Bioaugmentation of UASB reactors with immobilized <Emphasis Type="Italic">Sulfurospirillum barnesii</Emphasis> for simultaneous selenate and nitrate removal

Whole-cell immobilization of selenate-respiring Sulfurospirillum barnesii in polyacrylamide gels was investigated to allow the treatment of selenate contaminated (790 μg Se × L⁻¹) synthetic wastewater with a high molar excess of nitrate (1,500 times) and sulfate (200 times). Gel-immobilized S. barnesii cells were used to inoculate a mesophilic (30°C) bioreactor fed with lactate as electron donor at an organic loading rate of 5 g chemical oxygen demand (COD) × L⁻¹ day⁻¹. Selenate was reduced efficiently (>97%) in the nitrate and sulfate fed bioreactor, and a minimal effluent concentration of 39 μg Se × L⁻¹ was obtained. Scanning electron microscopy with energy dispersive X-ray (SEM–EDX) analysis revealed spherical bioprecipitates of ≤2 μm diameter mostly on the gel surface, consisting of selenium with a minor contribution of sulfur. To validate the bioaugmentation success under microbial competition, gel cubes with immobilized S. barnesii cells were added to an Upflow Anaerobic Sludge Bed (UASB) reactor, resulting in earlier selenate (24 hydraulic retention times (HRTs)) and sulfate (44 HRTs) removal and higher nitrate/nitrite removal efficiencies compared to a non-bioaugmented control reactor. S. barnesii was efficiently immobilized inside the UASB bioreactors as the selenate-reducing activity was maintained during long-term operation (58 days), and molecular analysis showed that S. barnesii was present in both the sludge bed and the effluent. This demonstrates that gel immobilization of specialized bacterial strains can supersede wash-out and out-competition of newly introduced strains in continuous bioaugmented systems. Eventually, proliferation of a selenium-respiring specialist occurred in the non-bioaugmented control reactor, resulting in simultaneous nitrate and selenate removal during a later phase of operation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Operation of packed-bed immobilized cell reactor featuring active β-galactosidase inclusion body-containing recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> cells

In this study, we developed a packed-bed immobilized cell reactor containing active β-gal (β-galactosidase) inclusion body (IB)-containing Escherichia coli (E. coli) cells in alginate beads. This packed-bed reactor was operated using a substrate feed solution 0.72 ∼ 38.4 mM ONPG (o-nitrophenyl-β-D-galactoside) prepared in Z buffer supplemented with chloroform and 0.1% SDS (sodium dodecyl sulfate). The production rate of ONP (o-nitrophenol) in the reactor containing cells that were incubated with α-MG (α-methyl D-glucospyranoside) or D-fucose after induction was superior to those prepared with cells that were not incubated with α-MG or D-fucose. The ONP production rate was increased proportionally with ONPG concentration in the substrate feed up to a concentration of 38.4 mM. However, as the ONPG concentration was increased in the substrate feed solution, galactose inhibition inside the alginate beads was increased. This most likely occurred due to problems with diffusion. In addition, partial breakage of alginate beads was observed during the later periods of operation. In this study, we demonstrated that active β-gal IB-containing E. coli cells were sustained in the immobilized cell reactor during operation. Particularly, these findings demonstrate the feasibility of using active IBs in an enzymatic reaction without the need for any purification step. In addition, we showed that these IB-containing cells could be directly used in an immobilized reactor.     

Heterogeneity of the populations of marine luminescent bacteria Photobacterium leiognathi under different conditions of cultivation

Manifestation of pleiotropic effects in the isogenic variants of the luminescent bacterium Photobacterium leiognathi 54 was investigated. The decrease or increase of the expression level of bioluminescence was caused by changes in lux operon regulation. The dynamics of the bioluminescence of dark and dim variants did not differ from the dynamics of the initial luminescent variant, but dependence of the level of luminescence intensity on the exogenous autoinducer of the lux operon was revealed. The investigated variants of P. leiognathi 54 inherited fairly stable morphological characteristics, colony architectonics, level of luminescence, and activity of some enzymes; variants with reduced bioluminescence formed colonies of the S type. Stable bright variants with S-and R-type colonies appeared both in the initial strain population and in the dark variant population, but with smaller frequency. Populations of the bright variant with R-type colonies were most heterogeneous; this can be determined by the lack of glucose repression of the bioluminescence in contrast to other investigated inherited variants of P. leiognathi.     

