specificity for the reverse reaction of ß-elimination and ortho- and meta-substituted phenols with small steric size were preferred substrates. The enzyme showed excellent catalytic performance for synthesis of L-tyrosine, 3-fluoro-L-tyrosine, and L-DOPA with a yield of 98.1%, 95.1%, and 87.2%, respectively. Furthermore, the fed-batch bioprocess displayed space-time yields of 9.6 g L-1 h-1 for L-tyrosine and 4.2 g L-1 h-1 for 3-fluoro-L-tyrosine with a yield of 67.4 g L-1 and 29.5 g L-1, respectively. These results demonstrated the great potential of Mm-TPL for industrial application.

Modulating the properties of the lipase from Thermomyces lanuginosus immobilized on octyl agarose beads by altering the immobilization conditions.

The lipase from Thermomyces lanuginosus (TLL) has been immobilized on octyl-agarose beads via interfacial activation under 16 different conditions (changing the immobilization pH, the ionic strength, the presence of additives like calcium, phosphate or glycerol) and using a low loading (1 mg/g support). Then, the properties of the different biocatalysts have been evaluated: stability at pH 7.0 and 70 °C and activity versus p-nitro phenyl propionate, triacetin and R- and S- methyl mandelate. Results clearly indicate that the immobilization conditions determine the final enzyme properties, altering enzyme stability (by 10 folds), activity (by 8 folds using R- methyl mandelate) and specificity (VR/VS changed from 0.7 to 2.3 using mandelate esters). For instance, the enzymes immobilized at pH 7.0 using 5 mM buffer were the most stable preparations, while the presence of 250 mM sodium phosphate greatly decreased the final enzyme stability. The biocatalyst stability of TLL increased with increasing NaCl in the immobilization buffer at pH 5. Fluorescence studies confirmed that the conformation of the different immobilized enzymes were different, despite being a physical and reversible immobilization method. Thus, the immobilization of TLL on octyl agarose beads under different conditions produced biocatalysts with different properties, the optimal condition depends on the studied reaction and condition.

Haloferax volcanii as immobilised whole cell biocatalyst: new applications for halophilic systems.

Enzyme-mediated synthesis of pharmaceutical compounds is a 'green' alternative to traditional synthetic chemistry, and microbial engineering opens up the possibility of using whole cells as mini-factories. Whole-cell biocatalysis reduces cost by eliminating expensive enzyme purification and cofactor addition steps, as well as resulting in increased enzyme stability. Haloferax volcanii is a model halophilic archaeon encoding highly salt and organic solvent tolerant enzymes such as alcohol dehydrogenase (HvADH2), which catalyses the reduction of aldehydes and ketone in the presence of NADPH/NADH cofactor. A H. volcanii strain for constitutive HvADH2 expression was generated using a strong synthetic promoter (p.syn). The strain was immobilised in calcium alginate beads and repeatedly used as a whole-cell biocatalyst. The reduction of acetophenone, used as test substrate, was very successful and high yields were detected from immobilised whole cells over repeated biotransformation cycles. The immobilised H. volcanii retained stability and high product yields after 1 month of storage at room temperature. This newly developed system offers halophilic enzyme expression in its native environment, high product yield, stability and reusability without the addition of any expensive NADPH/NADH cofactor. This is the first report of whole cell-mediated biocatalysis by the halophilic archaeon H. volcanii.

Contribution of cellulose synthesis, formation of fibrils and their entanglement to the self-flocculation of Zymomonas mobilis.

