Journal of Analytical & Bioanalytical Techniques
Open Access

Electro transformation (ET); Acetogens; Carbon Negative fuels; Inner membrane structures; High efficiency of ET of Acetones

Introduction

Much has been written on the electro transformation of Acetones [1-6]. However nothing has been written on the process of the electro transformation itself and on the specific details of the electric response of Acetones subjected to the high voltage pulse electric field of the electro transformation generators. Most likely that happen because the commercial electroporation generators do not allow neither the development of the electric response of the high voltage treated cells nor the records of the electric pulses applied to the target cells [3, 4]. We have to note that we used only the electrotrasformation Generator created by the Author in 1992 [1, 2]. We have never used the electroporation equipment every lab has due to the circumstances clearly elaborated in [3]. Said reference explains in detail why conventional electroporation equipmet is not applicable for electrotransformaion of Acetones (Figure 1). There is another circumstance not outlined at Figure 1. The Author has patented the electro transformation generator having the power capacitor 40 mkF and the power tetrode. The last element has the capability to amplify any current fluctuations in the treated sample since the treated sample is located at the anode part of said tetrode. The tetrad is a relatively high frequency current and voltage amplification element and it amplifies said fluctuations of the current passing through the treated sample (Figure 2). Said current fluctuations were recorded by the digital storage oscilloscope and appear only at the part of the applied 6 ms square pulse used for the optimization of the electro transformation parameters for Acetones [3-12] (Figure 3). Said secondary oscillations were of about 26 MHz as shown in (Figure 4). The image shown in Figure 3 was expanded to measure the duration of each oscillation shown in Figure 3 at the end of the electro transformation parameters optimization pulse shown in Figure 3 and 4 has that part of the 6 ms optimization pulse expanded to determine the duration of each of said secondary oscillations which became the approximate number of 0.0384615384615385 x 10-6 sec (5 Nano sec is the value of one unit of the scale in Figure 4. The Author has noticed the secondary pulse oscillations in 1997 as that has been reported at the 17 International Congress on Biochemistry and Molecular Biology in San Francisco. The most intriguing thing about said secondary oscillations is related to the secondary membrane structures detected in the cells producing alcohols and solvents toxic for producing them cells of bacteria. The Author has noticed said secondary electro transformation pulse oscillations while doing electro transformation of Clostridium acetobutylicum ATCC 824 at the laboratory of Prof. George N. Bennett at Rice University. Specifically alcohols do the following to the cell membranes: 1) solvents such as alcohols or acetone increase membrane permeability. Lipids dissolve in alcohol; therefore, the phospholipids in a cell membrane will easily dissolve in solutions such as ethanol. As a result, the cell membrane becomes more fluid and permeable as it starts to break down; 2) the effect on membrane permeability depends on solvent type. Certain types of solvent can cause a greater degree of membrane permeability than others, for example, ethanol causes greater membrane permeability than methanol; 3) Increasing solvent concentration increases membrane permeability. Solvent concentration and membrane permeability are directly correlated i.e. increasing solvent concentration increases membrane permeability. This is because as the solvent becomes more and more concentrated, it has a greater ability to dissolve phospholipids and disrupt the membrane structure, making it more permeable to external substances. The most damaging effect of his electro transformation generator has happened to the Author when he started to work of the cellulosic ethanol production for use it as fuel project at the laboratory of Prof. Lee Lynd at Thayer School of engineering at Dartmouth College in 2001-2006. Said Prof. Attempted to steal the Author’s intellectual property when attempted to patent already patented Electro transformation / Electro fusion Generator. His attempts made the Author to sue said College at the Federal Court and he will be suing again for the attempted murder of the Author committed by said Prof. at said College. That activity immediately has developed after the success the Author had with the electro transformation of Clostridium thermocellum strains [which none has achieved before and has expanded way beyond said stealing of the Author’s intellectual property by accepting venture funds from the criminal investor’s structure Khosla Ventures in 2006. Khosla Ventures funds nearly dead businesses attempting to collect funds given to said dying businesses by the Governmental agencies as DOE, etc. (already after departure of the Author back to Houston TX) to start the criminal business MAs coma Corporation which ended up with bankruptcy of said company, while MASCOMA Corporation has filed legal paperwork on protection from bankruptcy. In said particular case the successful electrotransfromation of C. thermocellum was accompanied by the secondary pulse oscillations at the frequency of about 24 MHz It is very important to note that the Author has discovered the secondary membrane structures in the cells of bacteria producing alcohols or solvents (acetone). The role of said secondary membrane structures is unclear, but it seems logical to assume that production of alcohols or other solvents is toxic for the inner cell content. However the cells produce said alcohols or solvents. That means said secondary membrane structures are intended for the protection of the inner cellular content from said alcohols or solvents. What this specifically means time- and experimentally wise? The time is needed to create said structures if the synthetic DNA construct encodes the biosynthesis of alcohols or solvents in the cells. That is why we have used additional time and temperature for electro transformation of Acetones to allow the synthesis of said secondary membrane structures. For the electro transformation of samples of Acetones the cells subjected to the application of the optimal electric pulse are then kept on ice for 10 min, then are exposed to the temperature 26 o C for three hours (water bath installed in the anaerobic chamber) and only after that are placed at the normal incubation temperature keeping in mind that after the application of the optimal electro transformation pulse the target cells are diluted at least ten times with the growth medium of the best possible composition. The same is noted for the electro transformation of cells of Clostridium acetobutylicum and C. thermocellum.

