Final Report: Biomineralization of Heavy Metals Within Fungal Mycelia A New Technology for Bioremediation of Hazardous Wastes

EPA Grant Number: R823341
Title: Biomineralization of Heavy Metals Within Fungal Mycelia A New Technology for Bioremediation of Hazardous Wastes
Investigators: Crusberg, Theodore C.
Institution: Worcester Polytechnic Institute
EPA Project Officer: Lasat, Mitch
Project Period: October 1, 1995 through September 1, 1998
Project Amount: $296,053
RFA: Exploratory Research - Engineering (1995) Recipients Lists
Research Category: Engineering and Environmental Chemistry , Land and Waste Management


The long-term goal of this research was to demonstrate feasibility of a process using an innovative fungal BIOTRAP for removal and recovery of heavy metal ions from wastewaters. This project dealt with developing a model system composed of a renewable fungal BIOTRAP to remove heavy metal ions from wastewaters and to recover them for possible reuse, and focused on studies which together would accomplish the goals of the project by:

1. Investigating the thermodynamics of heavy metal biomineralization within mycelia beads of Penicillium ochro-chloron BIOTRAP [using Cu++, Ni++, Pb++, and Cd++] at pH values from 2 to 5 and in the presence of competing ions and chelators (these metals are listed in 40CFR Part 433).

2. Determining the composition of biomineralized precipitates within the mycelia of the BIOTRAP.

3. Measuring intrabead pH, and oxygen consumption of mycelia of fungal beads and correlate cell viability in the core of the bead with these parameters and with the efficiency of the biomineralization process.

4. Studying periplasmic alkaline phosphatase in order to determine if levels of that enzyme correlate with the efficiency of the biomineralization process.

5. Demonstrating the feasibility, with a bench-scale model system, that this type of biomineralization process could be developed into a technology to aid in the removal and recovery of heavy metals from wastewaters.

6. Developing a mathematical predictive model to describe the bench-scale separation system for heavy metal removal and retrieval from various wastewaters.

Summary/Accomplishments (Outputs/Outcomes):

The long-term goal of this research was to demonstrate feasibility of a process using an innovative fungal biotrap for removal and recovery of heavy metal ions from wastewaters. Four bioreactor systems (shake flasks, packed column, trickling filter, and fluidized bed) were evaluated using 3-4 mm mycelia beads of the filamentous fungus Penicillium ochro-chloron (ATCC 36741) as a biological trap (biotrap) for remediation of heavy metal contaminants in industrial wastewaters.

Capillary ion electrophoresis was optimized for analysis of several ions (Cu, Ni, Zn and Au) at concentrations as low as 10 mg/L in nutrient media containing 540 mg/L Na, 270 mg/L K, 50 mg/L Mg and 27 mg/L Ca. The analysis used 400 mL samples, permitting aliquots to be taken during the course of a shake-flask experiment without introducing significant volumetric errors. Multiple analysis were made from the same sample. The method was applied for determination of metal uptake rates, optimizing nutritional requirements and microbial growth rates and the effect of varying environmental factors for metal-sorbing organisms. Analytical methodologies were confirmed using the spectrophotometric bathocuproine method for Cu and by inductively coupled plasma (ICP) mass spectroscopy for all metal ions.

In shake flasks (150 mL volumes) the fungus removed copper from surrogate wastewaters containing 100 mg/L Cu2+ by almost 99 percent. Shake flask batch cultures are very inconvenient and cumbersome for an industrial application, require long incubation periods up to 5 days for maximal metal ion removal and require a large "footprint", not to mention the energy costs associated with temperature control and continual shaking for aeration. Although shake flask cultures showed very promising results and were useful in establishing a baseline to understand the biological basis of metal ion removal this process was dismissed as a possible industrial application. Sodium azide pre-treatment prior to an incubation to test copper ion uptake by the fungi showed that a functional respiratory system was required in Penicillium ochro-chloron is to serve as a biotrap. Azide inhibited Cu2+ precipitation until the respiratory system recovered hours after treatment.

