Baris R. Mutlu

Silicon alkoxide cross-linked silica nanoparticle gels for encapsulation of bacterial biocatalysts

A method is developed for encapsulation of bacterial biocatalysts in silica gels formed by silica nanoparticles (SNP) and a silicon alkoxide crosslinker. Formulation of the gel was optimized by changing the SNP size, SNP to crosslinker ratio and crosslinker functionality. Hydrolysis and condensation reactions of silicon alkoxide were controlled by water to alkoxide ratio (r) and pH of the solution. FTIR analysis verified that a reactive and temporally stable silicon alkoxide crosslinker was obtained. As a case study, recombinant Escherichia coli (E. coli) cells expressing the atrazine dechlorinating enzyme AtzA were encapsulated. Synthesized catalytic biomaterials (silica gel encapsulated bacterial biocatalysts) were evaluated based on their gelation time, biocatalytic activity and mechanical strength. Diffusivity assays and SEM were used for characterization of the gel structure. We found that SNP to crosslinker ratio affected all the features of the gel, whereas crosslinker functionality primarily affected the gelation time and SNP size affected the mechanical strength and diffusivity. Based on systematic evaluation, we selected three gel formulations and subjected them to long-term activity measurements in a continuous-flow bioreactor for removing trace levels of atrazine. The effluent atrazine concentration was sustained below 30% of the influent concentration, <3 ppb, for 2 months.

Manufacturing of bioreactive nanofibers for bioremediation

Recombinant Escherichia coli (E. coli) cells were successfully encapsulated in reactive membranes comprised of electrospun nanofibers that have biocompatible polyvinyl alcohol (PVA)‐based cores entrapping the E. coli and silica‐based, mechanically sturdy porous shells. The reactive membranes were produced in a continuous fashion using a coaxial electrospinning system coupled to a microfluidic timer that mixed and regulated the reaction time of the silica precursor and the PVA solution streams. A factorial design method was employed to investigate the effects of the three critical design parameters of the system (the flow rate of the core solution, protrusion of the core needle, and the viscosity of the core solution) and to optimize these parameters for reproducibly and continuously producing high‐quality core/shell nanofibers. The feasibility of using the reactive membranes manufactured in this fashion for bioremediation of atrazine, a herbicide, was also investigated. The atrazine degradation rate (0.24 µmol/g of E. coli/min) of the encapsulated E. coli cells expressing the atrazine‐dechlorinating enzyme AtzA was measured to be relatively close to that measured with the free cells in solution (0.64 µmol/g of E. coli/min). We show here that the low cost, high flexibility, water insolubility, and high degradation efficiency of the bioreactive membranes manufactured with electrospinning makes it feasible for their wide‐spread use in industrial scale bioremediation of contaminated waters. Biotechnol. Bioeng. 2014;111: 1483–1493.

Bacterial Cyanuric Acid Hydrolase for Water Treatment

ABSTRACT Di- and trichloroisocyanuric acids are widely used as water disinfection agents, but cyanuric acid accumulates with repeated additions and must be removed to maintain free hypochlorite for disinfection. This study describes the development of methods for using a cyanuric acid-degrading enzyme contained within nonliving cells that were encapsulated within a porous silica matrix. Initially, three different bacterial cyanuric acid hydrolases were compared: TrzD from Acidovorax citrulli strain 12227, AtzD from Pseudomonas sp. strain ADP, and CAH from Moorella thermoacetica ATCC 39073. Each enzyme was expressed recombinantly in Escherichia coli and tested for cyanuric acid hydrolase activity using freely suspended or encapsulated cell formats. Cyanuric acid hydrolase activities differed by only a 2-fold range when comparing across the different enzymes with a given format. A practical water filtration system is most likely to be used with nonviable cells, and all cells were rendered nonviable by heat treatment at 70°C for 1 h. Only the CAH enzyme from the thermophile M. thermoacetica retained significant activity under those conditions, and so it was tested in a flowthrough system simulating a bioreactive pool filter. Starting with a cyanuric acid concentration of 10,000 μM, more than 70% of the cyanuric acid was degraded in 24 h, it was completely removed in 72 h, and a respike of 10,000 μM cyanuric acid a week later showed identical biodegradation kinetics. An experiment conducted with water obtained from municipal swimming pools showed the efficacy of the process, although cyanuric acid degradation rates decreased by 50% in the presence of 4.5 ppm hypochlorite. In total, these experiments demonstrated significant robustness of cyanuric acid hydrolase and the silica bead materials in remediation.

Silica ecosystem for synergistic biotransformation.

