Microencapsulated bacterial cells can be used to produce the enzyme feruloyl esterase: preparation and in-vitro analysis

Biotechnological production of ferulic acid, a precursor of vanillin, is an attractive alternative for various industries due to the high price and demand for natural ferulic acid. Feruloyl esterase has been identified as a key enzyme involved in microbial transformations of ferulic acid to vanillin. Several fungal feruloyl esterases have been purified and characterized for their use in the production of ferulic acid. This paper, for the first time, discusses the use of lactic acid bacteria for the production of ferulic acid. Specifically, we have used Lactobacillus cells and microencapsulation so that ferulic acid can be produced continuously using various types of fermentation systems. Bacteria were encapsulated in alginate-poly-l-lysine-alginate (APA) microcapsules, and the production of ferulic acid by lactobacilli was detected using a real-time high-performance liquid chromatography (HPLC)-based assay. Results show that ferulic acid can be produced using microencapsulated Lactobacillus fermentum (ATCC 11976) with significant levels of biological feruloyl esterase activity.     

Nitrate and nitrite reduction by liquid membrane-encapsulated whole cells.

Purified enzymes and cell-free homogenates encapsulated by liquid-surfactant membrane have been shown to retain their catalytic activity (see previously published literature). This paper describes the preparation and properties of liquid-surfactant membrane-encapsulated whole cells of Micrococcus denitrificansATCC 21909. Batch and continuous studies with this model system have demonstrated that encapsulated viable cells reduce nitrates and retain their catalytic activity over anextended period of time. In batch operation, the reactivity of the encapsulated whole cells has been investigated under a variety of experimental conditions. The system is capable of reducing NO₃^? or NO₂^?. Data obtained indicate that encapsulated live cells have a broad pH and temperature optimum range. The encapsulated cells remain viable and do not “escape” into the external aqueous phase, even after five days of constant stirring with nitrate-containing simulated wastewater. Pulsed substrate addition experiments have demonstrated that the encapsulated cells also effectively reduce NO₂^? with no significant reduction in activity, even after 5.5 days of incubation at 30°C. The membrane selectivity for ion transfer has been achieved by incorporating oil-soluble ion exchangers in the membrane. Because of the protection of the liquid membranes, the catalytic reduction of NO₂^? by the encapsulated whole cells is not inhibited by 1 × 10^?4 M mercuric chloride, which is otherwise extremely toxic to the cells, when present in the external aqueous phase. Continuous reduction of 20 ppm of NO₂^? by liquid membrane-encapsulated whole cells has been demonstrated in a constantly stirred reactor over a test period of about one week. In this paper we will discuss the reduction of NO₃^?and NO₂^? by the liquid membrane-encapsulated whole cells of M. denitrificansATCC 21909 mainly in batch runs undera variety of experimental conditions, such as cell and substrate concentrations, product and inhibitor permeation, pH and temperature, effect of oil-soluble ion exchangers on the substrate diffusion, etc.     

Transport and survival of alginate-encapsulated and free lux-lac marked Pseudomonas aeruginosa UG2Lr cells in soil

Transport and survival of alginate-encapsulated and unencapsulated Pseudomonas aeruginosa UG2Lr through soil microcosms was examined. Bacterial cells encapsulated in alginate beads or mixed with soil were introduced into soil microcosms. Microbial cell survival and cell transport were monitored by destructive sampling and selective plating of the microcosms over a 9-week period. Survival rates were greatest when using encapsulated P. aeruginosa UG2Lr cells. Water flow increased microbial cell dispersal from the site of inoculation. After 3 weeks, encapsulated and free cells showed similar distribution patterns. However, after 9 weeks microbial cell distribution was more extensive throughout the soil in the encapsulated treatments under all conditions. Therefore, alginate encapsulation is a suitable method to enhance survival and distribution of microbial inocula in the soil environment.     