Due to the unique Entner-Doudoroff pathway, Zymomonas mobilis has been acknowledged as a potential host to be engineered for biorefinery to produce biofuels and biobased chemicals. The self-flocculation of Z. mobilis can make the bacterial cells self-immobilized within bioreactors for high density to improve product productivities, and in the meantime enhance their tolerance to stresses, particularly product inhibition and the toxicity of byproducts released during the pretreatment of lignocellulosic biomass. In this work, we explored mechanism underlying such a phenotype with the self-flocculating strain ZM401 developed from the regular non-flocculating strain ZM4. Cellulase de-flocculation and the restoration of the self-flocculating phenotype for the de-flocculated bacterial cells subjected to culture confirmed the essential role of cellulose biosynthesis in the self-flocculation of ZM401. Furthermore, the deactivation of both Type I and Type IV restriction-modification systems was performed for ZM4 and ZM401 to improve their transformation efficiencies. Comparative genome analysis detected the deletion of a thymine from ZMO1082 in ZM401, leading to a frame-shift mutation for the putative gene to be integrated into the neighboring downstream gene ZMO1083 encoding the catalytic subunit A of cellulose synthase, and consequently created a new gene to encode a larger transmembrane protein BcsA_401 for more efficient synthesis of cellulose as well as the development of cellulose fibrils and their entanglement for the self-flocculation of the mutant. These speculations were confirmed by the morphological observation of the bacterial cells under scanning electron microscopy, the impact of the gene deletion on the self-flocculation of ZM401, and the restoration of the self-flocculating phenotype of ZM401 ΔbcsA by the gene complementation. The progress will lay a foundation not only for fundamental research in deciphering molecular mechanisms underlying the self-flocculation of Z. mobilis and stress tolerance associated with the morphological change but also for technological innovations in engineering non-flocculating Z. mobilis and other bacterial species with the self-flocculating phenotype.

Co-immobilized Whole Cells with ω-Transaminase and Ketoreductase Activities for Continuous-Flow Cascade Reactions.

An improved sol-gel process involving the use of hollow silica microspheres as a supporting additive was applied for the co-immobilization of whole cells of Escherichia coli with Chromobacterium violaceum ω-transaminase activity and Lodderomyces elongisporus with ketoreductase activity. The co-immobilized cells with two different biocatalytic activities could perform a cascade of reactions to convert racemic 4-phenylbutan-2-amine or heptan-2-amine into a nearly equimolar mixture of the corresponding enantiomerically pure R amine and S alcohol even in continuous-flow mode. The novel co-immobilized whole-cell system proved to be an easy-to-store and durable biocatalyst.

Production of R-Mandelic Acid Using Nitrilase from Recombinant E. coli Cells Immobilized with Tris(Hydroxymethyl)Phosphine.

Recombinant Escherichia coli cells harboring nitrilase from Alcaligenes faecalis were immobilized using tris(hydroxymethyl)phosphine (THP) as the coupling agent. The optimal pH and temperature of the THP-immobilized cells were determined at pH 8.0 and 55 °C. The half-lives of THP-immobilized cells measured at 35, 40, and 50 °C were 1800, 965, and 163 h, respectively. The concentration of R-mandelic acid (R-MA) reached 358 mM after merely 1-h conversion by the immobilized cells with 500 mM R,S-mandelonitrile (R,S-MN), affording the highest productivity of 1307 g L-1 day-1 and the space-time productivity of 143.2 mmol L-1 h-1 g-1. The immobilized cells with granular shape were successfully recycled for 60 batches using 100 mM R,S-MN as substrate at 40 °C with 64% of relative activity, suggesting that the immobilized E. coli cells obtained in this study are promising for the production of R-MA.

Immobilization in polyvinyl alcohol hydrogel enhances yeast storage stability and reusability of recombinant laccase-producing S. cerevisiae.

OBJECTIVES: To improve the storage stability and reusability of various yeast strains and species by immobilization in polyvinyl alcohol (PVA) hydrogel particles. RESULTS: Debaryomyces hansenii, Pichia sorbitophila, Saccharomyces cerevisiae, Yarrowia lipolytica, and Zygosaccharomyces rouxii were immobilized in PVA particles using LentiKats technology and stored in sterile water at 4 °C. The immobilization improved the survival of all species; however, the highest storage stability was achieved for S. cerevisiae and Y. lipolytica which survived more than 1 year, in contrast to free cells that survived for only 3 months. Tests of the reusability of immobilized recombinant laccase-secreting S. cerevisiae revealed that the cells were suitable for repetitive use (55 cycles during 15 months) even after storage in water at 4 °C for 9 months. A suitable method for killing immobilized laccase-secreting cells without affecting the produced enzyme activity was also developed. CONCLUSIONS: The immobilization of yeasts in PVA hydrogel enables long-term, cheap storage with very good cell viability and productivity, thus becoming a promising approach for industrial applications.

