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Hunting for Nanobacteria

The recent announcements in the BBC News Online (http://www.bbcnews.com) and New Scientist (newscientist.com) that a new and tiny form of life (spheres 30–100 nanometers in diameter) was found in medical wastes from two U.S. hospitals has rocked the worlds of medicine and science, but it turns out that this is merely the latest outburst in a controversy which has been going on behind the scenes for years, clear back to the late 1980s. In an amusing side note, there’s even disagreement over proper spelling. Standard scientific usage would be “nanobacteria,” as in “nanometer,” (a billionth of a meter) but the geologic community argues that it ought to be “nannobacteria,” in conformance with its other terms, such as “nannoplankton.” This has caused further confusion, leading to the bizarre use of multiple spellings within a single article. Maybe a little history would provide some perspectives?

The Concept of the Tiny

Readers of this magazine should by now be familiar with the Hermetic Principle: “As above, so below.” In essence, this holds that the lower forms of reality mirror the higher that what is real and true at the highest levels also holds down the line. Western philosophy provides its own proximal equivalents, including the first known exposition of the atom by the Greek philosopher Democritus and the much later philosophical novel Micromega, by Voltaire. Western science appeared to provide further confirmation via empirical observation and tests. Antoine van Leeuwenhoek’s discovery of what he called “animalcules” in a droplet of pond water with his primitive microscope added practical fuel to the conceptual fire, as did Pasteur’s much later discovery of germs. Later still came the discovery of viruses, the smashing of the atom, electron microscopy, the discovery of DNA by Watson and Crick, and the search for the almost mythical fundamental atomic particle called the quark. It seems particularly appropriate that the quark hunt at one point entailed going through tons of clam shells searching for a particle, while what purports to be a new life form in humans was allegedly found in surgical waste, specifically “calcified and non-calcified arteries, arterial plaques and heart valves.” If this seems a bit icky and off-putting, recall that phosphorus was discovered by boiling down huge quantities of urine (what a great job that must’ve been!) and that the mold from which comes penicillin was found growing on bread. Truly, great science comes from strange places!

Rock It to Me, Baby!

Before nanobacteria were reported in surgical waste, one Richard first hotly debated them following initial reports. Y. Morita in a seminal paper titled “Bioavailability of energy and starvation survival in nature,” Canadian Journal of Microbiology, 1988, 34:436-441. Writing in Natural Science (http://www.naturalscience.com/ns/articles/01-03/ns,folk.html) in his March 4, 1997 paper “Nanobacteria: surely not figments, but what under heaven are they?” Robert L. Folk, Ph.D., describes how a simple desire to stay in Italy and enjoy warm sunshine and good food led him to a path of ultimately profound discovery.

“The important role of nannobacteria in the mineralogical world was discovered through dumb luck, idle curiosity and random reading. There was no lifetime research plan or this can get me lots of national funding idea involved. I was simply looking for a good excuse to continue doing field work in Italy because I loved the food and the lifestyle, and hit upon the idea of working on the travertines of Rome (travertine is a whitish type of limestone, usually porous, formed in springs, lakes and streams, and has been used as building stone in Rome for 2000 years).”

Working with Professor Henry S. Chafetz of the University of Houston, Dr. Folk, of the Geological Department at the University of Texas, Austin, the study of Italian travertines began in 1979, as a result of which a “chance discovery” revealed the wholly unexpected role of “normal-sized” bacteria in precipitating travertine from Tivoli, Italy’s warm springs. This was particularly true of a type of bacteria called sulfur-oxidizers. He goes on to note: “Before this discovery neither Chafetz nor myself knew or cared anything about bacteria, as we were specialists in the microscopic examination of limestones.” Since all good things must end, Dr. Folk eventually returned to the classroom, but managed to indulge his passion again in 1988, returning to Italy, but this time going to the hot springs of Viterbo, some 50 km. northwest of Rome, to study the travertines there. It was while scanning his travertine samples at magnifications of much as 100,000 times that he got quite a surprise—loads of tiny bumps and balls. Of this surprise he says:

“At first I passed them off as artifacts of sample preparation or laboratory contamination, as had every other scientist who had studied minerals and rocks with the scanning electron microscope (SEM)… After a year of doubts, a little reading in Microbiology unearthed the fact that very small cells called ultramicrobacteria did in fact exist. With further SEM work, slowly the realization dawned that there really were entombed in minerals enormously abundant cells of this minute size… and in some examples the minerals seemed to be entirely made up of nannobacteria as closely packed as beans in a bag. Sometimes within a single crystal or mineral, part of the crystal would be crowded with nannobacteria and parts would be deserted, belying the idea of artifacts or ‘that’s the way minerals naturally dissolve.’ Their occurrence in chains and grape-like clusters further attested to their true living status.”

