Endolith, Extremophilic Organisms from Volcanic Rock: Biochip Precursors

International Biology Review, Vol. 1, Issue 2, August 2017 Endolith, Extremophilic Organisms from Volcanic Rock: Biochip Precursors

Authors: Abstract Robert E. Baier, PhD, PE Extremophilic microbes, Pseudomonas syzgii, from the ultrapure water of computer circuit fabrication Anne E. Meyer, PhD facilities, crystallize semiconductor matter around Robert L. Forsberg, MS themselves while remaining biologically active in a novel form of biochips. The search for extremophilic organisms with similar abilities was extended to Affiliation: volcanic rock from “wellness” water filters. A State University of New York at volcanic rock material called Taicho stone was Buffalo identified as harboring subterranean Sphingomonas species known to selectively destroy monoaromatic pollutants. They are also water-flow re-activated and Correspondence addresses: productive of substantial exopolymeric substances Robert E. Baier related to the claimed multiple health-related benefits E-mail: [email protected] of its filtered water. Attempts to convert these Sphingomonas sp. from the stone host to germanium Anne E. Meyer oxide crystalline host began with the filtered water E-mail: aemeyer@ buffalo.edu aggressively corroding germanium substrata, as with Pseudomonas syzgii. Although semiconductors have Robert L. Forsberg not yet stably incorporated these biota, their roles in E-mail: [email protected] accelerating superficial biocorrosion suggests their additional utility as non-abrasive biochemical/ mechanical polishing aids. Recognizing that extraterrestrial exobiology has posited numerous

similar microbes occluded in minerals, the possibilities for functional organism-based biochip fabrication are diverse when successful semiconductor incorporation and communication are achieved. The results suggest that the best sources for organism-based biochip

production will be from endolithic media.

Keywords: biochips, oxide electronics, germanium, extremophiles, electron acceptors, biocorrosion.

International Biology Review, Vol. 1, Issue 2, August 2017 Endolith, Extremophilic Organisms from Volcanic Rock: Biochip Precursors

1. Introduction bacterial adducts that could provide new generations of functional “biochips” It has been nearly two decades since specifically as extreme-condition sensors.2 an international attempt was mounted to Figure 1 is a low-voltage scanning electron identify and possibly eliminate microbial microscope image, not further coated, of contaminants in the ultra-pure rinse waters one of these biochips. Exposure of such of “Fabs” (= fabrication facilities; specimens to pure-water flow leads to “foundries”) for semiconductor chip organismal reproduction and initiation of manufacturing. The discovery then made of new biocorrosion sites on germanium in-dwelling extremophilic microorganisms surfaces downstream,2 suggesting the path that could also corrode and embed to desired biochip foundries for additional themselves into semiconductor materials, extremophilic organisms now being and specifically germanium—the first 1 explored with volcanic rock-derived transistor material —launched this Sphingomonas species. continuing search for other semiconductor/

Figure 1. Scanning electron micrograph of live bacteria in GeO2 microcrystal

The extremophile-embedded germa- convergence of these studies with the search nium oxide microcrystals so easily and for residual extraterrestrial life forms5 is abundantly produced from the Fabs’ clear and influential while seeking ultrapure water supplies, and later from less- additional test organisms. pure water sources seeded with these microorganisms, encourages their entry into the Thus, an alternative source of emerging field of oxide electronics3 where potentially GeO2-incorporable microbes has numerous similar circuitry challenges are been from an anomalous behavior of certain being addressed. Recognizing that the intact volcanic-rock-containing water filters claiming significant health and medically- biological processes of the embedded 6 bacteria almost certainly engage electron related benefits for its “purified” water, acceptor cascades,4 interrogation of these from which 16SrRNA genes identified cascades by modulated electron flow active Sphingomonas species S. subterranean (AB025014) and S. to/from the GeO2 chips on germanium semiconductors opens opportunities for aromaticivorans (U20774). The feed water numerous sensor and control devices. The to the filter unit, according to similar

International Biology Review, Vol. 1, Issue 2, August 2017 Endolith, Extremophilic Organisms from Volcanic Rock: Biochip Precursors

