Selective fungal bioprecipitation of cobalt and nickel for multiple-product metal recovery

There are a need for novel, economical and efficient metal processing technologies to improve critical metal sustainability, particularly for cobalt and nickel which have extensive applications in low-carbon energy technologies. Fungal metal biorecovery processes show potential in this regard and the products of recovery are also industrially significant. Here we present a basis for selective biorecovery of Co and Ni oxalates and phosphates using reactive spent Aspergillus niger culture filtrate containing mycogenic oxalate and phosphate solubilized from struvite. Selective precipitation of oxalates was achieved by adjusting phosphate-laden filtrates to pH 2.5 prior to precipitation. Co recovery at pH 2.5 was high with a maximum of ~96% achieved, while ~60% Ni recovery was achieved, yielding microscale polyhedral biominerals. Co and Ni phosphates were precipitated at pH 7.5, following prior oxalate removal, resulting in near-total Co recovery (>99%), while Ni phosphate yields were also high with a recovery maximum of 83.0%.     

Carbonate and Phosphate Precipitation by Chromohalobacter marismortui

The ability of Chromohalobacter marismortui to precipitate carbonate and phosphate minerals has been demonstrated for the first time. Mineral precipitation in both solid and liquid media at different salts concentrations and different magnesium/calcium ratios occurred whereas crystal formation was not observed in the control. The precipitated minerals were studied by X-ray diffraction, scanning electron microscopy and EDX, and were different in liquid and solid media. In liquid media aragonite, struvite, vaterite and monohydrocalcite were precipitated forming crystals and bioliths. Bioliths accreted preferentially close to organic pellicles, whereas struvite preferentially grows in microenvironments free of such pellicles. Magnesian calcite, calcian-magnesian kutnahorite, “proto-dolomite” and huntite were formed in solid media. The Mg content of the magnesian calcite and of Ca-Mg kutnahorite also varied depending on the salt concentration of the culture media. This is the first report on bacterial precipitation of Ca-Mg kutnahorite and huntite in laboratory cultures. The results of this research show the active role played by C. marismortui in mineral precipitation, and allow us to compare them with those obtained previously using other taxonomic groups of moderately halophilic bacteria.     

Removal of nutrients from piggery wastewater using struvite precipitation and pyrogenation technology

In this paper, removal of nutrients from piggery wastewater by struvite crystallization was conducted using a combined technology of low-cost magnesium source in struvite precipitation and recycling of the struvite pyrolysate in the process. In the present research, it was found that high concentrations of K⁺ and Ca²⁺ present in the solution significantly affected the removal of nutrients. When the struvite crystallization formed at the condition of dosing the magnesite pyrolysate at a Mg:N:P molar ratio of 2.5:1:1, and having a reaction time of 6 h, a majority of nutrients in piggery wastewater can be removed. Surface characterization analysis demonstrated that the main components of the pyrolysate of the obtained struvite were amorphous magnesium sodium phosphate (MgNaPO₄) and MgO. When the struvite pyrolysate was recycled in the process at the pH range of 8.0-8.5, the precipitation effect was optimum. When the struvite pyrolysate was recycled repeatedly at pH 8.5 or without any adjustment of pH, the outcome of the removal of the nutrients in both cases was similar. With the increase in the number of recycle times, the performance of struvite precipitation progressively decreased. An economic evaluation showed that the combination of using low-cost material and recycling of struvite was feasible. Recycling struvite for three process cycles could save the chemical costs by 81% compared to the use of pure chemicals.     