Optimization of alginate microcapsules containing cells overexpressing α-l-iduronidase using Box-Behnken design.

Mucopolysaccharidosis type I (MPS I) is an autosomal recessive disease caused by deficiency of α-l-iduronidase (IDUA), which results in the lysosomal accumulation of glycosaminoglycans (GAG) leading to widespread clinical manifestations. The microencapsulation of IDUA overexpressing recombinant cells has been considered as a promising strategy for the treatment of MPS I. This study aimed at the optimization of alginate microcapsules containing recombinant BHK (Baby Hamster Kidney) cells (rBHK) overexpressing IDUA produced by electrostatic extrusion technique. The alginate microcapsule (MC-A) optimization study was carried out by means of an experimental Box-Behnken Design that allowed the simultaneous evaluation of the influence of voltage (kV), alginate/cell suspension flow (mL/h), and alginate concentration (%) on size and IDUA activity. The optimal conditions of voltage (10kV), flow (25mL/h), and alginate concentration (1.3%) made possible to obtain the smallest microcapsules showing the highest IDUA activity. After optimization, the microcapsules were sequentially coated with PLL and alginate (MC-APA) to increase their stability. MC-A and MC-APA presented monodisperse populations (span<1.22) with an average diameter of less than 350µm. The coating increased the mechanical stability of MC-APA by about 6-fold and modulated the permeability to the enzyme. Surface analyzes of MC-APA showed the presence of PLL bands, suggesting that the last alginate layer appears to have only partially coated the PLL. After 30days of subcutaneous implantation of the MC-APA microcapsules containing rBHK cells in a MPS I murine model, a significant increase in IDUA activity was observed in the skin near the implant. Histological analysis revealed an inflammatory infiltrate at the application site, which did not prevent the release of the enzyme under the conditions evaluated. Taken together, the overall results demonstrate the feasibility of MC-APA as a potential alternative for local treatment of MPS I.

Enhanced Production of κ-Carrageenase and κ-Carrageenan Oligosaccharides through Immobilization of Thalassospira sp. Fjfst-332 with Magnetic Fe3O4-Chitosan Microspheres.

In this study, immobilized bacteria (IMB) microsphere was prepared by embedding κ-carrageenase-producing Thalassospira sp. Fjfst-332 (TF332) onto a magnetic Fe3O4-chitosan carrier. The performance of Fe3O4-chitosan carrier was optimized by comparing its bacteria immobilization capacity at different Fe3O4:chitosan ratios and temperatures, while the functions of IMB microspheres were characterized by examining their κ-carrageenase production at different temperatures, pH's, and reuse cycles. At the 1:1 (w:w) Fe3O4:chitosan ratio, the Fe3O4-chitosan carriers possessed sufficient anchoring capacity for bacterial immobilization without significant compromise of their magnetism for magnetic separation of IMB from culture media. The spectroscopic analysis of IMB microspheres indicated that the immobilization of TF332 might affect the amide groups in chitosan. Compared to free bacteria, IMB can produce κ-carrageenase at higher temperature, wider pH range, and faster rate. More importantly, the κ-carrageenase-producing activity was sustained for at least seven reuse cycles. The major κ-carrageenan degradation products of IMB-derived κ-carrageenase were the oligosaccharides containing two to six monosaccharide units. Overall, this Fe3O4-chitosan-TF-332 microsphere has the potential to become a stable and reusable platform for large-scale production of κ-carrageenan oligosaccharides.

Using concanavalinA as a spacer for immobilization of E. coli onto magnetic nanoparticles.

ConcanavalinA (conA) is a protein extracted from the concanavalin, which has specific recognition through mannose components on bacterial cell surfaces. A magnetic nanocarrier with the structure of a dopamine functionalized magnetic nanoparticles was grafted with conA, and was used for immobilization of recombinant Escherichia coli harboring glycerol dehydrogenase with the specific recognition between glycoconjugates and glycoprotein. The effect of various factors on the immobilization including temperature, pH, cell concentration and immobilization time were investigated. The highest immobilization yield of 91% was obtained under the conditions: enzyme/support 1.28mg/mg, pH 8.0, immobilization time 2h and temperature 4°C. The obtained immobilized cell was characterized and exhibited higher thermal stability compared with the free cell. After ten cycles, the immobilized cell remained 62% initial activity. These results indicate that the cell immobilized onto conA-grafted nanoparticles by specific recognition of glycoconjugates and glycoprotein is a potential method for preparation of stable cell, and the immobilized cell showed perspective applications in the biocatalysis and biosensors.

