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How did super engineered bacteria that "eat everything" by eating waste water, plastic, and metal waste bring about "green factories"

   Since the United Nations Sustainable Development Summit was held in 2015, global environmental issues and how to achieve sustainable development have become irreversible topics.

  A small number of rich people are busy "going to heaven", and most of the problems on the ground have to be solved. In addition to the fact that researchers are constantly pushing for technological progress, solving problems may also require the help of microorganisms.

  Since the early 19th century, people have discovered that some microorganisms can use their own metabolism to consume certain special chemicals. But they are usually inefficient, so metabolic engineering is used to improve the performance of microorganisms. Since the 1990s, the use of metabolic engineering to transform microbial cells for special purposes has become a hot topic in the biological world.

  In recent years, by combining with synthetic biology, metabolic engineering has become more powerful. Based on the advantages of synthetic biology design and construction of new biological functions and systems, combined with metabolic engineering to develop engineered microorganisms with various functions has become an effective helper in solving the earth’s dilemma.

  On the application side, scientists are no longer satisfied with simple biomolecule synthesis. Synthetic biology technology has gradually entered the fields of energy, environment, and heavy industry. Various microorganisms that have undergone gene editing and modified metabolism are trying to solve the problems of raw material collection and processing, waste treatment, and derived environmental pollution.

Reduce marine oil pollution


  The coast of Orange County in southern California was calm, and a black liquid slowly floated from the bottom of the sea to the sea. This was the "most serious" crude oil spill in the area in 10 years. The cause was a crack in an oil pipeline. This has also caused about 35 square kilometers of sea and some beaches to be polluted, seriously affecting the local ecological environment.

  In the past few decades, there have been many major oil spills, each time causing immeasurable environmental and ecological damage. The current methods for dealing with oil spills include salvage, physical barriers and chemical dispersants, but they still cannot prevent the chemical components in the oil from drifting to the depths of the ocean. Microbial-based bioremediation methods are due to their ecological compatibility and lower cost. And getting more and more attention.

  As early as the 1990s, many microorganisms capable of breaking down petroleum molecules have been identified, including bacteria, fungi and microalgae.


General scheme of bioremediation strategies involving different microbial taxa


  In different depth environments of the ocean, physical factors such as temperature, light, and pressure are different. Therefore, the types of free or deposited petroleum molecules are different, and different biodegradation schemes need to be designed.


A conceptual diagram of the metabolic processes involved in the degradation of hydrocarbons in marine sediments characterized by different redox conditions


  Common bacteria are generally Gamma Proteobacteria, including members of the Marine Spirochetes and Tautomonas. The halophilic characteristics of these bacteria also allow them to show a stronger ability to degrade hydrocarbons than terrestrial bacteria.

  In addition to the direct use of hydrocarbons, another strategy is to use microorganisms to produce surfactant compounds to improve the efficiency of bioremediation of polluted environments.


An overview of oil-degrading bacteria isolated from different marine ecosystems and their hydrocarbon specificity


  One of the factors limiting the biodegradation of hydrocarbons is their hydrophobicity, which leads to poor bioavailability. Surfactants are amphiphilic, that is, hydrophilic and lipophilic, which can effectively separate petroleum molecules in submarine sludge. , Can help improve the bioavailability of hydrocarbons by microorganisms.

  With the development of biomaterial technology, a new method of accelerating marine bioremediation has been born. The new material is used as a carrier for microorganisms, and the microorganisms are wrapped in "capsules" composed of calcium alginate and chitosan, which can float on the receiver. Oil contaminated sea water. This "capsule" has a nano-scale porous structure, allowing specific intra- and external exchanges of molecules, which not only ensures the long-term survival of microorganisms in highly polluted seawater, but also enables the degradation process of hydrocarbons for a long time. Field meso-scale experiments conducted in China have shown that most petroleum hydrocarbons (>98%) are removed from the seawater surface within 24 hours.