Environmental applications of immobilized microbial cells: A review

Immobilized microbial cells have been used extensively in various industrial and scientific endeavours. However, immobilized cells have not been used widely for environmental applications. This review examines many of the scientific and technical aspects involved in using immobilized microbial cells in environmental applications, with a particular focus on cells encapsulated in biopolymer gels. Some advantages and limitations of using immobilized cells in bioreactor studies are also discussed.     

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular weight metabolites in tissues of the treated host to achieve long-term survival. The semipermeable membrane allows engrafted encapsulated cells to avoid rejection by the immune system. The encapsulation procedure was designed to enable a controlled release of bioactive compounds, such as insulin, other hormones, and cytokines. Here we describe a method for encapsulation of catabolic cells, which consume lipids for heat production and energy dissipation (thermogenesis) in the intra-abdominal adipose tissue of obese mice. Encapsulation of thermogenic catabolic cells may be potentially applicable to the prevention and treatment of obesity and type 2 diabetes. Another potential application of catabolic cells may include detoxification from alcohols or other toxic metabolites and environmental pollutants.     

Immobilization in polyelectrolyte complex capsules: Encapsulation of a gluconate-oxidizing Serratia marcescens strain

In previous papers, it was shown that eukaryotic microbial systems can be encapsulated in polyelectrolyte complexes (PEC) prepared from sodium cellulose sulfate and poly(dimethyldiallylammonium chloride) with maintainance of vitality. In the present study, prokaryotic cells were successfully encapsulated in these PEC. Serratia marcescens B345 (IMET 11312) was chosen as a model organism. This strain converts gluconic acid to 2-ketogluconic acid. Since the 2-ketogluconic acid produced has very strong complexing properties, the number of applicable immobilization methods is restricted. Due to the high stability of PEC towards complexing agents, these problems can be overcome by the described method.

As already described in previous papers, a preimmobilization of cells in a PEC coprecipitate prior to capsule formation proved to be advantageous also for encapsulation of bacilli. The mean productivity of the encapsulated S. marcescens cells was 1–4.4 g l⁻¹ h⁻¹ in comparison to 5 g l⁻¹ h⁻¹ for free cells. The productivity was highly dependent on the flow rate of the reactor. The encapsulated cells were used for 1,200 h in a continuous biotransformation process for the production of 2-ketogluconic acid.     

Biooxidation of 2-phenylethanol to phenylacetic acid by whole-cell Gluconobacter oxydans biocatalyst immobilized in polyelectrolyte complex capsules

A high-performance biocatalyst in the form of encapsulated cells of Gluconobacter oxydans have been developed for production of phenylacetic acid (PAA) as a natural flavor component. Polyelectrolyte complex (PEC) capsules consisting of sodium alginate, cellulose sulfate, poly(methylene-co-guanidine), CaCl₂, and NaCl were used for highly controlled and mild encapsulation of cells. Utilization of encapsulated G. oxydans cells was a significant improvement on existing data on operational stability of cells and cumulative product concentration during biocatalytic production of PAA from 2-phenylethanol. Concerning operational stability, encapsulated cells were active over 12 cycles with a high biotransformation rate, while free cells were inactive after 7 cycles of use. The biocatalytic properties of encapsulated G. oxydans were tested in a bubble column reactor over 7 days with a final cumulative product concentration of 25 g/L. High cell viability (90%) was observed within PEC capsules by confocal laser scanning microscopy, performed before and after repetitive PAA production in the bubble column reactor. The surface microstructure of fully hydrated capsules with and without G. oxydans cells was investigated and compared using an environmental scanning electron microscope.     