Carbonate and Phosphate Precipitation by Saline Soil Bacteria in a Monitored Culture Medium

Carbonate and phosphate precipitation by bacteria isolated from a saline soil was studied in vitro in a liquid culture medium over 45 days. Physicochemical parameters of this medium were continuously monitored using both selective electrodes (continuous monitoring, CM) and individual measurements by other techniques on days 5, 10, 15, 20, 25, 35 and 45 (discontinuous monitoring, DM). In DM, the precipitated minerals were studied (XRD and SEM-EDX) and the saturation index of the mineral phases was analyzed (PHREEQC program). Using the CM and DM data it was possible to distinguish several temporary stages in which both the medium and the mineralogy changed: 1) 0 to 10 days: pH reaches 8.4; significant loss of Mg²⁺ (incorporated into the bacterial biomass) and Ca²⁺ (through mineral precipitation); formation of crystals, although not in sufficient quantity to be studied until day 10. 2) 10 to 25 days: pH decreases but remains above 8; appreciable loss of Mg²⁺ and Ca²⁺ due to formation of spherical carbonate bioliths with traces of phosphates occluded within these carbonates. 3) After 25 days: biomineralization slow down; pH returns to initial values and struvite is formed (idiomorphic prismatic crystals). These trends are in agreement with the findings of other workers, although with some peculiarities regarding stages and types of mineral precipitated. In some cases the struvite contained small quantities of K and Ca, possibly because these are intermediate mineral species between typic-struvite, K-struvite and Ca-struvite. The bacteria-mediated precipitation of carbonates of Ca and/or Mg and phosphates (struvite) by the bacteria from a saline soil is demonstrated. However, struvite was not found in the soils of origin of the bacteria, possibly because it is a metastable mineral in most soils.     

Biotransformation of struvite by Aspergillus niger: phosphate release and magnesium biomineralization as glushinskite

Struvite (magnesium ammonium phosphate-MgNH₄PO₄·6H₂O), which can extensively crystallize in wastewater treatments, is a potential source of N and P as fertilizer, as well as a means of P conservation. However, little is known of microbial interactions with struvite which would result in element release. In this work, the geoactive fungus Aspergillus niger was investigated for struvite transformation on solid and in liquid media. Aspergillus niger was capable of solubilizing natural (fragments and powder) and synthetic struvite when incorporated into solid medium, with accompanying acidification of the media, and extensive precipitation of magnesium oxalate dihydrate (glushinskite, Mg(C₂O₄).2H₂O) occurring under growing colonies. In liquid media, A. niger was able to solubilize natural and synthetic struvite releasing mobile phosphate (PO₄³⁻) and magnesium (Mg²⁺), the latter reacting with excreted oxalate resulting in precipitation of magnesium oxalate dihydrate which also accumulated within the mycelial pellets. Struvite was also found to influence the morphology of A. niger mycelial pellets. These findings contribute further understanding of struvite solubilization, element release and secondary oxalate formation, relevant to the biogeochemical cycling of phosphate minerals, and further directions utilizing these mechanisms in environmental biotechnologies such as element biorecovery and biofertilizer applications.     

Struvite Precipitation Induced by a Novel Sulfate-Reducing Bacterium Acinetobacter calcoaceticus SRB4 Isolated from River Sediment

This article presents a study of struvite formation in liquid medium induced by the sulfate-reducing bacterium Acinetobacter calcoaceticus SRB4, a strain isolated from river sediment. We identified the bacterial strain A. calcoaceticus SRB4 and analyzed its micromorphology. The minerals formed were studied with an electroprobe microanalyzer, Fourier transform infrared spectroscopy, high-resolution transmission electron microscopy, selected-area electron diffraction, X-ray diffraction, thermogravimetry, differential thermogravimetry, and differential scanning calorimetry. Acinetobacter calcoaceticus SRB4 was found to induce struvite precipitation, whereas sterile control cultures did not. Many transparent stick-shaped struvite precipitates were distributed at the bottom of the conical flasks in the experimental group. Most bacteria were spherical and a large quantity of spherical struvite particles (less than 200 nm in diameter) adhered to the bacterial surface. An electron probe microanalysis showed that the precipitates contained C, O, P, Mg, and other elements. Fourier transformation infrared spectra showed that the precipitates contained crystalline water, NH₄⁺, and PO₄^3? groups. X-ray diffraction spectra showed that the precipitates were struvite crystals, with preferential orientation and lattice distortion. Thermogravimetry showed that the weight loss was caused by the evaporation of crystalline water at temperatures lower than 136°C and the release of ammonia from struvite at temperatures of 136–228.5°C. In this article, we discuss the possible mechanism of struvite formation and the possible role played by A. calcoaceticus SRB4. Our study extends our understanding of the phosphate biomineralization mechanism and should prove useful in recycling phosphorus in wastewater.     