Decolorization of textile dye RB19 using volcanic rock matrix immobilized Bacillus thuringiensis cells with surface displayed laccase.

A triplicate volcanic rock matrix-Bacillus thuringiensis-laccase WlacD (VRMs-Bt-WlacD) dye decolorization system was developed. WlacD was displayed on the B. thuringiensis MB174 cell surface to prepare a whole-cell laccase biocatalyst by using two repeat N-terminal domains of autolysin Mbg (Mbgn)2 as the anchoring motif. Immunofluorescence microscopic assays confirmed that the fusion protein (Mbgn)2-WlacD was anchored on the surface of the recombinant B. thuringiensis MB174. After optimization by a single factor test, L 9(34)-orthogonal test, Plackett-Burman test, steepest ascent method, and Box-Behnken response surface methodology, the whole-cell specific laccase activity of B. thuringiensis MB174 was improved to 555.2 U L-1, which was 2.25 times than that of the primary culture condition. Optimized B. thuringiensis MB174 cells were further adsorbed by VRMs to prepare VRMs-Bt-WlacD, an immobilized whole-cell laccase biocatalyst. Decolorization capacity of as-prepared VRMs-Bt-WlacD toward an initial concentration of 500 mg L-1 of an textile dye reactive blue 19 (RB19) aqueous solution reached 72.36% at a solid-to-liquid ratio of 10 g-100 mL. Repeated decolorization-activation operations showed the high decolorization capacity of VRMs-Bt-WlacD and have the potential for large-scale or continuous operations.

Production of cyclodextrin glycosyltransferase by immobilized Bacillus sp. on chitosan matrix.

The whole-cell immobilization on chitosan matrix was evaluated. Bacillus sp., as producer of CGTase, was grown in solid-state and batch cultivation using three types of starches (cassava, potato and cornstarch). Biomass growth and substrate consumption were assessed by flow cytometry and modified phenol-sulfuric acid assays, respectively. Qualitative analysis of CGTase production was determined by colorless area formation on solid culture containing phenolphthalein. Scanning electron microscopy (SEM) analysis demonstrated that bacterial cells were immobilized on chitosan matrix efficiently. Free cells reached very high numbers during batch culture while immobilized cells maintained initial inoculum concentration. The maximum enzyme activity achieved by free cells was 58.15 U ml(-1) (36 h), 47.50 U ml(-1) (36 h) and 68.36 U ml(-1) (36 h) on cassava, potato and cornstarch, respectively. CGTase activities for immobilized cells were 82.15 U ml(-1) (18 h) on cassava, 79.17 U ml(-1) (12 h) on potato and 55.37 U ml(-1) (in 6 h and max 77.75 U ml(-1) in 36 h) on cornstarch. Application of immobilization technique increased CGTase activity significantly. The immobilized cells produced CGTase with higher activity in a shorter fermentation time comparing to free cells.

Effect of alginate microencapsulation on the catalytic efficiency and in vitro enzyme-prodrug therapeutic efficacy of cytosine deaminase and of recombinant E. coli expressing cytosine deaminase.

Cytosine deaminase (CD) catalyses the enzymatic conversion of the non-toxic prodrug 5-fluorocytosine (5-FC) to the potent chemotherapeutic form, 5-fluorouracil (5-FU). Intratumoral delivery of CD localises chemotherapy dose while reducing systemic toxicity. Encapsulation in biocompatible microcapsules immunoisolates CD and protects it from degradation. We report on the effect of alginate encapsulation on the catalytic and functional activity of isolated CD and recombinant E. coli engineered to express CD (E. coli(CD)). Alginate microcapsules containing either CD or Escherichia coli(CD) were prepared using ionotropic gelation. Conversion of 5-FC to 5-FU was quantitated in unencapsulated and encapsulated CD/E. coli(CD) using spectrophotometry, with a slower rate of conversion observed following encapsulation. Both encapsulated CD/5-FC and E. coli(CD)/5-FC resulted in cell kill and reduced proliferation of 9 L rat glioma cells, which was comparable to direct 5-FU treatment. Our results show that encapsulation preserves the therapeutic potential of CD and E. coli(CD) is equally effective for enzyme-prodrug therapy.

