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Topic: Production of Fusal Alcohol from Bacteria

= Abstract = Real world application of fuel production from microbial sources has proven to be difficult in recent years, for many reasons. Biomass is converted into a usable fuel in various organisms, limited to plants and animals. Recently, interest has spiked to introduce these synthetic bio fuel pathways on an industrial scale, but many challenges are faced. Storing bio fuels can be extremely difficult; in addition, scaling these production processes is another prevalent barrier for introduction in industry. Achieving efficient fuel production from microorganisms is still a developing process, many methods today are being researched and analyzed that show potential implementation in industry.

= Introduction = Bio fuels are a usable industrial fuel that are originally created from organic compounds, and molecularly they are primarily composed of hydrocarbons [6]. It has been known for decades that microbial organisms have the capability to make bio fuel, therefore introducing these fuel synthesis methods to an industrial scale has been a profound area for research and development over recent years [2]. Some examples of bio fuel include bio alcohols (ethanol, propanol, butanol), bio diesel, green diesel, vegetable oil/fat, bio gas, and various solid bio fuels [5]. The fuels are categorized into two major groups: 1st and 2nd generation bio fuels [6].. What separates the categories is the source of fuel, not the physical composition of fuel. 1st generation fuels are directly made from a food crop, while 2nd generation fuels require more advanced extraction techniques, and do not generally derive directly from a food crop source. 1st generation fuels face many limitations: producing too much fuel from food crop cuts into food stock and could possibly damage usable farmland [2]. In result, 2nd generation bio fuels are much more sustainable and hence the focus has been more concentrated on scaling 2nd generation biofuel synthesis methods versus 1st generation methods.

Biofuels are desired for distinct reasons, all of which indicate that the overall sustainability compared to petroleum-based fuels holds a more promising future for humanity. Many metabolomic engineers today are focusing research on discovering or improving 2nd generation (and further generation) biofuel synthesis methods.

Method I: Solar-To-Fuel Production
Solar power can be utilized to make biofuel through water splitting and yields hydrogen and oxygen [1]. Hydrogen is difficult to store on its own, so it is often bridged with carbon dioxide to make hydrocarbon fuels that have storage capability Reference. Since hydrogen storage holds many challenges, the technique is to fix CO2 directly into biomass and liquid biofuel via photovoltaic cell (PV). Traditionally, converting CO2 into liquid hydrocarbon fuel has seen terrible energy efficiency; plants only achieve 1% of the thermodynamic maximum Reference. Torella, Gagliardi, Chen, Bediako, Colón, Way, Silver, Nocera did a study on using water splitting catalysts and a strain of metabolically engineered bacteria, Rastolnia Eutropha, along with a PV to produce biomass and isopropyl alcohol, resulting in the highest bio electrochemical fuel yield reported yet [1].

Biofuel production requires many different reactions, some of which that utilize oxygen in key reactions called oxygen-evolution-reactions (OER’s). Many PV systems require an anaerobic bacterium, which can severely hinder the reaction rate for OER’s at the required reaction conditions; one method to overcome this hindrance is to introduce a metal catalyst to drive the reaction forward [1]. Even with introducing the metal catalysts, energy efficiency still is not maximized. The required cell potential to drive OER’s forward is substantially higher than the cell potential required for biological growth: 4.0 V-5.0 V compared to the required 1.23 V for biological growth [1]. In the study conducted by Torella and Gagliardi, the researchers overcome these barriers by incorporating a cobalt phosphate catalyst (CoPi)[1]. The CoPi catalyst can perform OER’s at low cell potentials because it shares many of the same qualities as the OER catalyst seen in photosystem II in plant organisms: it has similar structure, can self-repair, and can perform the necessary electron transfers required in water splitting.

The bioreactor system is designed so that water is split at the CoPi anode end of the PV, and then the electrons are transferred to the protons via a Nickel Molybdenum Zinc (NiMoZn) cathode [1]. Carbon dioxide is pumped into the cell during this entire process and the resulting reduced hydrogen is then oxidized by the bacterium Rastolnia Eutropha H16. Adenosine Triphosphate (ATP) and Nicotinamide adenine dinucleotide phosphate (NADPH) are produced and utilized as reducing equivalents which fix carbon via the Calvin Cycle to produce 3-phosphoglycerate (3PG). 3PG is used to produce biomass/bio-fuel, which can further react to create the fuel isopropanol [1].

Method II: Fungal-Bacteria Consortia
Microbial consortia often demonstrate phenomenal metabolic characteristics in nature which has sparked some interest in developing useful methods for biofuel production [3]. A sagacious barrier encountered while industrially scaling these methods is due to stability of the consortia as the population increases. The food and farm industry provide an abundance of lignin, which is a common source for the synthesis of biofuel. To convert lignocellulose into a usable biofuel, many biological interactions need take place. An organism, or multiple, are needed to produce the cellulase enzymes, hydrolyze lignocellulose into saccharides, and then metabolize the saccharides into biofuel Reference. Minty, Singer, Scholz, Bae, Ahn, Foster, Liao, and Lin assembled a microbial consortium consisting of Trichoderma reesei (T. reesei) and Escherichia Coli (E. Coli) [3]. T. reesei secretes enzymes to break down lignocellulose into saccharides that are then metabolized by E. Coli into the desired biofuel, which is usually isobutanol [3].