  In addition, fungi and microalgae are also key research organisms in marine bioremediation. Designing a biological combination of bacteria, fungi and microalgae may be a new strategy for the restoration of the marine environment in the future. However, different microbial taxa have different metabolic requirements and show different efficiencies in the biodegradation of petroleum hydrocarbons, which may also vary greatly due to the chemical structure and bioavailability of hydrocarbons and environmental conditions.

  Future research should focus on understanding the potential synergistic interactions between microbial groups and evaluating their potential to remove hydrocarbons after in-situ and ex-situ bioremediation applications.

  At the same time, other types of pollutants in the environmental matrix, such as heavy metals, should also be considered. In fact, biodegradation strategies may cause major changes in the mobility and bioavailability of heavy metals, which may increase environmental risks. Therefore, accurate risk analysis should be performed to assess the background impact of biological treatment, especially for marine sediments characterized by mixed chemical pollution (organic + inorganic pollutants).

Cycle from plastic to plastic


  Plastic pollution has become a global threat. According to data released on the PlasticsEurope website in 2020, the global plastic production scale has increased by 21% in the past six years, reaching 368 million tons in 2019. There are currently three main ways to treat plastic waste: landfill, incineration or recycling.

  Thanks to natural evolution, some microorganisms have evolved enzymes that degrade plastics. These microorganisms or enzymes have become the key to biodegradation of plastics. Biodegradation refers to the decomposition and transformation of substrates through the action of microorganisms, which are completely converted into carbon dioxide, water, minerals and biomass by aerobic microorganisms, or into carbon dioxide, methane and humus under the action of anaerobic organisms.


Synthetic biology helps the biological depolymerization and upgrading of waste plastic resources, synthetic biology


  Such microorganisms generally appear in places where plastic waste gathers. In 2016, Japanese scientist Shigerto Yoshida discovered the bacterium I. sakaiensis 201-F6, which has the ability to decompose PET plastic, from the sludge of a bottle recycling plant in Osaka. Under the reaction conditions of 30 degrees Celsius, it takes 6 weeks to completely degrade the low crystallinity PET film. It is currently known as the best bacteria for PET degradation.

  Since then, scientists have successively discovered microorganisms or enzymes that can degrade different types of plastics from garbage disposal sites, waste oil fields and other places. At present, for a large number of commonly used plastic types (PET, PE, PVC, PP, PS and PUR) on the market, scientists have found microorganisms that can degrade them one by one.


Hydrolyzable plastic depolymerase mining


  However, the efficiency of natural microorganisms or enzymes is extremely low, and the problems of low catalytic efficiency, poor stability, and low expression of the plastic depolymerase element store restrict the large-scale production and application of plastic depolymerase. In the time that microbes degrade a PET plastic bottle, there may be 100,000 more waste bottles in the world at the same time.

  With the blessing of synthetic biology technology, the use of protein engineering techniques such as rational design and directional transformation provides new solutions for improving the activity, stability and specificity of plastic depolymerases.

  At present, Carbios of France is at the forefront of the PET plastic recycling industry. Although it has been established for nearly ten years and has not made a year of profit, it depends on government relief. However, this year, its first PET plastic recycling industry demonstration plant is also the world's first. A demonstration plant was established in Clermont-Ferrand, France. The demonstration plant includes a 20 cubic meter depolymerization reactor capable of processing 2 tons of PET per cycle, which is equivalent to 100,000 plastic bottles.

  As early as June of this year, Carbios, L'Oréal, Nestlé, Pepsi and Suntory jointly announced that Carbios has successfully produced the world's first food-grade PET plastic bottle made of waste plastic. Carbios produced sample bottles for these partners. Carbios has also made bold claims that in the future, there may be no need to produce new PET plastics, and the existing plastics are sufficient to support recycling.


The first PET bottle made from waste plastic


  After 2 years, the Carbios R&D team has screened out a microorganism with potential for modification from 100,000 microorganisms. This microorganism was discovered from the leaves composted in autumn. The hydrolase produced by it can break down the leaf membrane of the leaves. Researchers modified it and finally obtained a highly efficient PET hydrolase and applied for a patent. The enzyme can decompose 97% of any kind of PET plastic within 16 hours, which is 10,000 times more efficient than any bioplastic recycling test so far.