He goes on to note that he carried on this research from his own resources and that his first presentation (1992) to his colleagues concerning his work was met with “stony silence.”

When Biologists Howl

To biologists, such claims are downright heretical, for what’s being discussed, as Dr. Folk describes it, is “about one tenth the diameter and 1/1000th the volume of ordinary bacteria.” In other words, it is 50 to 200 nanometers in size. The problem? A widely held belief that bacteria simply can’t bio-function once they hit the 200-nanometer threshold. Dr. Folk is flatly unimpressed, producing example after example in photomicrographs of nanobacteria, taken not only from his work with travertines and other stones, but some personally cultured in Austin, Texas tap water over a period of a week using “a stub of aluminum.” As if that weren’t enough to grasp, he then goes on to say that these look exactly like what he can find “in rocks and minerals as old as 2 billion years, or as recent as today, and are dead ringers for those occurring on Mars as to size, shape, and surface features.” Here he invokes the work of McKay et al., which appeared in Science, 273:924-926, in a paper titled “Search for past life on Mars: possible relic biogenic activity in Martian meteorite ALH84001.”

Let’s review. Dr. Folk is saying that nanobacteria are alive, can be cultured, and are found in not only the newest and the oldest terrestrial rocks but also in rocks once part of Mars.

Returning to his paper, we find three items of great value in understanding the controversy over nanobacteria in people. The first appears as part of a listing of rocks found to have active contributions to their formation by nanobacteria. There we read, “Preliminary studies imply that nannobacteria have an active role in the rusting of iron… the “greening” of copper, and the solubilization of aluminum. They also contribute to the plugging of pipes by mineral “scaling,” and appear to be involved in the construction of the CaCO3 shells of clams, foraminifera, and even birds’ eggs.”

The second lies in his account of how nanobacteria were missed so long:

“Probably the main reason is that microbiologists have little or no interest in the occurrence of any type of bacteria in soils or rock, and it has been standard microbiological dogma for fifty years that bacteria smaller than 0.2 micrometers (200 nanometers, Ed.) cannot exist.”

He comments that this limit corresponds to what can be seen through an optical microscope, used for counting bacterial cultures, and that it has long been presumed that such a filter would essentially strain out all the bacteria. Those few geologists actually investigating the interactions between bacteria and minerals naturally followed this methodology, and equally naturally interpreted those strange things in their SEM viewings as contaminated samples. His assessment is on target and biting: “You see what you are looking for and have faith in!” Per a colleague of his, “nannobacteria might be the Dark Matter of the Biological Universe—having enormous effects, but up to now not seen or recognized.”

The third important point to be gleaned from this is that Dr. Folk’s work on nanobacteria in carbonates prompted NASA’s Chris Romanek to apply similar techniques to the previously mentioned Martian meteorite, which was found in Antarctica. When Romanek’s claim for possible evidence of extraterrestrial life exploded on the world scene, it was jumped on by those Dr. Folk deliciously labels the “incognoscenti,” the unknowing ones who were clueless as to the six years of research Dr. Folk had done, hence were in a stampede to dismiss this, too, using the same old arguments (too small to be bacteria, “artifacts”) as before. Little did they know that he had unpublished nanobacteria images, taken from Sicilian volcanic clay, duplicating, feature for feature, those of Chris Romanek’s work. Further, using a different meteorite found in Allende, Mexico in 1969, and taking samples from deep within it, he and his colleagues now believe they have found grape-like nanobacteria clusters there as well. Having laid these foundations, Dr. Folk then summarizes the implications of the discoveries (excerpted here) and issues a call to action.

“If the idea of the ubiquity and overwhelming biomass of nannobacteria on Earth (and perhaps in space as well) is correct, this bears enormous implications for earth-surface chemistry, ore deposition, weathering, in microbiology and even medicine as a potential source of genes or hitherto unseen cause of diseases…

Or maybe the whole idea is nuts. but believe me, the tiny particles do indeed exist, whatever form of quasibiont they represent, and some biologist needs to tell us what they really are.