Figure 1: Distortion of the original square of exponential high voltage electric pulse in the widely used electroporation generators made by Bio-Rad, Inc. or BTX, Inc.

Figure 2: Electrotransformation / electrofusion generator invented by the author.

Figure 3: Secondary oscillations inside the standard square electric pulse (6 ms) generated by the cells of acetogens electro transformed using the author’s generator.

Figure 4: The secondary about 26 mhz oscillations noticed during the electrotransformation conditions optimization for the acetogens.

Materials and Methods

The cultures of Acetones for the electro transformation (ET) were grown in the fermenter similar to the 60 tons fermentation modules we use for biofuels manufacture but for the bench use with the total volume of 5 liters (Figure 5). The cells from the agar plate (10 colonies) were inoculated into the 3.5 liters of the liquid Acetone medium of the composition shown below. The total horizontal bioreactor volume was 5 liters. The liquid medium LM of the [1-12] composition given below was filled at the volume of 3.5 liters. The gas was pressed to the fermenter at the pressure of 15 psi via the setup of the gas blend 0.1 mkm filters. The culture of the Acetones was sub grown in the said fermenter for 3 h until the cell density at OD600 became around 0.52-0.55. The liquid medium for the growth of Acetones LM, g/L:

Figure 5: The horizontal bioreactor used to grow the cultures of acetogens preparing them for the electro transformation (ET).

CuSO4 x 5H2O                                0.0005

AlK (SO4)2 x12 H2O                        0.0005

H3BO3                                              0.0005

Na2MoO4 x 2 H2O                           0.0005

Distilled water to the volume of 1 L. pH 6.5

The Solid medium (SM) for the growth of single colonies and the selection of eletrotransformants were used, g/L:

Beef extract                      4.0

Yeast extract                   1.0

Peptone from meat           4.0

Glucose                         10.0

NaCl                               2.5

Heart infusion broth          10.0

Triptych soy broth          30.0

Agar-agar                      15.0

Distilled water to the volume of 1 L

pH before autoclaving at 121o C for 30 min was 6.5

After the ET the treated with an optimal ET pulse cells were diluted with 0.5 ml of the Acetogen ET recovery medium (RM) of the following composition, g/L:

Tryptic soy broth [40]                      30.0

Horse serum (heat inactivated)       200 ml*

Distilled water up to 1 L.

*Horse serum should be heat inactivated at 56ºC for 30 minutes.