Encapsulation of bacterial cells in electrospun microtubes

Encapsulation of whole microbial cells in microtubes for use in bioremediation of pollutants in water systems was the main focus of this investigation. Coelectrospinning of a core polymeric solution with bacterial cells and a shell polymer solution using a spinneret with two coaxial capillaries resulted in microtubes with porous walls. The ability of the microtube's structure to support cell attachment and maintain enzymatic activity and proliferation of the encapsulated microbial cells was examined. The results obtained show that the encapsulated cells maintain some of their phosphatase, β-galactosidase and denirification activity and are able to respond to conditions that induce these activities. This study demonstrates electrospun microtubes are a suitable platform for the immobilization of intact microbial cells.     

Proteomic Analysis of the Increased Stress Tolerance of Saccharomyces cerevisiae Encapsulated in Liquid Core Alginate-Chitosan Capsules

Saccharomyces cerevisiae CBS8066 encapsulated in semi-permeable alginate or alginate-chitosan liquid core capsules have been shown to have an enhanced tolerance towards complex dilute-acid lignocellulose hydrolysates and the lignocellulose-derived inhibitor furfural, as well as towards high temperatures. The underlying molecular reasons for these effects have however not been elucidated. In this study we have investigated the response of the encapsulation on the proteome level in the yeast cells, in comparison with cells grown freely in suspension under otherwise similar conditions. The proteomic analysis was performed on whole cell protein extracts using nLC-MS/MS with TMT® labelling and 2-D DIGE. 842 and 52 proteins were identified using each method, respectively. The abundances of 213 proteins were significantly different between encapsulated and suspended cells, with good correlation between the fold change ratios obtained by the two methods for proteins identified in both. Encapsulation of the yeast caused an up-regulation of glucose-repressed proteins and of both general and starvation-specific stress responses, such as the trehalose biosynthesis pathway, and down-regulation of proteins linked to growth and protein synthesis. The encapsulation leads to a lack of nutrients for cells close to the core of the capsule due to mass transfer limitations. The triggering of the stress response may be beneficial for the cells in certain conditions, for example leading to the increased tolerance towards high temperatures and certain inhibitors.     

Lack of Capsular Exopolymer Effects on the Biodegradation of Organic Compounds by Pseudomonas sp. Strains JS1 and JS150

Abstract Due to the increasing interest in the effects of exopolymers on microbial activities, two Pseudomonas sp. strains, JS1 (possesses capsular exopolymer) and JS150 (unencapsulated), were compared for their ability to degrade a variety of organic compounds under a number of different conditions. Degradation kinetics for citrate, salicylate, phenol, toluene, and 2,4,6-trinitrotoluene were identical for both strains in liquid media, regardless of cell density. JS1 and JS150 grew on citrate at the same rate in sand, sterile surface soil, and sterile subsurface sediments. The biodegradation curves for toluene by cells pregrown on citrate in any of the above matrices were indistinguishable. Twofold differences in water content and carbon-to-nitrogen ratios of 0.032 or 320 did not result in any apparent differences in phenol degradation by either encapsulated or unencapsulated cells in sand. These results indicate that generalizations about the effects of exopolymers on microbial processes may not be possible without further research. Received: 14 August 1996; Accepted: 19 November 1996     

In situ biobutanol recovery from clostridial fermentations: a critical review

Butanol is a precursor of many industrial chemicals, and a fuel that is more energetic, safer and easier to handle than ethanol. Fermentative biobutanol can be produced using renewable carbon sources such as agro-industrial residues and lignocellulosic biomass. Solventogenic clostridia are known as the most preeminent biobutanol producers. However, until now, solvent production through the fermentative routes is still not economically competitive compared to the petrochemical approaches, because the butanol is toxic to their own producer bacteria, and thus, the production capability is limited by the butanol tolerance of producing cells. In order to relieve butanol toxicity to the cells and improve the butanol production, many recovery strategies (either in situ or downstream of the fermentation) have been attempted by many researchers and varied success has been achieved. In this article, we summarize in situ recovery techniques that have been applied to butanol production through Clostridium fermentation, including liquid–liquid extraction, perstraction, reactive extraction, adsorption, pervaporation, vacuum fermentation, flash fermentation and gas stripping. We offer a prospective and an opinion about the past, present and the future of these techniques, such as the application of advanced membrane technology and use of recent extractants, including polymer solutions and ionic liquids, as well as the application of these techniques to assist the in situ synthesis of butanol derivatives.     