Chitosanases in the autolysis of Mucor rouxii

The mycelium of Mucor rouxii reached a 50% degree of lysis after 50 days incubation, and was then stable with the incubation time. The pH of the medium was 4.3 when autolysis began, rising to pH 7.6 after 6 days of autolysis and remaining there for the duration of the experiment. Maximum degradation of mycelium occurs during the first days of autolysis. Glucosamine is present in the culture liquid during all the autolytic process. Enzymes implicated in the degradation of chitosan and chitin were studied in the culture fluid during autolysis. An exochitosanase activity was detected after a day of autolysis, and its activity increased during 20 days of autolysis and afterwards remained constant until the end of the process. An endochitosanase activity was detected in the culture fluid from the beginning of the autolysis, having its maximum activity after 34 days of incubation. Both activities show an optimum pH of 5.5, but the pH range of activity for endochitosanase was broader than for exochitosanase. Both activities were not inhibited by 0.5 mM glucosamine. Activities of the enzymes B-N-acetylglucosaminidase and chitinase were not found. The chitosan content in the cell walls decreased with the incubation time. In these cell walls the chitin content experienced an increase at the beginning of the autolysis, decreasing afterwards. The enzymatic complex obtained from autolyzed cultures of M. rouxii hydrolyzed 2-day-old cell walls of this fungus. The hydrolysis was 21% after 24 h of incubation, liberating glucose and glucosamine. As a consequence protoplasts from M. rouxii germinated spores were obtained with its own lytic enzymes in adequate osmotic conditions. The involvement of chitosanases in the autolysis of this fungus have been studied.     

Bacterial formation of struvite

We studied the formation of exocellular precipitates of struvite (Mg NH₄PO4.6H₂O) by 96 kinds of calcite‐pro‐ducing bacterial strains isolated from soil. We also studied the influence of calcium ions on struvite precipitation. The number of strains producing struvite was 20. Only four consistently formed large amounts. These results seem to indicate that the bacterial precipitation of struvite is not a general phenomenon. The strains studied were taxonomically identified, and no relationship was found between the production of struvite and the taxonomic identity of such strains. Calcium, supplied as Ca acetate in the culture medium, appeared to inhibit the biological precipitation of struvite.     

Prediction by computer modelling of the precipitation of stone-forming solids from urine

The development and verification of an equilibrium speciation computer model of urine is described. The model is based on critically selected equilibrium constants and solubility products corrected where needed to T=37°C and I=0.2 mol dm⁻³. The model represents a slight improvement over previous equilibrium models in that a simpler and more reliable measure is used to represent the degree of supersaturation with respect to a stone-forming salt. A further improvement arises from an implicit allowance being made for certain kinetic factors governing the precipitation of calcium-phosphate solids. The model was verified by comparison with an analogous experimental procedure applied to a standard reference artificial urine. Good predictions were obtained for the precipitation of calcium oxalate monohydrate (COM), dicalcium phosphate dihydrate (DCPD), calcium hydroxyapatite (HAP) and uric acid, but not for struvite. The model indicates that normal urine in the physiological pH range of 5.5 to 7.0 contains precipitates of COM, DCPD and HAP. Precipitates of uric acid and struvite are predicted to occur below and above a pH of 6, respectively. Further computations yield results which are consistent with the view that DCPD may act as a precursor in the precipitation of HAP. Finally, the results indicate that COM precipitation is far more markedly augmented by an increase in oxalate concentration than by an increase in calcium concentration.     