The diluted Acetone cells were incubated at 26 o C for 3.5 hours to allow the building of the intracellular membranes essential for the protection of the intracellular content from the toxic fuel Isobutene [4] or the solvent acetone. The electotarnsformed samples were plated on the solid selective agar.

Results

In 72-96 h we have selected the recombinant transform ants. The fuel Isobutanol producing recombinants have used the pathway shown in [4,5,7,10,12] (Figure 6). The acetone producing recombinants have used the genetically engineered pathway shown in (Figure 7). The recombinants suitable for the carbon negative fuel Isobutanol or acetone biosynthesis in the 60 tons horizontal bioreactors of the same type as shown in Fig. 5 were obtained with the relative frequencies of about 6.9x105 - - 106 recombinants per the microgram of the recombinant DNAs used. The productivity of these recombinants has been published already.

Figure 6: Fuel Isobutanol biosynthesis from the air CO2.

Figure 7: Acetate biosynthesis from the air CO2.

Discussion

It is very important to note that the post-ET incubation at 26o C was crucial for the reported herein high ET efficiency. Said ET efficiency is the consequence of our proposition made before these experiments about the need to reach biosynthesis of the secondary membrane structures discussed in the Introduction section. That appears that in the selective conditions of the ET transform ants selection we were right and allowed the biosynthesis of that secondary membranes protecting inner cellular content from the toxic fuel Isobutene or the solvent Acetone. The time used 3 h has never been experimentally adjusted in both directions; to reduce or extend it. Apparently, the exact timing for the secondary membranes biosynthesis is very important since our recombinant constructs indeed encode the biosynthesis of the alcohol or solvent which are very toxic for the intracellular content. It is important also that the dilution of the ET cell targets was performed with the rich nutrient medium RM chilled to around 0o C. hre is the possibility that additional play might be important with the composition of this rich medium RM. We report our finding herein since we do not plan to adjust any of the cited herein ET conditions since we have reached the purpose of the ET - achieve the obtaining of the recombinants suitable for the bulk manufacturing of said Carbon Negative fuels [4,6,8]. In general we must admit that the ET efficiencies obtained as reported herein might be the maximal as that has been postulated before [2-4]. Why do we need the carbon negative fuels? The current problem is that the air CO2 concentration is very high. It is much higher of the reported earlier by NASA in 2010 when NASA called the air CO2 content of 400 ppm the “Point of No Return” to the healthy environmental restoration of our planet Earth. The air CO2 is among the heaviest gases composing the air gas blend. At th average air density of 1.22 g/m3 the density of the air CO2 is 1.97 g/m3. Therefore the air CO2 spreads on the Ground. Ground is the known refuge of the fresh water: fresh water from rains, melting ice and snow. From the Ground all the creeks and rivers start ending up at the ocean, lakes and ponds. Besides being the heaviest gas in th air blend of gases air CO2 has the known property - it absorbs the solar infra-red energy converting it to heat. As the consequence of that the ground becomes warmer. In the Southern US states the ground temperature might even exceed the water boiling temperature under the normal pressure. What that means? Heating of the Ground increases fresh water evaporation. The Author has come to Houston in 1998. He remembers deep bottomless blue Houston sky at that time. What do we have now in the same Houston? Rains are almost daily. One might say: “Yes this is good; this is the fresh water falling to the Ground.” Indeed this is true, but that also means that the amount of fresh water vapors in the air exceeds the critical for rain fall magnitude. That means that now we have so much fresh water in the air that in Houston rain is almost daily. The layer of the Earth atmosphere is only about 400 miles thick. The atmosphere is attached to the Ground by the gravity force. What is at the other end of the earth atmosphere? There is the Space vacuum. In 2010 NASA has found ice on the dark side of the Moon (-293o C) located in the distance of 220,000 miles from the Earth. Compare the diameters of the Earth and the Moon. Moon worked as the “cold trap” in the Space vacuum surrounding our planet. It collected the small portion of the coming from Earth to the outer Space vacuum fresh water vapors and they have formed the ice detected by NASA in 2010. What is going to happen if we constantly loose fresh water vapors sucked by the outer Space vacuum? In 10-25 years from now we are going to experience the shortness of the fresh water on Earth. The Earth fresh water reserves are shown in threw governmental picture (Figure 8). As you could see from this picture (Figure 8). The fresh water reserves of Earth are very limited. 