Armed with the above and a wonderful new word, quasibiont (that which is in some sense or degree alive), let us now return to the biomedical lists, where we find not only the same basic debate raging yet afresh, but apparent profound ignorance of the years of research and tests run by Dr. Folk and his fellow geologists.

The terms of the battle are different in a few places, seemingly made more “scientific” by genetic testing, but the fundamental mind-sets by the naysayers are as inelastic as ever. And who are the latest “nut cases” preaching scientific heresy? Why, doctors from that citadel of radical medicine, the Mayo Clinic, of Rochester, New York! And where did the team fling down its heretical gauntlet? In a painstakingly written, multiply revised, peer-reviewed article in the American Journal of Physiology: Heart and Circulatory Physiology titled, “Evidence of nanobacterial-like structures in human calcified arteries and cardiac valves. The authors are Dr. John Lieske, et. al. The paper basically says “minuscule cell-like structures were isolated from surgical waste” and “self-replicated in culture.” Further, they “could be identified with an antibody and a DNA strain.”

The reactions to a low-key paper based on four years of research, which concludes: “The evidence is suggestive” illustrate science at its worst. “I just don’t think this is real. It is the cold fusion of microbiology.” Note the clever damning of one “scientific heresy” by association with another in Jack Maniloff’s statement. He’s with the University of Rochester, Rochester, New York. Note further that he doesn’t address the issues. John Cisar of the NIH (National Institutes of Health) is equally dismissive: “There are always people who are trying to keep this alive. It’s like it is on. “

Jack Maniloff is far from being an unbiased observer, for the articles report that it was he and his team who found evidence that a cell can’t be smaller than 140 nanometers and still function properly, and both his and John Cisar’s gloves came off when in 1998 Olavi Kajander and Neva Ciftcioglu of the University of Kuopio, Finland, claimed to have “found nanobacteria, surrounded by a calcium-rich mineral called apatite, in human kidney stones.” They also claimed to have cultured it and to have identified a unique DNA sequence. Not only did Jack Maniloff enter the fray, but so did John Cisar, who with his team published a paper in 2000 claiming that the DNA found by the Finnish team was “contamination” from a “normal bacterium” and that the reported self-replication was a crystal growth phenomenon. The reply to that was swift and scathing. Says Jorgen Christoffersen, who studies biomineralization at the University of Copenhagen, Denmark, “They talk about self-propagating apatite. This is scientific nonsense.” Nevertheless, Cisar’s paper has been seized upon as holy writ to trash not only the Finnish team’s work but everything even remotely akin to it, a situation exacerbated by the fact that the Finnish team holds patents in and owns a business, Nanobac Life Sciences in Tampa, Florida, for nanobacterial detection kits. The Mayo Clinic team holds neither patents nor owns a business.

It found apparent nanobacteria only in calcified aneurysms, arterial plaques, and heart valves, and none where there was no calcification. The method was to puree the samples, put them through a 200-nanometer filter, then put the filtrate into culture medium. A few weeks later, the culture’s optical density had doubled, indicating self-replication. With no filtrate added, the medium’s density remained constant. When particles from the apparent growing culture were stripped of minerals and SEM examined, apparent nanobacteria were seen. The team also reported that the filtrate cultures absorbed urididine, one of the key parts of RNA (ribonucleic acid) and that the same sites on the particles which stained positive for DNA also “took” a proprietary antibody produced by Nanobac. In an interview, one Mayo team member, Virginia Miller, said the team had recovered RNA and DNA sequences, but wasn’t ready yet to present the scientific evidence. “We are a conservative group, and that has stood us in good stead.” In yet another twist, microbiologist Yossef Av-Gay, hired by Nanobac to find out how nanobacteria work, said this, “The story seems to be gearing towards the idea that these are not bacteria, but maybe a new life form.”

Meanwhile, we here at AR would love to know how it is that neither the biomedical community nor the science journalists have managed to make the rather obvious connection between the work of Dr. Folk regarding nanobacterial involvement in pipe plugging via mineral “scaling” and the much-studied findings of the Mayo Clinic team under Dr. John Lieske that only calcified aneurysms, arteries and arterial plaques have nanobacteria in them. No calcification, no nanobacteria found! How difficult can it be?

CAPTION: Did Martian nanobacteria once live in this Martian meteorite (ALH 84001)?

Researchers argue that relic nanobacteria in carbonate concretions like those shown here in the Martian meteorite ALH84001 demonstrate life elsewhere than on Earth for the first time.

Alternative Science

Sept/Oct 2004 – #47