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40<br />

Technology Watch<br />

Bio-remediation Without Caution<br />

A bacterium living inside plants could be improved for cleaning up environmental<br />

pollutants without genetic modification. Pr<strong>of</strong>. Joe Cummins<br />

and Dr. Mae-Wan Ho reveal that this seemingly beneficial development<br />

is beset with danger, as the bacterium concerned is a known pathogen.<br />

Water soluble and highly volatile <strong>organic</strong><br />

environmental pollutants, such as benzene,<br />

<strong>to</strong>luene, ethylbenzene and xylene compounds,<br />

chlorinated solvents and nitro<strong>to</strong>luene<br />

ammunition wastes, are being<br />

cleaned up using plants in combination with<br />

microorganisms that naturally live inside the<br />

plants (endophytes).<br />

Endophyte bacteria live within the tissue<br />

<strong>of</strong> the plant without harming it. <strong>The</strong>y are<br />

found in most plant species, and many can<br />

colonize the vascular system. <strong>The</strong> highest<br />

densities <strong>of</strong> bacteria are usually found in the<br />

roots, less in the stem, and least in the<br />

leaves. <strong>The</strong> plants take up the pollutants<br />

through their roots, and the bacteria break<br />

these down within the roots or in other parts<br />

<strong>of</strong> the plant.<br />

This natural process is inefficient<br />

because the compounds tend <strong>to</strong> get transported<br />

up the plant faster than the bacteria<br />

can break them down. Once transported up,<br />

the plants metabolize the contaminants, and<br />

some <strong>of</strong> the metabolites as well as the contaminant<br />

can be <strong>to</strong>xic. For example,<br />

trichloroethane is metabolized in<strong>to</strong><br />

trichloroacetic acid, both <strong>of</strong> which are <strong>to</strong>xic.<br />

Worse still, plants tend <strong>to</strong> release volatile pollutants<br />

and their metabolites in<strong>to</strong> the atmosphere<br />

via evaporation from the leaves, which<br />

turns bio-remediation in<strong>to</strong> bio-pollution.<br />

A recent article in Nature Biotechnology<br />

reports how this clean up process could be<br />

greatly improved by engineering an endophyte<br />

bacterium Burkholderia cepacia, a natural<br />

resident <strong>of</strong> the yellow lupine.<br />

Researchers from Linburgs University in<br />

Diepenbeek, Belgium and Brookhaven<br />

National Labora<strong>to</strong>ry in New York, USA, created<br />

a strain <strong>of</strong> B. cepacia that has enhanced<br />

ability <strong>to</strong> degrade <strong>to</strong>luene within the plant,<br />

enabling the plant <strong>to</strong> <strong>to</strong>lerate high levels <strong>of</strong><br />

<strong>to</strong>luene, and also substantially reduced the<br />

amount <strong>of</strong> <strong>to</strong>luene released in<strong>to</strong> the atmosphere.<br />

<strong>The</strong> engineered strain <strong>of</strong> the bacterium<br />

carries marker genes for kanamycin resistance<br />

and nickel resistance and is derived<br />

from the natural endophyte. By adding <strong>to</strong> this<br />

endophyte strain a <strong>to</strong>luene-degrading plasmid<br />

from another strain <strong>of</strong> B. cepacia that normally<br />

lives in the soil through natural conjugation<br />

(bacterial reproduction) between the<br />

strains, a new endophyte strain is created<br />

that can live in the plant and degrade <strong>to</strong>luene<br />

taken up by the plant.<br />

Plants inoculated with the engineered<br />

bacterium grew much better than plants that<br />

were not inoculated; or else inoculated either<br />

with the control strain lacking the plasmid, or<br />

with the strain that normally lives in the soil.<br />

More impressively, the plants inoculated with<br />

SCIENCE IN SOCIETY 23, AUTUMN 2004<br />

the engineered bacterium reduced <strong>to</strong>luene<br />

evaporation in<strong>to</strong> the atmosphere <strong>to</strong> about<br />

50% <strong>of</strong> the control. This looks very promising,<br />

and as the researchers point out, the experiment<br />

could have been done without any<br />

genetic modification. <strong>The</strong> plasmid containing<br />

all the <strong>to</strong>luene degrading enzymes belonged<br />

<strong>to</strong> a natural soil bacterium, and an endophyte<br />

host without the marker genes could easily<br />

have been used <strong>to</strong> receive the plasmid by<br />

conjugation.<br />

A non-GM bacterial endophytic strain<br />

created in this way may well be the very first<br />

really useful and beneficial product from the<br />

industry. So what's wrong?<br />

<strong>The</strong> research paper did not deal with<br />

safety. What metabolites <strong>of</strong> <strong>to</strong>luene are generated<br />

in the plant, and will they be <strong>to</strong>xic?<br />

How will the plants be disposed <strong>of</strong>? <strong>The</strong>re<br />

are three lupine species cultivated for fodder<br />

- blue, white and yellow - and there are also<br />

a number <strong>of</strong> wild species. <strong>The</strong> wild species<br />

contain alkaloid chemicals that are very <strong>to</strong>xic<br />

<strong>to</strong> cattle and sheep while the cultivated<br />

species are edible for farm animals, provided<br />

care is taken <strong>to</strong> treat the seeds in such a way<br />

as <strong>to</strong> remove the <strong>to</strong>xins. Lupines thrive on<br />