SPONTANEOUS AND STIMULATED BIOLUMINESCENCE OF THE DINOFLAGELLATE CERATOCORYS HORRZDA (PERIDINIALES)1

This is the first report of spontaneous bioluminescence in the autotrophic dinoflagellate Ceratocorys horrida von Stein. Bioluminescence was measured, using an automated data acquisition system, in a strain of cultured cells isolated from the Sargasso Sea. Ceratocorys horrida is only the second dinoflagellate species to exhibit rhythmicity in the rate of spontaneous flashing, flash quantum flux (intensity), and level of spontaneous glowing. The rate of spontaneous flashing was maximal during hours 2–4 of the dark phase [i.e. circadian time (CT)16–18 for a 14:10 h LD cycle (LD14:10)], with approximately 2% of the population flashing-min^?1, a rate approximately one order of magnitude greater than that of the dinoflagellate Gonyaulax polyedra. Flash quantum flux was also maximal during this period. Spontaneous flashes were 134 ms in duration with a maximum flux (intensity) of 3.1×10⁹ quanta-s^?1. Light emission presumably originated from blue fluorescent microsources distributed in the cell periphery and not from the spines. Values of both spontaneous flash rate and maximum flux were independent of cell concentration. Isolated cells also produced spontaneous flashes. Spontaneous glowing was dim except for a peak of 6.4× 10⁴quanta-s^?1 cell^?1, which occurred at CT22.9 for LD14:10 and at CT22.8 for LD12:12. The total integrated emission of spontaneous flashing and glowing during the dark phase was 4×10⁹ quantacell^?1, equivalent to the total stimulable luminescence. The rhythms for C. horrida flash and glow behavior were similar to those of Gonyaulax polyedra, although flash rate and quantum flux were greater. Spontaneous bioluminescence in C. horrida may be a circadian rhythm because it persisted for at least three cycles in constant dark conditions. This is also the first detailed study of the stimulated bioluminescence of C. horrida, which also displayed a diurnal rhythm. Cultures exhibited >200 times more mechanically stimulated bioluminescence during the dark phase than during the light phase. Mechanical stimulation during the dark phase resulted in 6.7 flashes. cell^?1; flashes were brighter and longer in duration than spontaneous flashes. Cruise-collected cells exhibited variability in quantum flux with few differences in flash kinetics. The role of dinoflagellate spontaneous bioluminescence in the dynamics of near-surface oceanic communities is unknown, but it may be an important source of natural in situ bioluminescence.     

Contributions of immobilized and free cells of salt-tolerantZygosaccharomyces rouxii andCandida versatilis to the production of ethanol and 4-ethylguaiacol

Summary The contribution of immobilized cells and free cells released from gel beads to ethanol production by the salt-tolerant yeastsZygosaccharomyces rouxii andCandida versatilis, and 4-ethylguaiacol (4-EG) production byC. versatilis were investigated using an airlift reactor. The amounts of ethanol produced by free cells were about 65% and about 90% of total ethanol in the reactor forZ. rouxii andC. versatilis, respectively. It was found that immobilized cells gave a much lower specific productivity of ethanol (ethanol production per hour per cell) than free cells of both yeasts, especially ofC. versatilis. 4-EG was produced mainly by immobilized cells ofC. versatilis; the amount of 4-EG produced by free cells was about 20% of the total 4-EG, in contrast to the results of ethanol production. However, the specific productivity of 4-EG (4-EG production per hour per cell) by immobilized and free cells was fairly similar.     

