December 1, 2011 (Vol. 31, No. 21)

Editor’s Note

One of the most promising business areas in biotech during the early and late 1980s was bioremediation. GEN even devoted a regular column to the topic as there were about 30–40 companies carrying out this type of work. In addition, one or two major bioremediation conferences were held each year.

The technology itself involved the use of naturally occurring bacteria to clean up all types of hazardous waste. Indeed, bioremediation still takes place on a broad scale all over the globe.

But the bioremediation proponents 25 to 30 years ago truly wanted to see the technology evolve into something larger than its current status. They planned to develop bioremediation into a major biotechnology sector and to eventually explore the use of genetically engineered microorganisms to handle hazardous waste more efficiently and economically. However, the bioremediation industry never reached the pinnacle of such expectations, for a number of scientific and economic reasons.

This issue’s Flashback article was one of GEN’s first stories on the use of bacteria to remediate waste. It also discusses the research of Ananda Chakrabarty, Ph.D., who received the first U.S. patent for a genetically engineered life form, a modified Pseudomonas bacterium.

—John Sterling, Editor in Chief

“As Seen in GEN”—Flashback Volume 1, Number 3, May/June 1981

Bacteria Combat Waste

While the Patent Office tackles the pile of applications brought on by the Chakrabarty ruling, Chakrabarty himself continues with the same field of research involved in that litigation—developing strains of microorganisms capable of degrading the toxic wastes of man. He also may be taking his place alongside many of his scientific colleagues, riding the biotechnology wave into commercial venture.

Neither his present work nor the work done for General Electric, which led to the Supreme Court involve recombinant DNA. Bacteria have the natural ability to pass plasmids back and forth between individuals within a species and between species. This phenomenon first was noted among bacteria under assault with antibiotics, when it was found that hereditary resistance to an antibiotic could be passed from one strain of bacterium to another. The means of transfer of resistance was found to be a ring of DNA occurring not in the nucleus but in the cytoplasm—the plasmid, the key to recombinant-DNA technology.

Plasmids are passed back and forth continually. Usually they confer no selective advantage on recipient cells, and either persist unnoticed or are lost. But if they do confer an advantage, their new hosts can multiply faster than sister cells in the colony, and eventually will take over the colony. Chakrabarty’s technique basically involves establishing a mixed colony of bacteria, then introducing a chemical stress to the colony by slow degrees. Eventually, he hopes, some cells will do better than others under the stress, due to a combination of their nontransferable chromosomal hereditary factors and hereditary factors on newly transferred plasmids.

The stress used may be increasing levels of crude oil, or larger and larger amounts of a potently toxic waste. If all goes well, a pure strain of microorganism evolves very rapidly in the chemostat under this intense selection pressure that is capable of metabolizing the waste efficiently. No gene splicing has been done. Instead, by random shuffling of natural genes on plasmids, a combination of cell type and foreign plasmids emerges that can exist cheerfully metabolizing the chemical stress. Often the chemical can serve as an energy source for the bacteria, in fact.

Prior to the development of the microorganism of the court decision, bacteria were known that could metabolize this or that fraction of crude oil. But none existed that could do the whole job until Chakrabarty bred a strain containing not one type of oil-degrading plasmid but four. Now he is working to nudge some bacteria toward pure cultures, that will degrade toxic herbicides like Agent Orange (2,4,5-T) and its contaminant dioxin, or polychlorinated biphenylis.

Speaking at the recent Batelle Conference in Washington, Chakrabarty said one of the major difficulties in using this approach for chlorinated compounds is that the pathways needed for degrading them are so far away from anything normally occurring in nature. But he told GEN that he already has developed a mixed culture that can degrade the 2,4,5-T fraction of Agent Orange, which is highly persistent in nature. After eight months of selection pressure, he has a mixture of some four species that acting together can completely metabolize experimental levels of 2,4,5-T within five days. This compound and its dioxin impurity have been blamed for cases of sterility, birth defects, and neurological disorders.

Chakrabarty obtained his original mixed culture from soil taken from sites heavily contaminated with 2,4,5-T, and added to it other bacteria known to have efficient degradative pathways. By reducing ordinary substrates for the colony while increasing levels of 2,4,5,-T, he selected for bacteria using the chemical as an energy source. After six months, 2,4,5-T was the only energy source available. Still to be determined, noted Chakrabarty at the Batelle meeting, is whether these specially evolved bugs can survive any more in their native habitat.

The problem may be, he explained, that in becoming so specialized, they may have lost the flexibility and other characteristics they need to compete with their wild cousins. So simply scattering these newly evolved bacteria around in areas of 2,4,5-T contamination may not be sufficient to clean up the chemical, as the bugs may not survive long enough to do the job.

Chakrabarty predicts that if he can produce a single strain, rather than a mixture, which can render the 2,4,5-T harmless, his “flash-evolving” process will provide the path to developing other detoxifying microorganisms. He told GEN he is interested in studying the enzymes and breakdown intermediates involved in the detoxification process. He said he expects that once his present mixed strains produce a single strain with the mixture’s degradative power, all the enzymes involved will be coded for on one plasma, although he admits multiple plasmids may be possible.

Two commercial ventures will be sharing Chakrabarty’s time with the University of Illinois Medical Center, where he presently works. He will be a consultant to and part owner of a new Chicago biotechnology firm, whose president is Al G. Swan and which currently is engaged in raising capital. He will also serve as a consultant to Applied Genetics International, a North Carolina company.

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