Biomineralization of carbonate and phosphate by moderately halophilic bacteria

We investigated the precipitation of carbonate and phosphate minerals by 19 species of moderately halophilic bacteria using media with variable Mg(2+)/Ca(2+) ratios. The precipitated minerals were calcite, magnesium (Mg) calcite, and struvite (MgNH(4)PO(4) x 6H(2)O) in variable proportions depending on the Mg(2+)/Ca(2+) ratio of the medium. The Mg content of the Mg-calcite decreased with increasing Ca(2+) concentration in the medium. According to the saturation indices, other minerals could also have precipitated. We observed important differences between the morphology of carbonate and phosphate, which may help us to recognize these minerals in natural systems. We studied the growth and pH curves of four bacteria in media specific for carbonate and struvite precipitation. We consider the biomineralization processes that produce carbonate and phosphate minerals, and propose a hypothesis for the lack of struvite in natural environments and ancient rocks.     

Application of struvite precipitation as a pretreatment in treating swine wastewater

In this study, the effect of the pretreatment of NH₄-N by struvite precipitation on biological nitrogen removal was investigated in treating swine wastewater. Evaluation was mainly focused on nitrification which occurred in the activated sludge system after struvite precipitation. Laboratory experiments were performed at four different hydraulic retention times (HRT), i.e., 48, 32, 24 and 16 h. Results of the long-term operation of systems showed that the struvite precipitation used as the pretreatment of raw swine wastewater enhanced the nitrification performance in activated sludge system by reducing the applied loading rates of NH₄-N and TCOD in all operating conditions. The reduction of the applied NH₄-N loading rate kept the levels of free ammonia (FA) concentration in biological reactors low and it prevented nitrite accumulation. In addition, the struvite precipitation elicited the biological denitrification reaction and PO₄-P removal by increasing the ratios of carbon-to-nitrogen and carbon-to-phosphorus of wastewater after struvite precipitation. The struvite precipitation also enhanced the biological TCOD removal performance by reducing the toxic effect of FA. Triplicate INT-dehydrogenase tests clearly showed that FA inhibited the degradation of organic matter in activated sludge system. Finally, the struvite precipitation contributed to high TCOD, T-N and PO₄-P removals of 83, 90, and 97% by facilitating biological reaction at a short HRT of 16 h.     

Autolysis of high-GC isolates of Pseudomonas putrefaciens

High-GC isolates of P. putrefaciens undergo extensive autolysis after growth, resulting in a marked decrease in turbidity and the release of high-molecular-weight DNA which imparts a high viscosity to culture broths. The native DNA released is resistant to attack by the exocellular DNase activity of the culture broths. Autolysis is inhibited by a pH of 6.0 and the presence of 0.001 m Mg⁺⁺ or Ca⁺⁺, and is enhanced by elevated pH values and temperatures. This autolytic phenomenon in broth cultures readily distinguishes high- from low-GC isolates. The latter do not exhibit autolysis.Paper No. 1093, Massachusetts Agricultural Experiment Station, University of massachusetts at Amherst. This research was supported in part from Experiment Station Project No. 194 and Public Health Service Research grant FD 00153-09.     

Isolation and metagenomic characterization of bacteria associated with calcium carbonate and struvite precipitation in a pure moving bed biofilm reactor-membrane bioreactor

A bench-scale pure moving bed bioreactor-membrane bioreactor (MBBR-MBR) used for the treatment of urban wastewater was analyzed for the identification of bacterial strains with the potential capacity for calcium carbonate and struvite biomineral formation. Isolation of mineral-forming strains on calcium carbonate and struvite media revealed six major colonies with a carbonate or struvite precipitation capacity in the biofouling on the membrane surface and showed that heterotrophic bacteria with the ability to precipitate calcium carbonate and struvite constituted ~7.5% of the total platable bacteria. These belonged to the genera Lysinibacillus, Trichococcus, Comamomas and Bacillus. Pyrosequencing analysis of the microbial communities in the suspended cells and membrane biofouling showed a high degree of similarity in all the samples collected with respect to bacterial assemblage. The study of operational taxonomic units (OTUs) identified through pyrosequencing suggested that ~21% of the total bacterial community identified in the biofouling could potentially form calcium carbonate or struvite crystals in the pure MBBR-MBR system used for the treatment of urban wastewater.     