Metabolic self‐organization of bioluminescent Escherichia coli

A possible reason for the complexity of the signals produced by bioluminescent biosensors might be self‐organization of the cells. In order to verify this possibility, bioluminescence images of cultures of lux gene reporter Escherichia coli were recorded for several hours after being placed into 8–10 mm diameter cylindrical containers. It was found that luminous cells distribute near the three‐phase contact line, forming irregular azimuthal waves. As we show, space–time plots of quasi‐one‐dimensional bioluminescence measured along the contact line can be simulated by reaction–diffusion–chemotaxis equations, in which the reaction term for the cells is a logistic (autocatalytic) growth function. It was found that the growth rate of the luminous cells (~0.02 s^?1) is >100 times higher than the growth rate of E. coli. We provide an explanation for this result by assuming that E. coli exhibits considerable respiratory flexibility (the ability of oxygen‐induced switching from one metabolic pathway to another). According to the simple two‐state model presented here, the number of oxic (luminous) cells grows at the expense of anoxic (dark) cells, whereas the total number of (oxic and anoxic) cells remains unchanged. It is conjectured that the corresponding reaction–diffusion–chemotaxis model for bioluminescence pattern formation can be considered as a model for the energy‐taxis and metabolic self‐organization in the population of the metabolically flexible bacteria under hypoxic conditions. Copyright © 2011 John Wiley & Sons, Ltd.     

Process and technoeconomics of ethanol production by immobilized cells

Summary A two-stage fermentation process has been developed for continuous ethanol production by immobilized cells of Zymomonas mobilis. About 90–92 kg/m³ ethanol was produced after 4 h of residence time. Entrapped cells of Zymomonas mobilis have a capability to convert glucose to ethanol at 93% of the theoretical yield. The immobilized cell system has functioned for several weeks, and experience indicates that the carrageenan gel apparently facilitates easy diffusion of glucose and ethanol.The simplicity and the high productivity of the plug-flow reactor employing immobilized cells makes it economically attrative. An evaluation of process economics of an immobilized cell system indicates that at least 4 c/l of ethanol can be saved using the immobilized cell system rather than the conventional batch system. The high productivity achieved in the immobilized cell reactor results in the requirement for only small reactor vessels indicating low capital cost. Consequently, by switching from batch to immobilized processing, the fixed capital investment is substantially reduced, thus increasing the profitability of ethanol production by fermentation.     

Immobilization Results in Sustained Calcium Transport in Nostoc calcicola Bréb

The uptake pattern of Ca²⁺ by the cyanobacterium Nostoc calcicola Bréb in its freely suspended and immobilized form is comprised of two distinct phages; (a) rapid uptake for 1^st 10 min followed by (b) slower transport at least up to 60 min. Entrapment of cyanobacterial cells in polyvinyl foam always maintained a higher Ca²⁺ profile over freely suspended cells. Also, the intracellular Ca²⁺ concentration was three times more in the former under similar experimental conditions. Whereas, illumination supported maximum Ca²⁺ transport in all the sets, darkness resulted in drastic reduction (90%) of Ca²⁺ uptake in freely suspended cells and least (15%) in polyvinyl entrapped cyanobacterial cells. Exogenously added ATP (10 μM) on the other hand, enhanced Ca²⁺ uptake in dark incubated freely suspended cells; ATP at the same concentration failed to bring out any significant enhancement in cation uptake in immobilized cells facing dark exposure. It was observed that these cells were still able to sustain sufficient ATP preserves to drive active transport of Ca²⁺ even in the dark. Furthermore, the immobilized cells exhibited remarkable Ca²⁺ transport rate even at the age of 20 and 50 days at which its free living counterpart took up insignificant Ca²⁺. These findings suggest the improved metabolic efficiency of polyvinyl foam entrapped cells over freely suspended cells in terms of Ca²⁺ accumulation and its possible use as a bioreactor for metal accumulation/removal in repetitive cycles without any measurable loss in cell biomass. Received: 21 May 2001 / Accepted: 27 June 2001     

Active protein and substrate flow effects in a tubular immobilized invertase reactor

Investigations of invertase (EC 3.2.1.26) immobilized inside modified nylon tubes showed that between 4% and 20% (w/w) of the protein exposed to binding sites on the tube was immobilized. An enhanced activity consistent with enzyme purification during immobilization was also evident, suggesting that, in scaled-up commercial applications, nylon tube invertase would be a more economical converter of sucrose than the free enzyme. The quantity and specific activity of the immobilized protein were not stochiometrical with the amount used in the coupling solution and, in the system studied, a concentration of 2 mg ml^?1 was optimal. K_m and V_max values confirmed higher rates of immobilized invertase catalysis when the rates of substrate flow through the reactor were higher. Higher rates of substrate flow imply a shortened residence time in the reactor and would lower the fractional conversion per pass of the substrate, reducing the efficiency of the reactor in flow-through situations. Thus, these higher catalysis rates, attributable at the higher flow rates to a reduction of the diffusion barrier between enzyme and substrate, would not translate into improved economy in the commercial flow-through processes at which the reactor is aimed.     