Optical detection of paraoxon using single-walled carbon nanotube films with attached organophosphorus hydrolase-expressed Escherichia coli.

In whole-cell based biosensors, spectrophotometry is one of the most commonly used methods for detecting organophosphates due to its simplicity and reliability. The sensor performance is directly affected by the cell immobilization method because it determines the amount of cells, the mass transfer rate, and the stability. In this study, we demonstrated that our previously-reported microbe immobilization method, a microbe-attached single-walled carbon nanotube film, can be applied to whole-cell-based organophosphate sensors. This method has many advantages over other whole-cell organophosphate sensors, including high specific activity, quick cell immobilization, and excellent stability. A device with circular electrodes was fabricated for an enlarged cell-immobilization area. Escherichia coli expressing organophosphorus hydrolase in the periplasmic space and single-walled carbon nanotubes were attached to the device by our method. Paraoxon was hydrolyzed using this device, and detected by measuring the concentration of the enzymatic reaction product, p-nitrophenol. The specific activity of our device was calculated, and was shown to be over 2.5 times that reported previously for other whole-cell organophosphate sensors. Thus, this method for generation of whole-cell-based OP biosensors might be optimal, as it overcomes many of the caveats that prevent the widespread use of other such devices.

Isolation of the stable strain Labrys sp. BK-8 for L(+)-tartaric acid production.

A novel cis-epoxysuccinate hydrolase (CESH) producing strain of Labrys sp. BK-8 for production of L(+)-tartaric acid was isolated and identified. After optimization, a maximum activity of 3597 ± 151 U/g was achieved in batch culture in a 10 L fermentor. When Labrys sp. BK-8 was immobilized on κ-carrageenan, the immobilized cells showed a high conversion rate (>99%), enantioselectivity (EE > 99.5%) and storage stability (>90 d). A conversion rate of 97% was still achieved after 10 repeat batches. The CESH was stable over a broad range of temperatures (up to 45°C) and pH values (4.0-10.0). The Labrys sp. BK-8 isolate provides a new alternative with good stability for the industrial biosynthesis of L(+)-tartaric acid.

Enhancement of nuclease P1 production by Penicillium citrinum YL104 immobilized on activated carbon filter sponge.

The efficiency of current methods for industrial production of the enzyme nuclease P1 is limited. In this study, we sought to improve fermentation methods for the production of nuclease P1. An immobilized fermentation system using an activated carbon filter sponge as a carrier was used for the production of nuclease P1. In an airlift internal loop reactor (ALR), the fermentation performance of three different fermentation modes, including free-cell fermentation, repeated-batch fermentation, and semi-continuous immobilized fermentation, were compared. The fermentation kinetics in the fermentation broth of the three fermentation modes, including dissolved oxygen (DO), pH value, cell concentration, residual sugar concentration, and enzyme activity, were tested. The productivity of semi-continuous immobilized fermentation reached 8.76 U/mL/h, which was 33.3 and 80.2% higher than that of repeated-batch fermentation and free-cell fermentation, respectively. The sugar consumption of free-cell, repeated-batch, and semi-continuous immobilized fermentations was 41.2, 30.8, and 25.9 g/L, respectively. These results showed that immobilized-cell fermentation by using Penicillium citrinum with activated carbon filter sponge in an ALR was advantageous for nuclease P1 production, especially in the semi-continuous immobilized fermentation mode. In spite of the significant improvement in nuclease P1 production in semi-continuous immobilized fermentation mode, the specific activity of nuclease P1 was almost equal among the three fermentation modes.

Biotransformation of 1,3-propanediol cyclic sulfate and its derivatives to diols by toluene-permeabilized cells of Bacillus sp. CCZU11-1.