Struvite precipitation in anaerobic swine lagoon liquid: effect of pH and Mg:P ratio and determination of rate constant

Because of increased concern about surface water eutrophication from nutrient-enriched agricultural runoff, many swine producers are encouraged to decrease application rates of waste-based P. Precipitation and subsequent removal of magnesium ammonium phosphate (MgNH(4)PO(4) x 6H(2)O), commonly known as struvite, is a promising mechanism for N and P removal from anaerobic swine lagoon effluent. The objectives of this research were to (i) quantify the effects of adjusting pH and Mg:P ratio on struvite precipitation and (ii) determine the rate constant pH effect for struvite precipitation in anaerobic swine lagoon liquid. Concentrations of PO(4)-P in liquid from two anaerobic swine lagoons were determined after 24 h of equilibration for a pH range of 7.5-9.5 and Mg:P ratios between 1:1 and 1.6:1. Struvite formation reduced the PO(4)-P concentration in the effluents to as low as 2 mgl(-1). Minimum concentrations of PO(4)-P occurred between pH 8.9 and 9.25 at all Mg:P ratios. Struvite precipitation decreased PO(4)-P concentrations by 85% within 20 min at pH 9.0 for an initial Mg:P ratio of 1.2:1. The rate of PO(4)-P decrease was described by a first-order kinetic model, with rate constants of 3.7, 7.9, and 12.3 h(-1) at pH 8.4, 8.7 and 9.0 respectively. Our results indicate that induced struvite formation is a technically feasible method to remove N and P from swine lagoon liquid and it may allow swine producers to recover nutrients for off-farm sale.     

Production of syngenetic minerals with struvite by myxococcus coralloides D

The formation under laboratory conditions of newberyite, schertelite, and taylorite in conjunction with struvite by the bacterium Myxococcus coralloides D is reported for the first time. The presence of these syngenetic minerals with struvite was only detected in static liquid cultures.     

Effects of different buffers on the thermostability and autolysis of a cold-adapted protease MCP-01

A cold-adapted protease MCP-01 was obtained from deep-sea psychrotrophic bacterium Pseudoaltermonas sp. SM9913. The effects of four different buffers, all at 50 mmol/l concentration, on its thermostability and autolysis were studied. The autolysis process of MCP-01 was studied by capillary electrophoresis. The thermostability of MCP-01 increased successively in the following order: carbonate < Tris < phosphate < borate. The optimum temperature for casein hydrolysis also increased in the same order. This suggested that the conformation of MCP-01 was flexible and its autolytic susceptibility was affected by some factors in the buffers such as charge and ionic species. The results also showed that different buffers, in addition to affecting the autolysis speed, gave different patterns of autolysis products. In carbonate buffer, Tris buffer, phosphate buffer and borate buffer, the autolysis patterns of MCP-01 were different. These results suggested that protease MCP-01 probably have different conformations in different buffers, thus exposing different autolysis sites on the enzyme surface. In addition, the loss of activity correlated with the speed of autolysis in the four different buffers, showing that autolysis may be a reason for the low thermostability of the enzyme.     

Protease A activity and nitrogen fractions released during alcoholic fermentation and autolysis in enological conditions

Determination of protease A activity during alcoholic fermentation of a synthetic must (pH 3.5 at 25°C) and during autolysis showed that a sixfold induction of protease A activity occurred after sugar exhaustion, well before 100% cell death occurred. A decrease in protease A activity was observed when yeast cell autolysis started. Extracellular protease A activity was detected late in the autolysis process, which suggests that protease A is not easily released. Evolution of amino acids and peptides was determined during alcoholic fermentation and during autolysis. Amino acids were released in early stationary phase. These amino acids were subsequently assimilated during the fermentation. The same pattern was observed for peptides; this has never been reported previously. During autolysis, the concentration of amino acids and peptides increased to reach a maximum of 20 and 40 mg N l⁻¹, respectively. This study supports the idea that although protease A activity seemed to be responsible for peptides release, there is no clear correlation among protease A activity, cell death, and autolysis. The amino acid composition of the peptides showed some variations between peptides released during alcoholic fermentation and during autolysis. Depending on aging time on yeast lees, the nature of the peptides present in the medium changed, which could lead to different organoleptic properties. Journal of Industrial Microbiology & Biotechnology (2001) 26, 235–240. Received 02 August 2000/ Accepted in revised form 15 December 2000     