Continuous phenol biodegradation in a simple packed bed bioreactor of calcium alginate-immobilized <Emphasis Type="Italic">Candida tropicalis</Emphasis> (NCIM 3556)

Phenol biodegradation in a continuous system of immobilized Candida tropicalis NCIM 3556 was studied. The bioreactor was simple, it had a feed inlet from the bottom and the effluent outlet from top, no supplementary oxygen was supplied, the reactor was operated continuously for 116 days. Initially the column was run continuously with a feed concentration of 2 g l⁻¹ for 42 days whence a degradation of >97% was achieved. The feed concentration was then increased to 3 g l⁻¹, for which a ~80% biodegradation was sustained for 90 days after which there was a steady decrease in the performance. When the phenol degradation was reduced to ~50% in 116 days, the reactor was stopped. The efficiency of free cells recycled every 24 h and immobilized cells were compared; it was estimated that repeated reuse of free cells in batch mode gave an overall efficiency of 0.102 g phenol degradation g⁻¹ cell wet weight in 12 days. In contrast, the immobilized system of the same biomass had a longer working lifetime of ~4 months indicating an efficiency of 3.72 g phenol g⁻¹ cell wet wt.     

Detection of living <Emphasis Type="Italic">Salmonella</Emphasis> cells using bioluminescence

ATP-based bioluminescence using mutant firefly luciferase was combined with an immunochromatographic lateral flow test strip assay for Salmonella enteritidis detection. In this combination method, the Salmonella-antibody–gold complex captured at the test line on the test strip was lysed by heat-treatment, and the ATP released from the cells was measured using mutant luciferase. This method resulted in approximately 1,000 times higher sensitivity in the detection of Salmonella (i.e. 10³ c.f.u./ml) compared to immunochromatographic lateral flow assay.     

Heterogeneity of the populations of marine luminescent bacteria Photobacterium leiognathi under different conditions of cultivation

Manifestation of pleiotropic effects in the isogenic variants of luminescent bacteria Photobacterium leiognathi 54 was investigated. The decrease or increase of the expression level of bioluminescence was caused by changes in lux operon regulation. The dynamics of the bioluminescence of dark and dim variants did not differ from the dynamics of the initial luminescent variant, but dependence of the level of luminescence intensity on the exogenous autoinductor of the lux operon was revealed. The investigated variants of P. leiognathi 54 inherited fairly stable morphological characteristics, colony architectonics, level of luminescence, and activity of some enzymes; variants with reduced bioluminescence formed colonies of the S type. Stable bright variants with S- and R-type colonies appeared both in the initial strain population and in the dark variant population, but with smaller frequency. Populations of the bright variant with R-type colonies were most heterogeneous; this can be determined by the lack of glucose repression of the bioluminescence in contrast to other investigated variants of P. leiognathi.     

Treatment of simulated wastewater containing toxic amides by immobilized <Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> NHB-2 using a highly compact 5-stage plug flow reactor

Biodegradation of toxic amides by immobilized Rhodococcus rhodochrous NHB-2 has been studied to generate data for future development of reactors for the treatment of simulated wastewater containing various toxic amides. The whole resting cells were immobilized in different matrices like agar, polyacrylamide and alginate. Agar gel beads were selected for the treatment of simulated wastewater containing 100mM each acetamide, propionamide, and 10mM of acrylamide and packed in a highly compact five-stage plug flow reactor. The immobilized bacterium worked well in a broad pH range from 5 to 10, with an optimum at 8.7. The apparent K _m-value for the turnover of acetamide for the resting cells was determined to be around 40mM at pH 8.5 and 55°C, whereas the K _m-value of the purified amidase was predicted to be about 20 mM. This organism exhibited greater turnover of aliphatic amides as compared to aromatic amides. Although these cells showed maximal amide-degrading activity at 55°C, simulated wastewater treatment was carried out at 45°C, because of the greater stability of the amidase activity at that temperature. Of note, indices for overall temperature stability, based on the temperature dependence of apparent first order kinetic temperature denaturation constants, were determined to be –7.9±1.1×10^–4, and –13.7±1.3×10^–4, –14.5±0.7×10^–4, and –13.7±0.8×10^–4°Cmin, for free cells and cells immobilized in alginate, agar and polyacrylamide respectively. After 250min the reactor showed maximum degradation of acetamide, propionamide and acrylamide of about 97, 100 and 90%, respectively by using 883 enzyme activity units per reactor stage. The results of this investigation showed that R. rhodochrous NHB-2 expressing thermostable amidase could be used for the efficient treatment of wastewater containing toxic amides. Therefore, we suggest that this microbe has a very high potential for the detoxification of toxic amides from industrial effluents and other wastewaters.     