16SrRNA analyses, showed only transmission capabilities throughout this Acinetobacter sp. ATCC17925 (accession work, allowing a multiplicity of analyses8 number Z93441). while serving simultaneously as the corroding substratum expected to 2. Methods and Materials incorporate extremophilic microbes in their re-crystallizing superficial oxides. An The major material of analysis was internal reflection infrared9 spectrum of a crushed volcanic rock (called Taicho stone), Taicho stone smear on a Ge test plate is of light green and pink hues, from Central given as Figure 3, and an energy-dispersive Japanese volcanoes,7 pictured spread over a X-ray spectrum is provided as Figure 4. germanium prism in the Figure 2 scanning These data illustrate the starting qualities of electron micrograph. the Taicho stone to be a majority aluminosilicate mineral having minor iron Germanium prisms were key materials and potassium contents, with trace for both their substance and infrared energy hydrocarbon matter present.

Figure 2. Taicho stone from water filter, spread over germanium surface

Figure 3. Internal reflection IR Figure 4. EDX-ray elemental analysis Spectrum of Taicho stone on Ge in of Taicho stone on Ge in Figure 2 Figure 2 (IR absorptions from stone are

circled)

International Biology Review, Vol. 1, Issue 2, August 2017 Endolith, Extremophilic Organisms from Volcanic Rock: Biochip Precursors

Water contact angle measurements on trapezoidal test plates; 20x50x1mm) reference-grade Teflon film8,10 were used to conducted tap water through 13x50x1mm ascertain whether the filtration material rectangular channels at 350 ml/min (10 added surface-active constituents to the dynes/cm2, 1 Pa) for increasing times before water. In this test, pure distilled water gives the Ge test plates were reanalyzed by an average measurement of approximately infrared spectroscopy and re-inspected by 120 degrees. In the experiments, both tap scanning electron microscopy and EDX-ray water and distilled water passed through the techniques. Operationally, then, it was filtration device that did not contain Taicho arranged that side-by-side filtration units, stone showed contact angles of between 113 one without Taicho stone and one with degrees and 119 degrees on the Teflon, the Taicho stone operated identically for long higher values after filtration. In contrast, the periods of time before inspecting the same water sources passed through the germanium prisms’ flow surfaces both filtration device containing the Taicho stone spectroscopically and microscopically. exhibited contact angles of only 107-109 degrees on the Teflon. This surface tension depression of water indicated a specific 3. Results elution from the Taicho stone of some As shown in Figure 5, equal exposure of surface-active, surface-tension-lowering clean Ge prisms to filtered water flow with material consistent with the trace organic and without Taicho stone in the filter device matter noted in the infrared spectrum (note showed dramatically different results. It is small absorption bands at 2850 and -1 also noteworthy that the presence of the 2950cm in Figure 3). Taicho stone correlated with a much-more- For longer-duration experiments, rapid, obviously biocorrosion-accelerated, analytical flow cells11 (each containing two rate of superficial erosion of the germanium clean, smooth Ge internal reflection prisms.

Figure 5. Transmission IR spectra comparing water deposits on Ge test plates after water passage through filter devices without (left) and with (right) Taicho stone.

International Biology Review, Vol. 1, Issue 2, August 2017 Endolith, Extremophilic Organisms from Volcanic Rock: Biochip Precursors

The water control (no exposure to into the water.12 Taicho stone) flowing over germanium in its most corrosive state left a superficially As revealed in Figure 7, however, the corroded Ge surface bearing no evidence of incorporation of the living, slime-producing organic matter. In contrast, that same extremophilic Sphingomonas organisms was corrosive stream passing over Taicho stone not retained in regular GeO2 microcrystals did acquire and subsequently deposit onto over the corroding Ge surface. This feature the Ge surfaces an increasingly dense is likely to require careful controls of both deposit of biofilm material that actually water velocity and purity, as already observed for the Pseudomonas syzgii diminished the corrosion. The kinetics of 2 this process are compared in the internal organisms. Figure 8 does reveal trace reflection infrared spectra of the filter carbon and phosphorous components to effluent (Figure 6). An abundant “slime” have been present in the Taicho stone- deposit formed on the Taicho stone and contributed surface layers on the germanium released glycoproteinaceous components surface early in the experiment.