Design of mutualistic microbial consortia for stable conversion of carbon monoxide to value-added chemicals

Carbon monoxide (CO) is a promising carbon source for producing value-added biochemicals via microbial fermentation. However, its microbial conversion has been challenging because of difficulties in genetic engineering of CO-utilizing microorganisms and, more importantly, maintaining CO consumption which is negatively affected by the toxicity of CO and accumulated byproducts. To overcome these issues, we devised mutualistic microbial consortia, co-culturing Eubacterium limosum and genetically engineered Escherichia coli for the production of 3-hydroxypropionic acid (3-HP) and itaconic acid (ITA). During the co-culture, E. limosum assimilated CO and produced acetate, a toxic by-product, while E. coli utilized acetate as a sole carbon source. We found that this mutualistic interaction dramatically stabilized and improved CO consumption of E. limosum compared to monoculture. Consequently, the improved CO consumption allowed successful production of 3-HP and ITA from CO. This study is the first demonstration of value-added biochemical production from CO using a microbial consortium. Moreover, it suggests that synthetic mutualistic microbial consortium can serve as a powerful platform for the valorization of CO.     

Liquid and gaseous fuels from biotechnology: challenge and opportunities

Abstract: This paper presents challenging opportunities for production of liquid and gaseous fuels by biotechnology. From the liquid fuels, ethyl alcohol production has been widely researched and implemented. The major obstacle for large scale production of ethanol for fuel is the cost, whereby the substrate represents one of the major cost components. Various scenarios will be presented for a critical assessment of cost distribution for production of ethanol from various substrates by conventional and high rate processes. The paper also focuses on recent advances in the research and application of biotechnological processes and methods for the production of liquid transportation fuels other than ethanol (other oxygenates; diesel fuel extenders and substitutes), as well as gaseous fuels (biogas, methane, reformed syngas). Potential uses of these biofuels are described, along with environmental concerns which accompany them. Emphasis is also put on microalgal lipids as diesel substitute and biogas/methane as a renewable alternative to natural gas. The capturing and use of landfill gases is also mentioned, as well as microbial coal liquefaction. Described is also the construction and performance of microbial fuel cells for the direct high-efficiency conversion of chemical fuel energy to electricity. Bacterial carbon dioxide recovery is briefly dealt with as an environmental issue associated with the use of fossil energy.     

T helper cell-dependent, microbial superantigen-induced murine B cell activation: polyclonal and antigen-specific antibody responses.

Microbial superantigens (SA), bound to human B cell surface MHC class II molecules, have been shown to promote direct, "cognate" interaction with SA-reactive autologous Th cells, resulting in polyclonal Ig production. To investigate the potential for microbial SA to support Th cell-dependent, Ag-specific antibody responses, we have extended our studies to the murine system. BALB/c Th cell lines (TCL), specific for either the Mycoplasma arthritis-derived SA or the Staphylococcus aureus-derived toxic shock syndrome toxin-1) were generated. These TCL cells are SA-specific, functionally noncross-reactive, and utilize distinct TCR V beta gene families. Coculture of SA-reactive TCL cells and syngeneic B cells bearing the relevant SA results in B cell proliferation and polyclonal IgM and IgG production. In contrast, Ag-specific (SRBC-specific) antibody-forming cells are only generated in cultures that also contain SRBC. Thus, microbial SA-mediated Th-B cell interactions induce both polyclonal B cell activation and provide selective help for the proliferation and/or differentiation of B cells that have encountered specific Ag. In additional studies, we determined that the in vivo administration of toxic shock syndrome toxin-1 to young, athymic (nude) BALB/c mice results in SA binding to splenic B cells, rendering these B cells effective stimulators of and targets for SA-reactive helper TCL cells. Taken together, these results demonstrate that microbial SA mediate productive Th-B cell interactions analogous to those that occur during allospecific Th-B cell interactions in vitro and GVHD in vivo. These findings are consistent with the hypothesis that microbial SA represent environmental factors that may trigger autoimmune disease in the genetically susceptible host.     