Stabilization of water-in-oil emulsion by <Emphasis Type="Italic">Rhodococcus opacus</Emphasis> B-4 and its application to biotransformation

Rhodococcus opacus B-4, which has recently been isolated as an organic solvent-tolerant bacterium, stabilized water-in-oil (w/o) emulsions by inhibition of droplet coalescence when the cells were dispersed in 90% (v/v) organic solvents. Confocal microscopy revealed that many bacterial cells assembled at the interface between oil and water droplets, though free cells were also detectable at the inside of water droplets. Bacterial cells in the w/o emulsion were capable of utilizing both a water-soluble (glucose) and an oil-soluble substrate (oleic acid) as an energy source. Availability of the w/o emulsion as an immobilized cell system in organic solvents was demonstrated using production of indigo from indole and production of o-cresol from toluene as model conversions. When glucose and oleic acid were simultaneously supplied as energy sources, the w/o emulsion culture of R. opacus B-4 produced indigo and o-cresol at levels of 0.217 and 2.12 mg ml⁻¹, respectively, by 12 h.     

Optimization of lactic acid production by immobilized <Emphasis Type="Italic">Lactococcus lactis</Emphasis> IO-1

Production of lactic acid from glucose by immobilized cells of Lactococcus lactis IO-1 was investigated using cells that had been immobilized by either entrapment in beads of alginate or encapsulation in microcapsules of alginate membrane. The fermentation process was optimized in shake flasks using the Taguchi method and then further assessed in a production bioreactor. The bioreactor consisted of a packed bed of immobilized cells and its operation involved recycling of the broth through the bed. Both batch and continuous modes of operation of the reactor were investigated. Microencapsulation proved to be the better method of immobilization. For microencapsulated cells at immobilized cell concentration of 5.3 g l⁻¹, the optimal production medium had the following initial concentrations of nutrients (g l⁻¹): glucose 45, yeast extract 10, beef extract 10, peptone 7.5 and calcium chloride 10 at an initial pH of 6.85. Under these conditions, at 37 °C, the volumetric productivity of lactic acid in shake flasks was 1.8 g l⁻¹ h⁻¹. Use of a packed bed of encapsulated cells with recycle of the broth through the bed, increased the volumetric productivity to 4.5 g l⁻¹ h⁻¹. The packed bed could be used in repeated batch runs to produce lactic acid.     

Degradation of <Emphasis Type="Italic">p</Emphasis>-Toluenesulfonate by Immobilized <Emphasis Type="Italic">Comamonas testosteroni</Emphasis> BS1310 (pBS1010) Cells

Parameters of degradation of p-toluenesulfonate (TS) by free and agar-embedded Comamonas testosteroni BS1310 (pBS1010) cells were determined. The maximum rate of TS degradation was 25% lower in immobilized than free cells, equaling 11 nmol min^?1 mg^?1 cells. Degradation of TS by both free and immobilized cells was associated with molecular oxygen consumption (molar ratio 1 : 2). In a plug-flow reactor, the degradation rate was 10.4 nmol min^?1 mg^?1 cells. The results can be applied to designing reactors for TS degradation in sewage and developing biosensors.     