3% of the Earth’s water is fresh. 2.5% of the Earth’s fresh water is unavailable: locked up in glaciers, polar ice caps, atmosphere, and soil; highly polluted; or lies too far under the earth’s surface to be extracted at an affordable cost. 0.5% of the earth’s water is available fresh water. Where such water came from? This is great question with no definite answer. Did it come from Space? Likely, but a research is needed top say so. As the Author considers we do anticipate the fresh water shortage on Earth in just 10-25 years from now. What that means? That means shortage of crops and livestock production. In 10-25 years from now we would be facing the Global starvation era. Many educated venture capitalists already know that. Jeff Bezos blasted into space on his rocket company’s first flight with people on board becoming the second billionaire in just over a week ride his own spacecraft. Jeff Bezos own a company Blue Origin with the purpose to develop the spacecraft capable of outer Space travelling to find a suitable for living of human beings planet with Oxygen in the atmosphere and fresh water reserves. Elon Musk is developing a vehicle that could be a game-changer for space travel. Starship, as it’s known, will be a fully reusable transport system capable of carrying up to 100 people to the Red Planet. Mr. Musk owns his own spacecraft manufacturing company Space X. Mr. Musk attempts to reduce the costs associated with the Space travel. We are here, on Earth, and we attempt to save our planet, to save its fresh water. We have done in that direction much. If we would be able to reduce the air CO2 level to the level of the air CO2 much before the wide discovery of petroleum and its distillation to manufacture gasoline and Diesel fuel along with the fuels for the aircrafts we would be able to not only reduce the level of the air CO2 but also preserve the Earth’s fresh water. We are developing carbon negative genetically engineered foods for the time which that will take to get rid of the world’s international petroleum corporations and replace them with only the Carbon Negative fuels manufacturing corporations. So, the Earth’s future is in our hands. Declaration of Interests Statement Ethics approval and consent to participate The Author has received all the proper documents granting the Ethical Approval and the Consent to Participate from the State of Texas officials. The Author has made sure that the ethical approval and his consent to participate in preparation and submission for publication of this article were properly approved by the respective authorities of the State of Texas. Consent for Publication The Author has expressed his complete consent to participate in work with this article and its publication in this Journal. Availability of Data and Materials The Author makes all his data and materials herein available for any third party. The data and materials might be obtained from the Author at PO Box 300230, Houston, TX, 77340. Email drmtyurin64@gmail. com. If any third party needs any materials used to publish this article, please, do contact the Author. The Author has changed his second name from “Vladimirovich” to “Vladislavovich” after his farther Professor Dr. Vladimir Inljich Tyurin has changed his first name after the death of his wife the mother of the Author Professor Aleksandra Nikolaevna Tyurina in 2017. Competing Interests This article is another step in the war which has started by SHELL International Petroleum Corporation some time back by attempted murder of the Author. The people hired by SHELL have totaled the Author’s corporate car and did some other illegal things to the Author which has concluded the Author that the war with the international petroleum corporations has started already. No Houston FBI or Houston Police investigation of said attempted murder has happened and this is behind the responsibility of the Texas Governor with all the legal remedies the Author might use in his fight to sue the State and its Governor Gregory Abbott. The attempted murder has the statute of limitations 20 years, and there are jurisdictions above the level of the State of TEXAS. The Author had certain legal problems with the at that time Attorney General of the State of Texas in 2013, now Texas Governor, Greg Abbott and the Author is going to resolve all the legal problems with the Texas Governor, regardless would he be dismissed or not. The corporate Author’s website http:// syngasbiofuelsenergy.com was destroyed by the person, the Attorney of Hirsch and Wertheimer Law Firm, PC by the last name Levy. She belongs to the same family which owned or owns the grocery stores chain named “Fiesta” in Houston Texas. The Author was unable to find a lawyer in Texas to file the respective federal law suit and recover his corporate website, but the Author has the US Constitutional right to recite this website in the references herein. Therefore the Author has multiple legal problems with the State of Texas and the Author is going to resolve said legal