Synergistical bacterial species can perform more varied and complex transformations of chemical substances than either species alone, but this is rarely used commercially because of technical difficulties in maintaining mixed cultures. Typical problems with mixed cultures on scale are unrestrained growth of one bacterium, which leads to suboptimal population ratios, and lack of control over bacterial spatial distribution, which leads to inefficient substrate transport. To address these issues, we designed and produced a synthetic ecosystem by co-encapsulation in a silica gel matrix, which enabled precise control of the microbial populations and their microenvironment. As a case study, two greatly different microorganisms: Pseudomonas sp. NCIB 9816 and Synechococcus elongatus PCC 7942 were encapsulated. NCIB 9816 can aerobically biotransform over 100 aromatic hydrocarbons, a feat useful for synthesis of higher value commodity chemicals or environmental remediation. In our system, NCIB 9816 was used for biotransformation of naphthalene (a model substrate) into CO2 and the cyanobacterium PCC 7942 was used to provide the necessary oxygen for the biotransformation reactions via photosynthesis. A mathematical model was constructed to determine the critical cell density parameter to maximize oxygen production, and was then used to maximize the biotransformation rate of the system.

Non-equilibrium Inertial Separation Array for High-throughput, Large-volume Blood Fractionation

Microfluidic blood processing is used in a range of applications from cancer therapeutics to infectious disease diagnostics. As these applications are being translated to clinical use, processing larger volumes of blood in shorter timescales with high-reliability and robustness is becoming a pressing need. In this work, we report a scaled, label-free cell separation mechanism called non-equilibrium inertial separation array (NISA). The NISA mechanism consists of an array of islands that exert a passive inertial lift force on proximate cells, thus enabling gentler manipulation of the cells without the need of physical contact. As the cells follow their size-based, deterministic path to their equilibrium positions, a preset fraction of the flow is siphoned to separate the smaller cells from the main flow. The NISA device was used to fractionate 400 mL of whole blood in less than 3 hours, and produce an ultrapure buffy coat (96.6% white blood cell yield, 0.0059% red blood cell carryover) by processing whole blood at 3 mL/min, or ∼300 million cells/second. This device presents a feasible alternative for fractionating blood for transfusion, cellular therapy and blood-based diagnostics, and could significantly improve the sensitivity of rare cell isolation devices by increasing the processed whole blood volume.

Performance of a composite bioactive membrane for H2 production and capture from high strength wastewater

In this study, a composite bioactive membrane was developed and tested to generate and capture hydrogen (H2) during the process of wastewater treatment. Hollow fiber membranes were coated with encapsulated acetogenic bacteria to simultaneously produce and capture H2 from waste feedstocks. Acetogens were encapsulated with cast poly(vinylalcohol) or electrospun microfibers. Under anaerobic conditions the poly(vinylalcohol) and electrospun composite membranes produced an average of 44.6 ± 11.3 mL H2 g−1 hexose (0.33 ± 0.08 mol H2 mol−1 hexose) and 21.2 ± 4.8 mL H2 g−1 hexose (0.16 ± 0.04 mol H2 mol−1 hexose), respectively, and captured 73 ± 12% and 57 ± 11%, respectively, of the total H2 produced in bioreactors fed synthetic high strength wastewater. The H2 capture efficiency of the electrospun composite membrane was improved by coating the modules with a thin film of polymeric silica gel, improving the H2 production to 28.3 ± 2.3 mL H2 per hexose (0.21 ± 0.02 mol H2 mol−1 hexose) and the H2 capture efficiency to 73 ± 15%. Final composite membranes were built by immobilizing bacteria directly onto the membrane surface, again improving H2 yields from high strength synthetic wastewater to a maximum of 48.4 ± 9.4 mL H2 g−1 hexose (0.36 ± 0.07 mol H2 mol−1 hexose) with a maximum H2 capture efficiency of 86 ± 9%. The optimized composite membranes were also capable of generating and capturing H2 from real wastewaters, with yields and capture efficiencies of 19.2 ± 3.0 mL H2 g−1 hexose (0.14 ± 0.02 mol H2 mol−1 hexose) and 99.1 ± 0.2%, and 46.0 ± 15.5 mL H2 g−1 hexose (0.34 ± 0.12 mol H2 mol−1 hexose) and 79 ± 19% when tested with a feed of sugar beet wastewater and dairy production wastewater, respectively. After further optimization, the composite membrane system could allow the extraction of high-quality energy from wastewater.