In this study, it was the first report that Bacillus sp. CCZU11-1 was used for the biotransformation of 1,3-propanediol cyclic sulfate (1,3-PDS) and its derivatives. The catalytic performance of Bacillus sp. sulfatase in the biotransformation of 1,3-PDS was significantly improved by biocatalyst permeabilization and immobilization. Using cell permeabilization, the hydrolytic activity of the whole-cell biocatalyst was increased by 3.5-fold after 1.5 h of pretreatment with 10 % (v/v) toluene at 30 °C and pH 7.0. Biotransformation of 20 mM 1,3-PDS for 24 h, 1,3-propanediol (1,3-PD) could be obtained in the yield of 97.4 % under the optimized reaction condition. Additionally, the immobilized biocatalysts, permeabilized cells entrapped in calcium alginate, and cross-linked enzyme aggregates were further employed to biotansform 1,3-PDS. Moreover, the total operational time of the immobilized biocatalysts could reach above 240 h with high conversion rate (>90 %).

Hydrogen photoproduction by immobilized n2-fixing cyanobacteria: understanding the role of the uptake hydrogenase in the long-term process.

We have investigated two approaches to enhance and extend H2 photoproduction yields in heterocystous, N2-fixing cyanobacteria entrapped in thin alginate films. In the first approach, periodic CO2 supplementation was provided to alginate-entrapped, N-deprived cells. N deprivation led to the inhibition of photosynthetic activity in vegetative cells and the attenuation of H2 production over time. Our results demonstrated that alginate-entrapped ΔhupL cells were considerably more sensitive to high light intensity, N deficiency, and imbalances in C/N ratios than wild-type cells. In the second approach, Anabaena strain PCC 7120, its ΔhupL mutant, and Calothrix strain 336/3 films were supplemented with N2 by periodic treatments of air, or air plus CO2. These treatments restored the photosynthetic activity of the cells and led to a high level of H2 production in Calothrix 336/3 and ΔhupL cells (except for the treatment air plus CO2) but not in the Anabaena PCC 7120 strain (for which H2 yields did not change after air treatments). The highest H2 yield was obtained by the air treatment of ΔhupL cells. Notably, the supplementation of CO2 under an air atmosphere led to prominent symptoms of N deficiency in the ΔhupL strain but not in the wild-type strain. We propose that uptake hydrogenase activity in heterocystous cyanobacteria not only supports nitrogenase activity by removing excess O2 from heterocysts but also indirectly protects the photosynthetic apparatus of vegetative cells from photoinhibition, especially under stressful conditions that cause an imbalance in the C/N ratio in cells.

BdlA, DipA and induced dispersion contribute to acute virulence and chronic persistence of Pseudomonas aeruginosa.

The human pathogen Pseudomonas aeruginosa is capable of causing both acute and chronic infections. Differences in virulence are attributable to the mode of growth: bacteria growing planktonically cause acute infections, while bacteria growing in matrix-enclosed aggregates known as biofilms are associated with chronic, persistent infections. While the contribution of the planktonic and biofilm modes of growth to virulence is now widely accepted, little is known about the role of dispersion in virulence, the active process by which biofilm bacteria switch back to the planktonic mode of growth. Here, we demonstrate that P. aeruginosa dispersed cells display a virulence phenotype distinct from those of planktonic and biofilm cells. While the highest activity of cytotoxic and degradative enzymes capable of breaking down polymeric matrix components was detected in supernatants of planktonic cells, the enzymatic activity of dispersed cell supernatants was similar to that of biofilm supernatants. Supernatants of non-dispersing ΔbdlA biofilms were characterized by a lack of many of the degradative activities. Expression of genes contributing to the virulence of P. aeruginosa was nearly 30-fold reduced in biofilm cells relative to planktonic cells. Gene expression analysis indicated dispersed cells, while dispersing from a biofilm and returning to the single cell lifestyle, to be distinct from both biofilm and planktonic cells, with virulence transcript levels being reduced up to 150-fold compared to planktonic cells. In contrast, virulence gene transcript levels were significantly increased in non-dispersing ΔbdlA and ΔdipA biofilms compared to wild-type planktonic cells. Despite this, bdlA and dipA inactivation, resulting in an inability to disperse in vitro, correlated with reduced pathogenicity and competitiveness in cross-phylum acute virulence models. In contrast, bdlA inactivation rendered P. aeruginosa more persistent upon chronic colonization of the murine lung, overall indicating that dispersion may contribute to both acute and chronic infections.