Figure 6. IR spectra of effluents from devices without (left) and with (right) Taicho stone. Controlled volumes of the effluents were evaporated on Ge test plates prior to analysis. Note glycoproteinaceous character of Taicho stone slime effluent (* Amide I & II, + carbohydrate).

International Biology Review, Vol. 1, Issue 2, August 2017 Endolith, Extremophilic Organisms from Volcanic Rock: Biochip Precursors

Figure 7. Biocorroded Ge surface; not yet Figure 8. EDX-ray elemental analysis of crystallized initial deposit (note trace carbon and phosphorous components)

4. Discussion addition of organic surface-active ingredients. With increasing flow durations, 4.1. The Water Supply the filter effluent was shown to become Numerous beneficial but peculiar increasingly enriched in slime-producing features have been anecdotally claimed in matter having the characteristics of a glycoproteinaceous biofilm long associated popular, consumer literature for water 12 produced by “wellness” filter units (e.g. with Sphingomonas microorganisms. better plant growth, smoother hair, Samples of the emerging water revealed that elimination of skin rashes, greater extremophilic Sphingomonas species had solubilities, and more), drawing attention to become active and both they and their the filter beds. Especially interesting were metabolic products had “fertilized” the reports that these properties all improved waters. Analyses by genetic 16SsRNA with water-flow duration. Thus, water flows methods indicated their best homology was through each and then all of the filter beds’ with S. aromaticivorans, known to be a components were examined, over increasing potent reducer of groundwater pollutants,13,14 and S. subterranean as often times. All but the final filter layer behaved 15 conventionally to produce effluent that was found in deep ground layers. Prior to organic-free and of slightly higher surface release of the glycoproteinaceous slime tension than its feed supply waters. The from the organisms, the initial superficial final filter layer was a crushed volcanic rock corrosion of clean, smooth germanium over called Taicho stone having pink or green which this water flowed was even more hues. aggressive than that of the control water passing through Taicho stone-free filters. Unlike the other filter components, the Thus, these effluent waters were tested in layer of Taicho stone in contact with the the flow cell model that had produced the filtered water caused that water to diminish pioneer biochips from similarly corrosive in surface tension in a manner suggesting water sources.2

International Biology Review, Vol. 1, Issue 2, August 2017 Endolith, Extremophilic Organisms from Volcanic Rock: Biochip Precursors

Although volcanoes first erupt as the 4.2. Rock-encased Organisms Earth’s tectonic plates diverge, with fluid Amazing micro-Computerized Tomo- molten rock (magma) flows arising from the graphic (µCT) images of microbes encased voids between the plates, geothermal water in sedimentary rocks have been shown,16 that originates from cold rainwater does and confirmed by conventional transmission seep through the resulting crust. electron microscopy. Many prior reports Nevertheless, any originally entrapped exist about the microbial diversity and organisms must truly be in the extremophile abundance of subterranean17,18 (and possibly category because water temperatures of 150- extraterrestrial19) extremophilic organisms. 200C have been reported in 1000m-deep Thus, there is a clear understanding that fields across volcanic belts. It is clear that, there can actually be “life in rock” of a whenever they entered the rock, the considerable range.20 However, until our Sphingomonas species are capable of re- prior discovery that these microbes could be activation by clear water flows. Carbon subsequently transferred to and encased dioxide outgassing during volcanic events within microcrystals of solid-state may have entrapped sufficient carbon to germanium (reminiscent of the first invented sustain minimal metabolic events. It is transistors), this intriguing new definition speculated that the source of organic carbon for true organism-based biochips wherein might have been lignite from plant material electronic flow-modulated information buried within the rock,23 both the gaseous transfer might occur was not anticipated. and organic sources being coupled to What has the Taicho stone now added to reduction of electron acceptors. Via such these prospects? properties, members of the Sphingomonas genus are well-recognized for their 4.3. Taicho Stone Origins and degradation of many exotic organic Emergent, Functioning Bacteria compounds of aromatic and halogenated chemistry.13,23 Their use in bioremediation Taicho stone is claimed to be a rare of groundwater pollutants is well-known,14 volcanic (igneous) mineral substance as is their isolation from Finnish and formed by the cooling and crystallization of Swedish drinking water distribution molten volcanic rock between 1100F and systems,15 which often employ selective 2200F temperature and from ground level to growth media.24 With regard to their depths greater than 98,000 feet. It is provision of special features that may be apparently named after a sixth century (CE) encapsulated in semiconductor oxides, it has Nara period monk in Japan, said to be the already been noted that the exploitation of first person to reach the top of Mount Haku Sphingomonas sp. metabolic capabilities 7,21 in the Ryohaku mountains. It has been could provide important biotechnological employed since 1984 in so-called “wellness benefits.25 filters” endorsed by the Kitazato Medical School of Public Health in Tokyo and since 4.4. Oxide Electronics 1995 by the Japanese Ministry of Health. The filters have been installed in numerous It is clearly established that electron hospitals as well as in NEC Corporation acceptor features at or within oxide office buildings throughout Japan since electronic materials are especially important 2000. It was shown to produce filtered water within the “roadmap” to important new with both a lowered surface tension and generations of sensors and switches.3,26 In a increased solubilization capability.6,22 novel approach to describe primary