These results however were not duplicated for a packed bed column bioreactor configuration. A packed bed column consisting of fungal beads was unable to support metal ion removal, due primarily to channeling of the medium around the beads as it was pumped through the column. Flow though the packed bed column exhibited characteristics very much like a column filled with solid polystyrene beads of similar size.

A closed-loop trickling filter bioreactor was constructed and tested under conditions of 10-100 mg/L Cu2+. A residence time of no more than 24 hr. using this configuration insured that the system did not clog, but in such a brief time period little copper ion could be removed. The trickling filter was able to remove just less than 30 mg Cu2+ from solution per g dry wt mycelia, far less than other bioreactor systems.

The results from the fluidized bed reactor showed that the fungus is capable of removing copper down to approximately 5ppm if the conditions were favorable. These results supported similar shake flask studies where 95 percent copper removal was achieved in 100 ppm solutions. The fluidized bed bioreactor resulted in 97 percent copper removal, with a capacity of up to 149 mg Cu/g dry weight biomass, under conditions of at least 50 percent (saturation) dissolved oxygen. Below the critical oxygen concentration for the fungus (20 percent saturation) there was minimal copper removal and respiration was required for optimal copper removal.

Mixing studies in the fluidized bed reactor showed that the system was diffusion limited and the first order rate constant under those conditions for Cu2+ removal was 0.031 hr-1, which was dependent upon the dissolved oxygen concentration. A second order rate equation suggested that there are possibly factors regulating copper uptake other than oxygen.

Scanning electron microscopy (SEM) coupled with energy dispersive x-ray (EDX) microanalysis showed that in some instances the copper removed from solution was retained within the fungal mycelia as porous spherical extracellular precipitates of insoluble copper phosphate. Smooth spherical precipitates also deposited in the mycelia appear to be the oxalate salt of copper as inferred from EDX analysis. Precipitation of copper as insoluble cupric phosphate suggested a role for the periplasmic enzyme alkaline phosphatase (AP) whose function is to hydrolyze the phosphate group from extracellular organophosphates. Biochemical studies on this enzyme were complemented with attempts to sequence its gene. A biochemical profile of periplasmic AP suggested that the enzyme was not very functional at pH 4, the pH at which the metal ion uptake studies were carried out. Only a partial sequence of the gene for the enzyme was obtained. Tranformation studies using several antibiotic resistance markers were not successful. It is concluded that copper uptake by the fungus is due to two mechanisms. The first process is precipitation of insoluble copper phosphate which appears to occur early during copper uptake. Porous microspheres of this precipitate are detected by SEM and EDX early in the fermentation within the mycelial beads. As glucose is utilized later in the fermentation then insoluble copper oxalate forms on the surface of the mycelia also deduced by SEM and EDX. Smooth microspheres of this precipitate and smooth surface coatings on the mycelia are observed later in the fermentation.

Removal of heavy metal contaminants from industrial wastewater discharges is necessary for many industries to maintain a competitive economic edge, due to environmental concerns and federal regulations. The use of a renewable biotrap for the removal and recycling of heavy metals could prove more economical than currently used physio-chemical processes. A fluidized bed bioreactor with high aeration appears to be the best configuration for further studies on the remediation of highly contaminated wastewaters, yet no system studied was able to reliably remove Cu2+ from aqueous solution at initial metal ion levels below 100 mg/L. That a high initial concentration of Cu2+ (100 mg/L) was a requirement for optimal metal ion uptake was not able to be explained through these experiments.