Slow-Release Inoculation Allows Sustained Biodegradation of γ-Hexachlorocyclohexane

This study investigated the feasibility of a slow-release inoculation approach as a bioaugmentation strategy for the degradation of lindane (γ-hexachlorocyclohexane [γ-HCH]). Slow-release inoculation of Sphingomonas sp. γ 1-7 was established in both liquid and soil slurry microcosms using open-ended silicone tubes in which the bacteria are encapsulated in a protective nutrient-rich matrix. The capacity of the encapsulated cells to degrade lindane under aerobic conditions was evaluated in comparison with inoculation of free-living cells. Encapsulation of cells in tubes caused the removal of lindane by adsorption to the silicone tubes but also ensured prolonged biodegradation activity. Lindane degradation persisted 2.2 and 1.4 times longer for liquid and soil slurry microcosms, respectively, than that for inoculation with free cells. While inoculation of free-living cells led to a loss in lindane-degrading activity in limited time intervals, encapsulation in tubes allowed for a more stable actively degrading community. The loss in degrading activity was linked to the loss of the linA gene, encoding γ-HCH dehydrochlorinase (LinA), which is involved in the initial steps of the lindane degradation pathway. This work shows that a slow-release inoculation approach using a catabolic strain encapsulated in open-ended tubes is a promising bioaugmentation tool for contaminated sites, as it can enhance pollutant removal and can prolong the degrading activity in comparison with traditional inoculation strategies.     

Quantification of depth-dependent microbial growth in encapsulated systems

Encapsulated systems have been widely used in environmental applications to selectively retain and protect microorganisms. The permeable matrix used for encapsulation, however, limits the accessibility of existing analytical methods to study the behaviour of the encapsulated microorganisms. Here, we present a novel method that overcomes these limitations and enables direct observation and enumeration of encapsulated microbial colonies over a range of spatial and temporal scales. The method involves embedding, cross-sectioning, and analysing the system via fluorescence in situ hybridization and retains the structure of encapsulants and the morphology of encapsulated colonies. The major novelty of this method lies in its ability to distinguish between, and subsequently analyse, multiple microorganisms within a single encapsulation matrix across depth. Our results demonstrated the applicability and repeatability of this method with alginate-encapsulated pure (Nitrosomonas europaea) and enrichment cultures (anammox enrichment). The use of this method can potentially reveal interactions between encapsulated microorganisms and their surrounding matrix, as well as quantitatively validate predictions from mathematical models, thereby advancing our understanding of microbial ecology in encapsulated or even biofilm systems and facilitating the optimization of these systems.     

Bioreactor systems for in vitro production of foreign proteins using plant cell cultures

Plant cells have been demonstrated to be an attractive heterologous expression host (using whole plants and in vitro plant cell cultures) for foreign protein production in the past 20years. In recent years in vitro liquid cultures of plant cells in a fully contained bioreactor have become promising alternatives to traditional microbial fermentation and mammalian cell cultures as a foreign protein expression platform, due to the unique features of plant cells as a production host including product safety, cost-effective biomanufacturing, and the capacity for complex protein post-translational modifications. Heterologous proteins such as therapeutics, antibodies, vaccines and enzymes for pharmaceutical and industrial applications have been successfully expressed in plant cell culture-based bioreactor systems including suspended dedifferentiated plant cells, moss, and hairy roots, etc. In this article, the current status and emerging trends of plant cell culture for in vitro production of foreign proteins will be discussed with emphasis on the technological progress that has been made in plant cell culture bioreactor systems.     