International Biology Review, Vol. 1, Issue 2, August 2017 Endolith, Extremophilic Organisms from Volcanic Rock: Biochip Precursors olfaction, electron tunneling of odorant pyroplastic flows of volcanoes, which molecules is invoked, and electron tunnel deposits cover large areas of that planet’s spectroscopy is proposed for sensing surface. Small chips of such rock have various molecules27 that might be especially shown a green-colored horizon beneath the important in military challenges. In the rock surfaces (similar to Taicho stone) and context of semiconductors, their ability for also illustrate life-describing biological electron flow to be easily modulated might features and morphologies when suspended be exploited by biological features of in distilled water.19 Can this entrapment of embedded functional microbes. The under- extremophilic life forms be converted to utilized but extraordinarily sensitive similar entrapment in functional “contact potential” method could be re- semiconductor oxides by the corrosion visited to detect and analyze polarization mechanism? features of adsorbed liquids28,29 or vapors,30 thereby providing a hardy electronic method 4.6. Biocorrosion Benefits of detecting impurities in the air. Application of this method to In the course of these investigations, it monomolecular organic films on the same was found that the microbe-induced types of germanium prisms used here and in corrosion of germanium substrata was at a previous extremophile work2 has already dramatically enhanced rate (prior to slime shown microvolt sensitivity to configuration formation) when compared to even microbe- changes of model proteins.31 Can these free ultrapure water, suggesting a new method of “biochemical-mechanical” findings be relevant to oxide electronic 34 features of the same materials bearing planarization of semiconductors, without corrosion-induced entrapped extremophiles? the negative consequences of abrasion from currently used oxide slurries. This technical Perhaps more immediately, successes use, by itself, could have significant with luminescence measurements of commercial and military benefits. extraordinary “molecular thermometers” for measurements at the microcrystal scales are 5. Conclusion now producible and might be exploited.32,33 Extremophilic organisms released into 4.5. Extremophilic Organisms flowing pure water are capable of aggressively corroding the surface of pure It is intriguing that these studies are germanium. In one prior example2, so- also entwined with curiosities about entrapping those reproducing microbes into extraterrestrial life. For example, specific to GeO2 “biochips” suggested the process here the volcanic origin of microorganisms partially reproduced with another emergent described here, two species of Picrophilius extremophile and setting the stage for future microbes isolated from volcanically heated, 5 “foundries” of true Biochips. Endolithic dry soils in Japan should be tested for their extremophiles are the best probable future amenability to GeO2 encapsulation while source from which the microbes will be retaining their acid-loving and thermophilic selected. qualities. Remaining studies must address the Especially important to note is that rates of water flow over the corroding substrata for endolithic life have already surfaces, and the water purity levels been explored in the search for life on Mars, necessary to produce abundant, functional examining further the rocks from microcrystals as done previously2 for