problems at the respective Court level in the US. The Author was already involved into the war which has been started by the International Petroleum Corporation SHELL. The Author has approached SHELL offering them for the commercialization the proprietary technology of Gasoline or Diesel fuel manufacture from the air CO2, not from petroleum which SHELL and other international petroleum corporations use. SHELL representative met the Author, got his draft of the at that time in publication article on creation Gasoline from the air CO2 [12]. SHELL hired two Mexicans to kill Dr. The Author in the car accident, paying the possibly around $6,000 for this dirty job, which has been done in Houston TEXAS at the US59 down South (9494 Southwest Fwy). The Houston FBI and the Houston Police were contacted by the Author multiple times over his mobile phone however there was no any detailed and thorough Police or the FBI investigation of said attempted murder of the Author since as the Author trusts, both Houston FBI and Houston Police are totally corrupted by the international petroleum corporations. Therefore any law is not used in the State of Texas to hurt said corporations. Therefore the Author has to look for the law enforcement outside of the State of Texas, at the National level and he will do that to affect the Texas Governor Abbott for his actions of 2013 and have him resign from his position. Therefore the Author has conflict of interests with the State of Texas, TEXAS Governor Gregg Abbott, Texas FBI and Texas Police. Since that time more scientific publications by the Author on the Gasoline and Diesel fuel production from the air CO2 came out. The US patents are extremely expensive; therefore the Author uses instead of patents scientific publications. Publications are as good as the US patents, each the US patent has to claim something better than the Author already did in his scientific publications. The Author working on the Acetonesbiocatalysts has no any competition in the world because of his prior invention, the electroporation / electro fusion Generator, already sold as a sample (with no right for reproduction) to the US corporation BTX, Inc. / Getronics, Inc. (San-Diego, CA). Said Generator and the invented by the Author genome tailoring technology make him with no any competition in the whole World. The Author has conflict of interest with his Ph. D. Supervisor, Professor Boris A. Shenderov, who worked for the KGB in the former Soviet Union. In 1992, when the Author has applied for his the first International Passport of the USSR, we went to the local Police department and set in line much before the International Passport department started working . When the Author has entered said local Police Department he immediately was arrested and jailed in the local prison without any announcement of his fault why he was arrested. The Author has changed his job in 1992 leaving Professor Shenderov and joining the USSR Research Institute GNIIGENETICA. Therefore Prof. Shenderov started making every possible effort to harm the Author with the use of KGB. Therefore the Author instead of getting his first International Passport of the USSR was arrested and without any notification of the crime he has committed he was arrested and forwarded with the machine guns armed Police workers to the local jail. In the local jail the Author was placed with another five persons, criminals, who knew what for they were arrested and jailed and smoked tobacco heavily right in the prison jail cell with the size of about 5.5 x 5.5 x 5.5 meters (about 10 x 10 x 10 feet). Because of said tobacco smoke the Author became very sick and in five hours after being jailed he asked the God he never prayed to (Dr. MV Tyurin is the atheist) to take the Author to haven. The Author wanted to die at said moment. His blood pressure and heart pulse were astonishingly high, and the Author was really sick at said time. He started knocking inside the jail cell to the cell entrance door trying to get attention of the Policemen on duty. But that was not effective and no medical help was rendered by the Police to the Author at the Author’s requests. He asked the God to make him dead, he asked God to take him to Haven. Suddenly the Author has heard the God’s voice right in the Author’s head. The God said in pure English: “It is not your time yet to die. You have not done your major business yet. Calm down, try to relax and wait till the Police understand that they were mistaken with you and will release you”. Indeed in a few days the Author was released and has received his first International passport of the USSR. In 2001 the Author has discovered that he has being doing wrong things at his work and started working on the carbon negative fuels and genetically engineered foods on the base of Acetones. That was the major business the Russian God told to the Author in 1992 at the jail cell when the Author asked his God to take him to Haven and die. Therefore the Author has the conflict of interest with his former Ph. D. Supervisor Prof. B. A. Shenderov.

