Encapsulation of Bacteria for Biodegradation of Gasoline Hydrocarbons

Peyman Moslemy, Serge R. Guiot, and Ronald J. Neufeld


Gasoline hydrocarbons are a common source of contamination to soil and aquifer systems. Biodégradation of these contaminants by hydrogel-encapsulated bacteria is a novel technique for bioremediation of contaminated sites. Hydrogel capsules provide a stable, consistent, and protective microenvironment for prolonged survival and metabolic activity of encapsulated cells. This monograph presents the materials and methods for encapsulation of gasoline-degrading bacteria in gellan gum microspheres. The microencapsulation process is based on a two-phase dispersion technique, termed emulsification-internal gelation. The sphere formation process involves the dispersion of two immiscible liquid phases resulting in a water-in-oil (w/o) emulsion. A suspension of viable cells in an aqueous solution of gellan gum (disperse phase) is emulsified in a hydrophobic phase such as canola oil (continuous phase). The gelation of the small droplets of the dispersed phase is subsequently initiated by decreasing the emulsion temperature. Changing the emulsion conditions can alter the size distribution of microspheres. The emulsification-internal gelation method is applicable to encapsulation of other microbial degraders in macro- or microspheres subject to the employment of proper emulsion conditions.

Key Words: Biodegradation; gasoline hydrocarbons; bacteria; encapsulation; emulsi-fication-internal gelation; gellan gum.

1. Introduction

The release of petroleum products such as gasoline from leaking underground storage tanks is a common source of contamination to soil and groundwater (1-3). Gasoline is a light distillate and may contain C4 to Cn alkane, cycloalkane, alk-ene, and aromatic hydrocarbons (4). These compounds are sufficiently soluble in water to pose a major treat to groundwater quality. Aromatic components such as benzene, toluene, ethylbenzene and xylenes (ortho-, meta-, and para-xylene), collectively referred to as BTEX, comprise between 20% and 30% of commercial

From: Methods in Biotechnology: Immobilization of Enzymes and Cells, Second Edition Edited by: J. M. Guisan © Humana Press Inc., Totowa, NJ

gasoline. The BTEX compounds are typically the targets of regulatory concern and the United States Environmental Protection Agency has classified them as hazardous waste (5). The BTEX compounds are relatively water-soluble and mobile, and therefore, even a small discharge of gasoline has the potential to render large portions of groundwater unfit for drinking.

Gasoline hydrocarbon components may be transferred from the fuel liquid phase to the air, water, and soil phases by a variety of physical, chemical, and biological processes that can occur in the subsurface. Upon the release of gasoline into a subsurface environment, a minor fraction of gasoline hydrocarbons may be subject to evaporative and photodegradative losses. However, the majority of gasoline hydrocarbons will be subject to vertical and horizontal infiltration at a rate determined primarily by the stratification and permeability of the soil materials. Infiltrated hydrocarbons migrate by gravity downward through unsaturated soil layers (vadose zone) and may eventually reach the soil water table (capillary zone) where they form a floating light nonaqueous phase liquid (LNAPL). A small portion of hydrocarbons is dissolved in groundwater (saturated zone), forming a contaminant plume, which spreads within the direction of bulk groundwater flow. Residual floating hydrocarbons may spread out laterally and act as a long-term source of groundwater contamination.

It has been known since the 1940s that some microorganisms are capable of degrading petroleum hydrocarbons (6). However, it was only after the accidental release of massive volumes of gasoline and crude oil into aquatic environments in the late 1960s (7) and the early 1970s (8) that biodegradation was actively considered as a remedial strategy to clean up contaminated groundwater and marine systems.

The biodegradation of gasoline hydrocarbons as the common environmental pollutants has been widely studied in the laboratory (9-17) and field studies (1822). Aromatic components of gasoline (BTEX) have also been the main focus of several biodegradation studies (23-29). The microbial degradation of hydrocarbons is strongly influenced by physical and chemical factors such as temperature, oxygen, nutrients, salinity, water activity, pH, and concentration and availability of the contaminant, and also by biological factors such as the composition, adaptability, and physiological capabilities of the microbial populations.