International Biology Review, Vol. 1, Issue 2, August 2017 Endolith, Extremophilic Organisms from Volcanic Rock: Biochip Precursors organisms from chip-fabrication facility Detection and Control of Micro- waters. Interrogation of these new Biocontamination in Ultra Pure Water manufactured Biochips will utilize the Processes. emerging techniques of oxide electronics26, and usher in an extraordinarily sensitive The 16SrRNA assays identifying the generation of functional biotransistors for rock-released Sphingomonas strains and feed use in medicine, commerce, and military water strain were performed by Dr. Leonid settings. A. Kulakov, Queen’s University of Belfast, Northern Ireland. Acknowledgments The “Wellness Filters” with and without Taicho stone were kindly provided The original experimental work by James S. Bazan, Chief Operating Officer described here was accomplished with the of D2O, Inc., Oneonta, NY at the request of support of National Science Foundation Dr. Russell W. Bessette, then Executive Award Number 9815211, TIE Project: Director of the New York State Science and Industry/University Cooperative Research Technology Foundation. Center for Microcontamination Control:

International Biology Review, Vol. 1, Issue 2, August 2017 Endolith, Extremophilic Organisms from Volcanic Rock: Biochip Precursors

References 1. History of the transistor. website 11. Baier RE. Initial events in microbial https://en.wikipedia.org/wiki/History_of_the film formation. In: Costlow JD, Tipper RC, _transistor Updated June 22, 2017. Accessed eds. Marine Biodeterioration: An June 23, 2017. Interdisciplinary Study. United States Naval Institute; 1984. 2. Sah VR, Baier RE. Bacteria inside semiconductors as potential sensor elements: 12. Ashtaputre AA, Shah AK. Studies on a Biochip progress. Sensors. 2014; viscous, gel-forming exopolysaccharide 14(6):11225-44. from Sphingomonas paucimobilis GS1. Appl Environ Microbiol. 1995; 61(3):1159-1162. 3. Ramirez AP. Oxide electronics emerge. Science. 2007; 315(5817):1377-1378. 13. Fredrickson JK, Balkwill DL, Drake, GR, Romine MF, Ringelberg DB, White 4. Dopson M, Ni G, Sleutel THJA. DC. Aromatic-degrading Sphingomonas Possibilities for extremophilic isolates from the deep subsurface. Appl microorganisms in microbial Environ Microbiol. 1995; 61(5):1917-1922. electrochemical systems. FEMS Microbiology Reviews. 2015; 40(2):164- 14. Farhadian M, Vachelard C, Duchez D, 181. Larroche C. In situ bioremediation of monoaromatic pollutants in groundwater: A 5. Cavicchioli R. Extremophiles and the review. Bioresource Technol. 2008; search for extraterrestrial life. Astrobiology. 99(13):5296-5308. 2002; 2(3):281-292. 15. Koskinen R, Ali-Vehmas T, Kampfer P, 6. Water for Wellness. website Laurikkala M, Tsitko I, Kostyal E, Atroshi http://www.waterforwellness.us . 2007. F, Salkinoja-Salonen M. Characterization of Accessed April 29, 2017. Sphingomonas isolates from Finnish and Swedish drinking water distribution systems. 7. Nakamura H. Chemical composition of J Appl Microbiol. 2000; 89(4):687-696. the mantle under the Ryohaku mountains. Geochimica et Cosmochimica Acta. 2003; 16. Alba-Tercedor J, Ascaso C, Wierzchos Supplement 67: 325. J. Using micro-CT to explore pore contents in the ignimbrite, a volcanic rock in the 8. Craver CD. Polymer Characterization Atacama Desert with endolithic microbial Interdisciplinary Approaches. New York; communities: the microhabitat potentially Plenum Press; 1971. expected on Mars. Bruker Micro–CT Users Meeting 2014. 2014. 9. Harrick NJ. Internal Reflection Spectroscopy. New York; Interscience 17. Heim C, Lausmaa J, Sjovall P, Toporski Publishers, John Wiley & Sons; 1967. J, Dieing G, Simon K, Hannen RT, Kronz A, Arp G, Reitner J, Thielo V. Ancient 10. Baier RE, Gott VL, Feruse A. Surface microbial activity recorded in fracture chemical evaluation of thromboresistant fillings from granitic rocks (Aspo Hard Rock materials before and after venous Laboratory, Sweden). Geobiology. 2012; implantation. Trans Amer Soc Artif Int 10(4):280-297. Organs. 1970; XVI:50-57.