Involvement of an N-Acetylglucosaminidase in Autolysis of Propionibacterium freudenreichii CNRZ 725

Propionibacterium freudenreichii plays an important role in Swiss cheese ripening (it produces propionic acid, acetic acid, and CO₂). Moreover, autolysis of this organism certainly contributes to proteolysis and lipolysis of the curd because intracellular enzymes are released. By varying external factors, we determined the following conditions which promoted autolysis of both whole cells and isolated cell walls of P. freudenreichii CNRZ 725: (i) 0.1 M potassium phosphate buffer (pH 5.8) at 40°C and (ii) 0.05 to 0.1 M KCl at 40°C. We found that early-exponential-phase cells possessed the highest autolytic activity. It should be emphasized that the pH of Swiss cheese curd (pH 5.5 to 5.7) is near the optimal pH which we determined. Ultrastructural observations by electron microscopy revealed a 16-nm-thick homogeneous cell wall, as well as degradation of the cell wall that occurred concomitantly with cell autolysis. In the presence of 0.05 M potassium chloride, there was a great deal of isolated cell wall autolysis (the optical density at 650 nm decreased 77.5% ± 7.3% in 3 h), and one-half of the peptidoglycan material was released. Finally, the main autolytic activity was due to an N-acetylglucosaminidase activity.     

Role of hydrolytic enzymes and oxidative stress in autolysis and morphology of <Emphasis Type="Italic">Blakeslea trispora</Emphasis> during β-carotene production in submerged fermentation

The role of hydrolytic enzymes (proteases and chitinase) and oxidative stress in the autolysis and morphology of Blakeslea trispora during β-carotene production from a chemically defined medium in shake flask culture was investigated. The process of cellular autolysis was studied by measuring the changes in biomass dry weight, pH, concentration of β-carotene, specific activity of the hydrolytic enzymes and micromorphology of the fungus using a computerized image analysis system. In addition, the phenomenon of autolysis was associated with high concentrations of reactive oxygen species (ROS). The accumulation of ROS produced during fermentation causes oxidative stress in B. trispora. Oxidative stress was examined in terms of the activities of two key defensive enzymes: catalase (CAT) and superoxide dismutase (SOD). The profile of the specific activities of the above enzymes appeared to correlate with the oxidative stress of the fungus. The high activities of CAT and SOD showed that B. trispora is found under oxidative stress during β-carotene production. The culture began to show signs of autolysis nearly in the growth phase and autolysis increased significantly during the production phase. The morphological differentiation of the fungus was a result of the degradation of the cell membrane by hydrolytic enzymes and oxidative stress. Increased β-carotene production is correlated with intense autolysis of clumps, which has as a consequence the increase of the freely dispersed mycelia.     

Enhanced production of manganese peroxidase from immobilized <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis> due to the increased autolysis of chlamydospore-like cells

The autolysis of chlamydospore-like cells in Phanerochaete chrysosporium immobilized in polyurethane foam correlated with the production of manganese peroxidase (MnP). The maximum specific activity of MnP was 1055 U g dry mycelium^–1 in the immobilized culture, compared with 260 U g dry mycelium^–1 in the submerged culture. Scattered mycelial pellets were formed in the immobilized culture in which almost all of the chlamydospore-like cells were subject to autolysis. However, highly crowded pellets were formed in the free culture, in which only the chlamydospore-like cells in the exterior were subject to autolysis. We propose that the enhanced production of MnP in immobilized cultures of P. chrysosporium is due to increased autolysis of the chlamydospore-like cells.