Figure 8: Fresh water reserves of earth.

Funding

The Author has received no funding. The investors of the Author have read this article draft to his investors and they have decided not to include no funding information as they have stated to the Author. They stated the Author should write “No Funding” per this original article. No funding was used to prepare and publish this original article. The available investors have declined to provide their information for this publication. They have suggested noting that the article was prepared and published with no any funding source.

Authors’ Contributions

The Author has conducted all the experiments himself. The Author has planned, wrote this original article and edited the written text, including proper placement of the illustrations mentioned. The Author read, edited and approved the final manuscript. The Author is the only owner of all materials disclosed in this original article. The Author has plans to distribute his proprietary products after their approval as needed. The Author might be contacted for the data and materials at PO Box 300230, Houston, TX, 77230, drmtyurin64@gmail.com. The Author contributed to the study conception and design. Material preparation, data collection and analysis were performed by the Author. The first draft of the manuscript was written by the Author. The Author read and approved the final manuscript.

Acknowledgement

The Author does have the other parties to acknowledge. Ms. Joany Kerr, the International Sales Director of BTX, Inc./ Getronics, Inc., and the CEO of BTX, Inc./ Getronics, Inc. Dr. Gunter A. Hoffman for giving to the Author his copy of the Electro fusion / Electro transformation Generator prior to that sold to BTX, Inc./ Getronics, Inc. The Author confirms herein the help of the Third Secretary of the US Embassy in Moscow at that time (1995) Mr. James Wink Elman for his help to receive funds for the transportation of said Generator from Moscow to San-Diego via UPS in 1995. The Author herein confirms help of Dr. Nikita Tamm from the Moscow State University for his help in receiving cash in $ in Moscow, the Russian Federation, from San-Diego, CA in 1995.

Data Availability Statement

The Author has made all the data per this manuscript available to any third party requesting said data. The Author might be contacted by any third party requesting said data via PO Box 300230, Houston TX 77230 and via Email in addition to the written request of said data to the mentioned address above.

Authors’ information

The Author has the B. Sci. In Biological Engineering (1984), M. Sci. In Biology (1985), M. D. in Internal Medicine (1986) (all from Saratov State Medical University, the USSR) in addition to the Ph. D. in Molecular Biology, Molecular Pharmacology, Microbiology (1990) from the USSR Research Institute for Antibiotics (Moscow, the USSR). The Author Tyurin Michael Vladislavovich has changed his second name from “Vladimirovich” to “Vladislavovich” after his father, Professor, Dr. Tyurin Vladimir Iljich has changed his first name from “Vladimir” to “Vladislav” in 2017. The Author at his corporations can be reached at PO Box 300230 in Houston TX 77230 and via Email drmtyurin64@ gmail.com.

Originality-Significance Statement

The Author has wrote this original article based on his originality of the business approach and the existing resistance of the International Petroleum corporation to the technology of manufacturing carbon negative fuels to replace their production of fuels originating from petroleum. The reduction of the air CO2 levels towards the prepetroleum era of the year 1900 is paramount, since our planet loses fresh water to the outer Space vacuum. NASA has confirmed that in 2010 sating that the Earth has reached the “Point of No Return” to the healthy environmental conditions suitable for human life on our planet. The new family of the gas stations selling not only carbon negative fuels but also foods for cooking at home and commodity chemicals for the households will save a lot of time for the customers of said new gas stations.

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