International Biology Review, Vol. 1, Issue 2, August 2017 Endolith, Extremophilic Organisms from Volcanic Rock: Biochip Precursors

18. Schafer N, Schmidt BC, Queric NV, Salgueiro D, Martins R, Carlos E, Liu A, Roring B, Reitner J. Organic compounds and Shan FK. The 2016 oxide electronic conditioning films within deep rock fractures materials and oxide interfaces roadmap. J of the Aspo Hard Rock Laboratory, Sweden. Physics D: Appl Physics. 2016; Geomicrobiology J. 2015; 32(3-4):231-242. 49(43):433001.

19. Wierzchos J, Davila AF, Artieda O, 27. Turin L. A spectroscopic mechanism for Cámara-Gallego B, de los Ríos A, Nealson primary olfactory reception. Chemical KH, Valea S, García-González MT, Ascaso Senses. 1996; 21(6):773-791. C. Ignimbrite as a substrate for endolithic life in the hyper-arid Atacama Desert: 28. Bewig KW, Zisman WA. Surface implications for the search for life on Mars. potentials and induced polarization in Icarus. 2013; 224(2):334-346. nonpolar liquids adsorbed on metals. J Phys Chem. 1964; 68(7):1804-1813. 20. Fredrickson JK, Onstott TC. Microbes deep inside the earth. Scientific American. 29. Timmons CO, Zisman WA. A study of 1996; 275(4):68-73. autophobic liquids on platinum by the contact potential method. J Phys Chem. 21. Taicho. website 1964; 68(6):1336-1342. https://en.wikipedia.org/wiki/Taicho. Updated February 22, 2016. Accessed June 30. Phillips G. An electronic method of 23, 2017. detecting impurities in the air. J Sci Instruments. 1951; 28(11):342-348. 22. Bazan JS. private communication, D2O Inc. Oneonta, NY. October 27, 2000. 31. Baier RE, Loeb GI. Multiple parameters characterizing interfacial films of a protein 23. Fredrickson JK, Balkwill DL, Romine analogue, polymethylglutamate. In: Craver MF, Shi T. Ecology, physiology, and CD, ed. Polymer Characterization phylogeny of deep subsurface Interdisciplinary Approaches. New York; Sphingomonas sp. J Indus Microbiol and Plenum Press; 1971. Biotechnol. 1999; 23(4-5):273-283. 32. Katsnelson A. Tiny temperature sensors. 24. Yim MS, Yau YCW, Matlow A, So JS, ACS Cent Sci. 2017; 3(5):364-366. Zou J, Flemming CA, Schraft H, Leung KT. A novel selective growth medium-PCR 33. Brites CDS, Lima PP, Silva NJO, assay to isolate and detect Sphingomonas in Millan A, Amaral VS, Palacio F, Carlos LD. environmental samples. J Microbiol A luminescent molecular thermometer for Methods. 2010; 82(1):19-27. long term absolute temperature measurements at the nanoscale. Adv Mater. 25. White DC, Sutton SD, Ringelberg DB. 2010; 22(40):4499-4504. The genus Sphingomonas: physiology and ecology. Curr Opin Biotechnol. 1996; 34. Babu S. Advances in Chemical 7(3):301-306. Mechanical Planarization (CMP). Woodhead Publishing; 2016. 26. Lorenz M, Rao MR, Venkatesan T, Fortunato E, Barquinha P, Branquinho R,