Over the years, a wide variety of engineered in situ and ex situ bioremediation technologies have been developed (5,30). These technologies are mainly designed to increase the availability of electron acceptors (oxygen or nitrate), electron donors (organic substrates), nutrients (nitrogen, phosphorous, or potassium), or degrading microbial communities. The last approach is generally referred to as bioaugmentation, which is defined as the process of adding specifically adapted exogenous microorganisms to a system to stimulate biodegradation and enhance the rate and/or extent of biodegradation.

Bioaugmentation of subsurface polluted environments (soil and groundwater) or surface bioreactors with pre-isolated microorganisms capable to degrade certain pollutants is an alternate remedy for decontamination of sites or industrial waste effluents (31,32). Successful application of living microorganisms to bioaugmentation schemes depends on inoculum density and formulation, mode of application (single or multiple introductions), mode of operation, biotic and abi-

Table 1

Characteristics of Microbial Supports for Environmental Applications


Size (surface area-to-volume ratio)



Biocompatibility Cell mass loading capacity Retention capacity Performance quality

Stability (chemical, biological, mechanical, and thermal)

Ease of production



Cost (economic feasibility)

otic environmental effects, rate of survival, and the extent of distribution and transport of added microorganisms (e.g., through the soil matrix). Both biotic and abiotic factors play critical roles in determining the survival of introduced microorganisms. In subsurface applications, biological factors include predation by protozoans, lower level of starvation resistance of introduced microbes, and lack of suitable soil niches for extended cell survival. Moisture content, pH, texture, and oxygen and nutrients availability are abiotic factors controlling the survival of microorganisms.

Encapsulation has emerged as a promising solution to overcome practical limitations of using free cell formulations. The polymeric matrix of the support material provides a defined, stable, consistent, and protective microenvironment, where cells can survive and metabolic activity can be maintained for extended periods of time without the immediate release of large number of cells. The entrapped cells can better tolerate numerous environmental stresses, and may be released after adaptation to surrounding environmental conditions. The main characteristics of microbial supports for environmental applications are summarized in Table 1. A variation of these characteristics may be considered in the selection of an appropriate microbial support for a specific application. For instance, micrometer-sized carriers are required for subsurface injection (33) whereas macrometer-sized supports may be desired for surface bioreactor applications or for constructing subsurface permeable reactive barriers (34).

The use of encapsulated cells for biodegradation of petroleum and gasoline hydrocarbons has been explored in a number of studies, as listed in Table 2. However, most of these investigations have been performed at the laboratory scale, and practical uses of encapsulated cells in the open environment have yet to be realized. Both natural and synthetic polymer gels have been used for microbial encapsulation. Criteria for gel supports to be used for soil applications are different from those required for controlled bioreactor systems. Although the long-term stability, low porosity, and small pore sizes afforded by synthetic polymers can be

Table 2

Examples of the Use of Encapsulated Microbial Cells in Biodégradation Studies

Compound Microorganism Carrier Ref.


Crude oil Crude oil Crude oil

Petroleum products Gasoline

Aliphatic hydrocarbons

Hexadecane Mixed culture Urea-formaldehyde 38

n-Alkanes (C14-C16) Prototheca zopfi Alginate 42

Monoaromatic hydrocarbons (MAHs)

Benzene Pseudomonas putida Polyacrylamide 43

Benzene Rhodococcus spp. Alginate 44

Polyaromatic hydrocarbons (PAHs)

Naphthalene Pseudomonas spp. Alginate 45

Phenanthrene Pseudomonas sp. Alginate 46

Phenanthrene Pseudomonas sp. Alginate 47

Phenanthrene Mixed culture Urea-formaldehyde 38

Mixed culture

Yarrowia lipolytica Yarrowia lipolytica

Urea-formaldehyde 38 Agar-Alginate 39

Polyurethane 40

Mixed culture

Gellan Gum

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