poor soil and provide ground cover and<br />

green manure as well as fodder for animals.<br />

More importantly, the research report<br />

failed <strong>to</strong> mention that B. cepacia has the ability<br />

<strong>to</strong> cause fatal disease in humans.<br />

<strong>The</strong> groundwater <strong>of</strong> Wichita, Kansas<br />

was found <strong>to</strong> be polluted with the chemical<br />

solvents dichloroethylene and trichloroethylene,<br />

and was cleaned up using a natural<br />

strain <strong>of</strong> B. cepacia. But no special public<br />

health measures or follow up seemed <strong>to</strong><br />

have been implemented after the clean up.<br />

<strong>The</strong> United States Environmental<br />

Protection Agency (EPA) has considered the<br />

problems associated with approval <strong>of</strong> B.<br />

cepacia as a plant pesticide, for, not only is<br />

the bacterium used <strong>to</strong> fight plant pests but is<br />

itself a pest as it is a disease agent in<br />

humans. EPA, through a Scientific Advisory<br />

Panel (SAP), reviewed B. cepacia as a plant<br />

pesticide and acknowledged that it is linked <strong>to</strong><br />

human disease. <strong>The</strong> SAP risk assessment<br />

peculiarly noted, "Bc [B. cepacia] has been<br />

referred <strong>to</strong> as an opportunistic human<br />

pathogen. However, as might be expected,<br />

the strains registered or proposed for use as<br />

biopesticides were isolated from the soil or<br />

plant roots, rather than from human patients".<br />

<strong>In</strong> reality, the SAP comment <strong>of</strong>fered cold<br />

comfort because the B. cepacia strains isolated<br />

from patients proved essentially undistinguishable<br />

from strains isolated from the<br />

roots <strong>of</strong> crops such as corn. <strong>The</strong> American<br />

Phy<strong>to</strong>pathological Society produced a useful<br />

review <strong>of</strong> the risks from plant disease or<br />

human disease along with the benefits in<br />

cleaning up chemical pollution and fighting<br />

some plant diseases. Unfortunately, there<br />

has been no clear and simple way <strong>to</strong> differentiate<br />

between the 'evil' and the beneficial<br />

strains <strong>of</strong> B. cepacia, and no way <strong>of</strong> preventing<br />

the two from exchanging genes.<br />

B. cepacia has an unusual genetic<br />

makeup; it has a relatively large amount <strong>of</strong><br />

DNA (about twice that <strong>of</strong> E. coli) and unlike<br />

most bacteria, which usually have a single<br />

chromosome, B. cepacia strains have as<br />

many as five large replicons (chromosomes)<br />

and the different chromosomes are rich in<br />

insertion sequences that allow for extensive<br />

gene exchange between different strains,<br />

and insertion <strong>of</strong> disease related genes from<br />

other bacterial species. B. cepacia is a prominent<br />

cause <strong>of</strong> death among cystic fibrosis<br />

patients, the bacterium frequently reaches<br />

epidemic proportions among such patients<br />

and an epidemic related strain was identified<br />

in soil samples in the USA. It is believed <strong>to</strong> be<br />

a complex species made up <strong>of</strong> seven distinct<br />

genomic subspecies all <strong>of</strong> which are capable<br />

<strong>of</strong> infecting humans; and all <strong>of</strong> the diseaserelated<br />

subspecies were isolated from maize<br />

rhizosphere (root zone). <strong>The</strong> disease is difficult<br />

<strong>to</strong> contain because disease bacteria may<br />

be replenished continually from the soil and<br />

plant material.<br />

Hospital acquired B. cepacia epidemics<br />

appeared among patients with diabetes,<br />

malignancy, heart failure and chronic obstructive<br />

pulmonary disease. One such B. cepacia<br />

outbreak appeared in an intensive pediatric<br />

care unit, and B. cepacia infection was common<br />

among renal transplant patients.<br />

Different B. cepacia clones showed different<br />

infectivity among cystic fibrosis patients and<br />

patients with different complaints. Antibiotic<br />

resistant B. cepacia infection was the most<br />

common cause <strong>of</strong> death among lung transplants<br />

for cystic fibrosis patients. B. cepacia<br />

causes feared infections because the strains<br />

tend <strong>to</strong> be antibiotic resistant. Bacteria isolated<br />

from different infections were found <strong>to</strong> be<br />

resistant <strong>to</strong> all seven tested antibiotics but<br />

were sensitive <strong>to</strong> treatment with honey.<br />

Do lupines pose a threat <strong>to</strong> people with<br />

compromised immune systems or cystic<br />

fibrosis? Yellow lupines, and perhaps the<br />

other commercial species as well, contain<br />

potentially disease-causing B. cepacia endophytes,<br />

so their presence in hospitals and<br />

homes <strong>of</strong> compromised people is unwise.<br />

<strong>The</strong> bacteria may be transferred by direct<br />

contact with broken plant stems or petals<br />

along with the dust and debris associated<br />

with the plant; a gift <strong>of</strong> lupines could be fatal.<br />

<strong>The</strong>re is clearly a large literature on the<br />

threat <strong>of</strong> B. cepacia infection and its death <strong>to</strong>ll<br />

among compromised patients. <strong>The</strong> existing<br />

evidence indicates that the bacterial infections<br />

may pass from the ecosystem <strong>to</strong> the<br />

hospital ward and there seems no way <strong>of</strong><br />

ensuring that the B. cepacia strains used in<br />

biotechnology are unable <strong>to</strong> infect compromised<br />

humans.<br />

SiS

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