Pancreatic cell immobilization in alginate beads produced by emulsion and internal gelation

Alginate has been used to protect transplanted pancreatic islets from immune rejection and as a matrix to increase the insulin content of islet progenitor cells. The throughput of alginate bead generation by the standard extrusion and external gelation method is limited by the rate of droplet formation from nozzles. Alginate bead generation by emulsion and internal gelation is a scaleable alternative that has been used with biological molecules and microbial cells, but not mammalian cells. We describe the novel adaptation of this process to mammalian cell immobilization. After optimization, the emulsion process yielded 90 ± 2% mouse insulinoma 6 (MIN6) cell survival, similar to the extrusion process. The MIN6 cells expanded at the same rate in both bead types to form pseudo‐islets with increased glucose stimulation index compared to cells in suspension. The emulsion process was suitable for primary pancreatic exocrine cell immobilization, leading to 67 ± 32 fold increased insulin expression after 10 days of immobilized culture. Due to the scaleability and broad availability of stirred mixers, the emulsion process represents an attractive option for laboratories that are not equipped with extrusion‐based cell encapsulators, as well as for the production of immobilized or encapsulated cellular therapeutics on a clinical scale. Biotechnol. Bioeng. 2011;108: 424–434. © 2010 Wiley Periodicals, Inc.     

Microbial preparation of metal-substituted magnetite nanoparticles

A microbial process that exploits the ability of iron-reducing microorganisms to produce copious amounts of extra-cellular metal (M)-substituted magnetite nanoparticles using akaganeite and dopants of dissolved form has previously been reported. The objectives of this study were to develop methods for producing M-substituted magnetite nanoparticles with a high rate of metal substitution by biological processes and to identify factors affecting the production of nano-crystals. The thermophilic and psychrotolerant iron-reducing bacteria had the ability to form M-substituted magnetite nano-crystals (M(y)Fe(3-y)O(4)) from a doped precursor, mixed-M iron oxyhydroxide, (M(x)Fe(1-x)OOH, x< or =0.5, M is Mn, Zn, Ni, Co and Cr). Within the range of 0.01< or =x< or =0.3, using the mixed precursor material enabled the microbial synthesis of more heavily substituted magnetite compared to the previous method, in which the precursor was pure akaganeite and the dopants were present as soluble metal salts. The mixed precursor method was especially advantageous in the case of toxic metals such as Cr and Ni. Also this new method increased the production rate and magnetic properties of the product, while improving crystallinity, size control and scalability.     

Deciphering interactions between the marine dinoflagellate Prorocentrum lima and the fungus Aspergillus pseudoglaucus

The comprehension of microbial interactions is one of the key challenges in marine microbial ecology. This study focused on exploring chemical interactions between the toxic dinoflagellate Prorocentrum lima and a filamentous fungal species, Aspergillus pseudoglaucus, which has been isolated from the microalgal culture. Such interspecies interactions are expected to occur even though they were rarely studied. Here, a co-culture system was designed in a dedicated microscale marine-like condition. This system allowed to explore microalgal–fungal physical and metabolic interactions in presence and absence of the bacterial consortium. Microscopic observation showed an unusual physical contact between the fungal mycelium and dinoflagellate cells. To delineate specialized metabolome alterations during microalgal–fungal co-culture metabolomes were monitored by high-performance liquid chromatography coupled to high-resolution mass spectrometry. In-depth multivariate statistical analysis using dedicated approaches highlighted (1) the metabolic alterations associated with microalgal–fungal co-culture, and (2) the impact of associated bacteria in microalgal metabolome response to fungal interaction. Unfortunately, only a very low number of highlighted features were fully characterized. However, an up-regulation of the dinoflagellate toxins okadaic acid and dinophysistoxin 1 was observed during co-culture in supernatants. Such results highlight the importance to consider microalgal–fungal interactions in the study of parameters regulating toxin production.