Engineering of a silica encapsulation platform for hydrocarbon degradation using Pseudomonas sp. NCIB 9816-4

Industrial application of encapsulated bacteria for biodegradation of hydrocarbons in water requires mechanically stable materials. A silica gel encapsulation method was optimized for Pseudomonas sp. NCIB 9816‐4, a bacterium that degrades more than 100 aromatic hydrocarbons. The design process focused on three aspects: (i) mechanical property enhancement; (ii) gel cytocompatibility; and (iii) reduction of the diffusion barrier in the gel. Mechanical testing indicated that the compressive strength at failure (σf) and elastic modulus (E) changed linearly with the amount of silicon alkoxide used in the gel composition. Measurement of naphthalene biodegradation by encapsulated cells indicated that the gel maintained cytocompatibility at lower levels of alkoxide. However, significant loss in activity was observed due to methanol formation during hydrolysis at high alkoxide concentrations, as measured by FTIR spectroscopy. The silica gel with the highest amount of alkoxide (without toxicity from methanol) had a biodegradation rate of 285 ± 42 nmol/L‐s, σf = 652 ± 88 kPa, and E = 15.8 ± 2.0 MPa. Biodegradation was sustained for 1 month before it dropped below 20% of the initial rate. In order to improve the diffusion through the gel, polyvinyl alcohol (PVA) was used as a porogen and resulted in a 48 ± 19% enhancement in biodegradation, but it impacted the mechanical properties negatively. This is the first report studying how the silica composition affects biodegradation of naphthalene by Pseudomonas sp. NCIB 9816‐4 and establishes a foundation for future studies of aromatic hydrocarbon biodegradation for industrial application. Biotechnol. Bioeng. 2016;113: 513–521.

Oscillatory inertial focusing in infinite microchannels

Significance Inertial microfluidics is a widely used technology which enables label-free manipulation of particles in microchannels. However, this technology has been limited to bioparticles larger than RBCs, due to the strong correlation between the inertial lift forces and the particle size. This paper presents a method to extend the capabilities of inertial microfluidics to smaller bioparticles, of which a plethora of clinically relevant types exist in the human body. Therefore, this method can be integrated with microfluidic devices for inertial manipulation of bioparticles that have defied all prior attempts, enabling a variety of applications in clinical diagnosis including cytometry of micron-scale bioparticles, isolation and characterization of pathogens and extracellular microvesicles, or phenotyping of cancer or stem cells at physiological shear stresses. Inertial microfluidics (i.e., migration and focusing of particles in finite Reynolds number microchannel flows) is a passive, precise, and high-throughput method for microparticle manipulation and sorting. Therefore, it has been utilized in numerous biomedical applications including phenotypic cell screening, blood fractionation, and rare-cell isolation. Nonetheless, the applications of this technology have been limited to larger bioparticles such as blood cells, circulating tumor cells, and stem cells, because smaller particles require drastically longer channels for inertial focusing, which increases the pressure requirement and the footprint of the device to the extent that the system becomes unfeasible. Inertial manipulation of smaller bioparticles such as fungi, bacteria, viruses, and other pathogens or blood components such as platelets and exosomes is of significant interest. Here, we show that using oscillatory microfluidics, inertial focusing in practically “infinite channels” can be achieved, allowing for focusing of micron-scale (i.e. hundreds of nanometers) particles. This method enables manipulation of particles at extremely low particle Reynolds number (Rep < 0.005) flows that are otherwise unattainable by steady-flow inertial microfluidics (which has been limited to Rep > ∼10−1). Using this technique, we demonstrated that synthetic particles as small as 500 nm and a submicron bacterium, Staphylococcus aureus, can be inertially focused. Furthermore, we characterized the physics of inertial microfluidics in this newly enabled particle size and Rep range using a Peclet-like dimensionless number (α). We experimentally observed that α >> 1 is required to overcome diffusion and be able to inertially manipulate particles.

Adsorption and Biodegradation of Aromatic Chemicals by Bacteria Encapsulated in a Hydrophobic Silica Gel

An adsorbent silica biogel material was developed via silica gel encapsulation of Pseudomonas sp. NCIB 9816-4, a bacterium that degrades a broad spectrum of aromatic pollutants. The adsorbent matrix was synthesized using silica precursors methyltrimethoxysilane and tetramethoxysilane to maximize the adsorption capacity of the matrix while maintaining a highly networked and porous microstructure. The encapsulated bacteria enhanced the removal rate and capacity of the matrix for an aromatic chemical mixture. Repeated use of the material over four cycles was conducted to demonstrate that the removal capacity could be maintained with combined adsorption and biodegradation. The silica biogel can thus be used extensively without the need for disposal, as a result of continuous biodegradation by the encapsulated bacteria. However, an inverse trend was observed with the ratio of biodegradation to adsorption as a function of log Kow, suggesting increasing mass-transport limitation for the most hydrophobic chemicals used (log Kow > 4).