Cloning of two SOS1 transporters from the seagrass <Emphasis Type="Italic">Cymodocea nodosa</Emphasis>. SOS1 transporters from <Emphasis Type="Italic">Cymodocea</Emphasis> and <Emphasis Type="Italic">Arabidopsis</Emphasis> mediate potassium uptake in bacteria

Two cDNAs isolated from Cymodocea nodosa, CnSOS1A, and CnSOS1B encode proteins with high-sequence similarities to SOS1 plant transporters. CnSOS1A expressed in a yeast Na⁺-efflux mutant under the control of a constitutive expression promoter mimicked AtSOS1 from Arabidopsis; the wild type cDNA did not improve the growth of the recipient strain in the presence of Na⁺, but a cDNA mutant that expresses a truncated protein suppressed the defect of the yeast mutant. In similar experiments, CnSOS1B was not effective. Conditional expression, under the control of an arabinose responsive promoter, of the CnSOS1A and CnSOS1B cDNAs in an Escherichia coli mutant defective in Na⁺ efflux was toxic, and functional analyses were inconclusive. The same constructs transformed into an E. coli K⁺-uptake mutant revealed that CnSOS1A was also toxic, but that it slightly suppressed defective growth at low K⁺. Truncation in the C-terminal hydrophilic tail of CnSOS1A relieved the toxicity and proved that CnSOS1A was an excellent low-affinity K⁺ and Rb⁺ transporter. CnSOS1B mediated a transient, extremely rapid K⁺ or Rb⁺ influx. Similar tests with AtSOS1 revealed that it was not toxic and that the whole protein exhibited excellent K⁺ and Rb⁺ uptake characteristics in bacteria.     

Bioreactor microbial ecosystems for thiocyanate and cyanide degradation unravelled with genome‐resolved metagenomics

Gold ore processing uses cyanide (CN^?), which often results in large volumes of thiocyanate‐ (SCN^?) contaminated wastewater requiring treatment. Microbial communities can degrade SCN^? and CN^?, but little is known about their membership and metabolic potential. Microbial‐based remediation strategies will benefit from an ecological understanding of organisms involved in the breakdown of SCN^? and CN^? into sulfur, carbon and nitrogen compounds. We performed metagenomic analysis of samples from two laboratory‐scale bioreactors used to study SCN^? and CN^? degradation. Community analysis revealed the dominance of Thiobacillus spp., whose genomes harbour a previously unreported operon for SCN^? degradation. Genome‐based metabolic predictions suggest that a large portion of each bioreactor community is autotrophic, relying not on molasses in reactor feed but using energy gained from oxidation of sulfur compounds produced during SCN^? degradation. Heterotrophs, including a bacterium from a previously uncharacterized phylum, compose a smaller portion of the reactor community. Predation by phage and eukaryotes is predicted to affect community dynamics. Genes for ammonium oxidation and denitrification were detected, indicating the potential for nitrogen removal, as required for complete remediation of wastewater. These findings suggest optimization strategies for reactor design, such as improved aerobic/anaerobic partitioning and elimination of organic carbon from reactor feed.     

Biosensitive element in the form of immobilized luminescent photobacteria for detecting ecotoxicants in aqueous flow‐through systems

We demonstrated the possibility of long‐term and efficient application of a biosensitive element (BE) in the form of Photobacterium phosphoreum photobacteria immobilized in poly(vinyl alcohol) (PVA) cryogel for detecting various ecotoxicants ( Zn ^{2 +} , Cu ^{2 +} , Hg ^{2 +} , Pb ^{2 +} , 2,4‐dichlorophenoxyacetic acid, 2,6‐dimethylphenol, pentachlorophenol, coumaphos, malathion, chlorpyrifos and methyl parathion) in flow‐through media. The range of detectable concentrations of ecotoxicants was determined at 1 × 10 ^?8 to 1 × 10 ^?4 M for heavy metal ions and at 1 × 10 ^?8 to 1 × 10 ^?5 M for phenol derivatives and organophosphorus pesticides. Immobilized cells of photobacteria quantitatively reacted with these ecotoxicants; cell sensitivity exhibited no flow rate dependence in the range from 45 to 180 mL/h. At a constant concentration of ecotoxicant in the flow, the bioluminescence quenching profile of immobilized cells demonstrated an integral response. The BE could remain in a flow‐through medium for at least 10 days while retaining 95% of luminescent activity in the absence of ecotoxicants. The BE tested in this work was demonstrated to have a long shelf life (> 60 weeks) at ?80°C without changes in the baseline level of bioluminescence. Copyright © 2016 John Wiley & Sons, Ltd.