  Similar to Carbios, the domestic Tianjin Enbohua Technology Co., Ltd. also has a patented PET recycling technology. Its core depolymerase is the most commonly used cutinase in PET degradation research, which can degrade plastic products into oligomers or monomers. Recycle it again.

  However, the efficiency of natural microorganisms or enzymes is extremely low, and the problems of low catalytic efficiency, poor stability, and low expression of the plastic depolymerase element store restrict the large-scale production and application of plastic depolymerase. In the time when microorganisms degrade a PET plastic bottle, there may be 100,000 more waste bottles in the world at the same time.

  With the blessing of synthetic biology technology, the use of protein engineering techniques such as rational design and directional transformation provides new solutions for improving the activity, stability and specificity of plastic depolymerases.

  At present, Carbios of France is at the forefront of the PET plastic recycling industry. Although it has been established for nearly ten years and has not made a year of profit, it depends on government relief. However, this year, its first PET plastic recycling industry demonstration plant is also the world's first. A demonstration plant was established in Clermont-Ferrand, France. The demonstration plant includes a 20 cubic meter depolymerization reactor capable of processing 2 tons of PET per cycle, which is equivalent to 100,000 plastic bottles.

  As early as June of this year, Carbios, L'Oréal, Nestlé, Pepsi and Suntory jointly announced that Carbios has successfully produced the world's first food-grade PET plastic bottle made of waste plastic. Carbios produced sample bottles for these partners. Carbios has also made bold claims that there may not be a need to produce new PET plastics in the future, and the existing plastics are sufficient to support recycling.

  After 2 years, the Carbios R&D team has screened out a microorganism with potential for modification from 100,000 microorganisms. This microorganism was discovered from the leaves composted in autumn. The hydrolase produced by it can break down the leaf membrane of the leaves. Researchers modified it and finally obtained a highly efficient PET hydrolase and applied for a patent. The enzyme can decompose 97% of any kind of PET plastic within 16 hours, which is 10,000 times more efficient than any bioplastic recycling test so far.

  Similar to Carbios, the domestic Tianjin Enbohua Technology Co., Ltd. also has a patented PET recycling technology. Its core depolymerase is the most commonly used cutinase in PET degradation research, which can degrade plastic products into oligomers or monomers. Recycle it again.

The old mine waste will become a "treasure"


  Biometallurgy is a process that uses microorganisms to extract metals from low-grade ore or mine waste, also known as microbial mining. According to the main principle of action, this process also includes biological leaching, biological oxidation and so on.

  Simply put, biological leaching refers to the conversion of target metals into soluble form by microorganisms, and its essence is the stepwise oxidation and decomposition of ore. For example, metals such as copper are usually found in sulfide minerals, and some microorganisms are particularly good at oxidizing sulfide minerals and releasing copper ions. Due to the acidity of the reaction medium, copper ions can remain in the solution and then be enriched on the electrode through an electrochemical reaction.


Stepwise oxidation model of sulfide ore established by Central South University


  In contrast, if the impurities are dissolved and the target metal is concentrated in the solid, it is called biological oxidation. It is only conceptually different from bioleaching. In addition, microorganisms used for mining laterite minerals under reducing conditions have also entered application scenarios.

  Microbial-based metallurgical methods can effectively increase revenue while limiting the use of toxic chemicals in traditional processes. In addition, it can also reduce carbon dioxide emissions and reduce the carbon footprint and water footprint of the entire process. At the same time, biological mining technology can also be used to clean up contaminated mines and recover metals from industrial residues and waste.

  Copper and gold mines are the most important industrial applications in this field. Globally, the amount of copper extracted by biological leaching method has reached 10% to 15%; and the yield of biologically oxidized gold is about 5%. In addition to copper and gold, biometallurgy has expanded to cobalt, nickel, zinc, uranium and rare earth elements.