During the course of investigating the fungal biotrap we were also undertaking a study of highly viscous fermentations of a Bacillus licheniformis strain in which the high molecular weight polymer poly-g-linked-glutamic acid (g-PGA, a polyamide) ) was produced. This polymer was purified with a yield of 30-40 g/L in a 40L fermenter. Since g-PGA above pH 4 is a polyanion it was evaluated as a potential biosorbent material for use in the removal of heavy metals from aqueous solution. Metal-biopolymer stability constants under varying conditions of pH and temperature were determined for native g-PGA using a specially designed multi-chamber equilibrium dialysis apparatus. Modeling the experimental data using the Langmuir adsorption isotherm showed that g-PGA had a copper capacity approaching 77.9 mg/g. The kinetics of copper removal and the effect of cations and ionic strength on copper uptake capacity were also investigated. Not satisfied with the binding capacity of g-PGA for Cu2+ in the equilibrium dialysis system we used an ultrafiltration system to form a membrane of the polyanion and constructed a polymer-enhanced diafiltration (PEDF) system which proved exceptionally efficient in removing Cu2+ ions from dilute aqueous solutions (10 mg/L).


This work has demonstrated promising results using Penicillium ochro-chloron for the removal of copper ions from aqueous solutions. The results obtained from the study proved that the fungus has the potential to remove copper to relatively low levels if provided with favorable conditions. The fungus requires oxygen above its critical oxygen concentration for optimal removal. When comparing the levels of copper remaining in experimental media to the standards set by the EPA, it is clear that the fungus would meet at least some of those standards, if used in a recovery process. The current values for the discharge copper concentration for the metal finishing industry is an average of 2.7 mg/L copper/day (40 CFR, 1998). However, for any fresh waterway, the maximum copper concentration is 17 µg/L (0.017 mg/L) (EPA, 1995), implying the fungus could not be used to detoxify existing bodies of water. The fungus can however remove copper at very high concentrations (100 mg/L), and can therefore be valuable in industrial wastewater treatment applications. Comparing it to other organisms known to absorb and bind copper it has a rather high binding capacity (148.7 mg Cu/g dry weight) and can tolerate higher initial concentrations of copper than most shown. Potential use, therefore, could be in the electroplating industries or any other industry which produce high copper levels in wastewaters. Any potential use of the fungus as an industrial wastewater treatment would require further research into the mechanism of uptake beyond oxygen availability in order to guarantee sustained uptake and eliminate re-release back into the waste stream. It is now possible to use the fungal biotrap in a fluidized bed reactor to reduce Cu2+ at high concentrations (100 mg/L) in industrial wastewaters to virtually 0 by coupling this process to the PEDF technology.

Journal Articles on this Report : 2 Displayed | Download in RIS Format

Other project views: All 8 publications 2 publications in selected types All 0 journal articles
Type Citation Project Document Sources
Book Chapter Crusberg TC, Mark SS. Biosorption of heavy metals: bacteria, yeast, fungi and plants, in metals: mine drainage, removal and toxicity. In: Britz M, Robbins N, Roddick F, eds. Part of the series, Current Issues in Global Environmental Biotechnology, Wise DL, ed. Elsevier Science 1997. R823341 (Final)
not available
Book Chapter Wise DL. Global environmental biotechnology. Elsevier Science 1997. R823341 (Final)
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Supplemental Keywords:

scanning electron microscopy, SEM, energy dispersive x-ray analysis, EDX, feasibility, demonstration, waste management, hazardous wastes, innovative technology, biomineralization, heavy metals, bioremediation, fungal mycelia, BIOTRAP, industrial wastewater, thermodynamics, metal recovery process, predictive model., Scientific Discipline, Waste, Hydrology, Physics, Environmental Chemistry, Chemistry, Bioremediation, Engineering, fate and transport, electron microscope, periplasmic phosphate activities, industrial wastewater, biomineralization of heavy metals, biodegradation, chelators, fungal mycelia, chemical transport, biotrap, biotechnology, hazardous waste cleanup, environmental engineering, contaminant release, copper, mathematical models, heavy metals

Progress and Final Reports:

Original Abstract
  • 1996
  • 1997