  As early as the 1950s, based on the discovery of Thiobacillus ferrooxidans, the concept of biometallurgy was first proposed. In the decades since, researchers have been experimenting, but progress has been slow.

  Part of the reason is that mining companies are reluctant to invest in upgrading infrastructure, but the bigger problem is that the time cost of microbial extraction of metals was too long: traditional methods of extracting metals require hours or days, while microbes may require weeks or months. Even longer. This is obviously fatal to the mining industry, which regards economic benefits as the primary criterion.

  Therefore, accelerating mining speed and increasing yield through technologies such as microbial screening and genetic modification have always been the focus of development in this field.

  In 2002, Codelco, the world's number one copper mining company located in Chile, established a joint venture company BioSigma Sa, dedicated to the development and promotion of faster and higher yield biometallurgical technologies. Its proprietary microbial technology completed pilot scale trials in 2005.

  Around 2010, the mindset of the mining industry has undergone a significant change, and many well-known mining companies around the world have successively shown their interest in biometallurgy. The reason is that the depletion of high-content ore resources and increasingly expensive energy prices are the main driving forces.

  Barry Johnson, a microbiologist at Bangor University in Wales, once said that ore with a copper content of less than 5% was not worth mining. But now, those mining waste slurries containing 0.4% or 0.5% copper left over from the past have become treasures.

  On the other hand, biometallurgy, which can be carried out under normal temperature and pressure, is also full of fatal appeal to many "energy-consuming giants": for example, the traditional refining process of nickel requires 800 degrees Celsius, while the microbial metallurgy process only requires 30 degrees Celsius.

  In 2014, Codelco officially announced that it has begun to use biometallurgical technology to obtain products on a large scale. The company stated that it will use its proprietary bacteria to extract copper from chalcopyrite for the first time. Prior to this, standard bioleaching methods could not do anything for chalcopyrite.


Reales shows nails and screws dissolved in a metal-eating bacteria tank



  A few days ago, Chilean scientist Nataque Reales improved the metal recovery rate in hydrometallurgy by using an extracted Helicobacter ferrospira.

  After two years of experimentation, Reales found that the Hunger Helicobacter ferrospira can decompose metals significantly faster, and the time it takes to decompose an iron nail is shortened from the first two months to three days. In addition, biochemical tests have shown that the bacteria is harmless to humans and the ecological environment, and it is expected to be used in large quantities to extract metals such as copper.

  In our country, biometallurgy is also a long-term development project. In the same period of the 1950s, Professor He Fuxi of Central South University of Mining and Metallurgy (now Central South University) established a Biometallurgical Laboratory. In 1997 and 2001, Central South University built two biological copper extraction heap leaching plants of more than 1,000 tons in Jiangxi Dexing Copper Mine and Fujian Zijinshan Copper Mine.

  In recent years, with the rapid development of biological technology, more and more technologies such as genes and genomes and metagenomics have been applied in the field of biometallurgy. Especially the application of genome technology has accelerated the research level of microorganisms in this field, and the mining efficiency and scope of use have been further expanded.


Researchers take water samples after the Talvivaara mine leaked


  Not only Central South University, but also the State Key Laboratory of Microbial Technology in the School of Life Sciences, Shandong University, has also been engaged in molecular research on extreme eosinophilic autotrophic microorganisms for a long time and obtained a number of patents.

  It should be noted that although biometallurgical technology effectively limits toxic agents, it cannot completely avoid the huge environmental pollution that may be caused by the mining industry. Its by-product sulfuric acid and target metal intermediate liquid are still deadly potential sources of pollution and need to be strictly monitored.

  In 2012, a waste liquid leak occurred in Talvivaara Sotkamo, the largest bio-mining mine in Europe, and the mine operating company Ahtium Plc filed for bankruptcy in 2018.

  Bio-mining is a niche market, but with resource constraints, environmental pressures and increasing regulations, more and more mining companies are actively joining this field.


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