To render the process more efficient, the goal was to design a method to combine the three separate steps of producing cellulase enzymes, hydrolyzing them, and subsequently metabolizing them, into one step via genetic engineering [3]. The engineering strategies also have many barriers before they can be utilized efficiently. Synthetic consortia are often very fragile, so when they are introduced to diverse communities of microbes they often dominate the population, which prevents necessary metabolic functions to occur within the population that result in a hindered ability to scale the consortia industrially [3]. One of the key challenges in the synthesized T.reesei/E.Coli consortia is that the fungus and bacteria are competing for hydrolyzing the saccharides produced from lignocellulose, which indicates the relationship between hydrolysis rate and biofuel yield [3].

The consortia can be improved by applying ecology theory to predict the stability and interactions within the population. For example, for the T.reesei/E.Coli consortia, the two organisms follow cooperator and cheater dynamics [3]. T. reesei cooperates by expending energy to hydrolyze saccharides and E. Coli cheats by metabolizing the saccharides at no energy cost [3]. Understanding these dynamics can give insight on how to improve reaction rates and therefor biofuel yield.

1.     Torella, Joseph P.; Gagliardi, Christopher J.; Chen, Janice S.; Bediako, D. Kwabena; Colón, Brendan; Way, Jeffery C.; Silver, Pamela A.; Nocera, Daniel G. (2015-02-24). "Efficient solar-to-fuels production from a hybrid microbial–water-splitting catalyst system". Proceedings of the National Academy of Sciences. 112 (8): 2337–2342. doi:10.1073/pnas.1424872112. ISSN 0027-8424. PMID 25675518

2.     "Partial Correlation in SPSS Statistics - Procedure, assumptions, and output using a relevant example". statistics.laerd.com. Retrieved 2018-03-13.

3.     Minty, Jeremy J.; Singer, Marc E.; Scholz, Scott A.; Bae, Chang-Hoon; Ahn, Jung-Ho; Foster, Clifton E.; Liao, James C.; Lin, Xiaoxia Nina (2013-09-03). "Design and characterization of synthetic fungal-bacterial consortia for direct production of isobutanol from cellulosic biomass". Proceedings of the National Academy of Sciences. 110 (36): 14592–14597. doi:10.1073/pnas.1218447110. ISSN 0027-8424. PMID 23959872

"Extracellular electron transfer from cathode to microbes: application for biofuel production"doi10.1186/s13068-016-0426-0ISSN1754-6834PMC4717640 PMID26788124

5.     "Biofuels - Types of Biofuels". biofuel.org.uk. Retrieved 2018-04-24.

6.     http://www.igem.org.uk/media/405523/biofuels%20final%20report.pdf

Ajayi-Oyakhire, Olu; Mohammed, Mohsin (2016). “Biofuels: Analysis of the Various Biofuel Types Including Biomass, Bioliquids, Biogas, and Bio-SNG”. Institution of Gas Engineers and Managers

 THIS IS WHERE MY ARTICLE ENDS AND THE MATERIAL BELOW IS JUST NOTES 

= Notes on Sources = Summary of Intro


 * When designing a solar-to-fuel system(SFE), a lot of the time the system depends on an anaerobic organism. The organism can be difficult to incorporate in the system because of the oxygen producing anode - it must be kept away from that region in order to be effective
 * Other issues with designing SFE's are also caused by the catalyst. Water splitting typically requires a cell potential of only 1.2V, but with metal catalysts such as platinum or indium, much higher cell potentials are observed.
 * Photosystem II - Cobalt catalyst can achieve water splitting at low potentials because it has many properties that are seen in the catalyst that splits oxygen in photosystem II.
 * Qualities of the photosystem include self repair, self assembly, and assisting electron transfer in water-splitting
 * Oxygen-evolving complex

Results Portion

Schematic Summary: Water is split at the cobalt phosphate anode, and then the electrons are transferred to the protons via NiMoZn. Carbon dioxide is pumped into the cell during this whole process. The reduced hydrogen are then oxidized by the bacterium R. Eutropha H16. ATP and NADPH reducing equivalent are produced, then fix carbon via the Calvin Cycle to produce 3-phosphoglycerate (3PG). 3PG is used to produce biomass/bio-fuel, which is can further react to create the fuel isopropanol. The cell potential for water splitting is higher than the system mentioned above. This is due to the resistivity of the growth media being higher than normal conditions. Also, the oxygen evolution reactions (OER) and hydrogen evolution reactions (HER) overpotential are higher in the growth media. (But why?) -Why is cell viabiltity lost below 2.7V?
 * Calvin Cycle
 * 3-Phosphoglyceric acid
 * Overpotential
 * R. Eutropha H16 growth was encouraged at cell potentials greater than 2.7V, even though it is achievable at 2.3V followed by an extended lag-phase
 * R. Eutropha H16 and NiMoZn have reciprocal compatibility
 * The bacteria and catalyst cathodes promoted biological growth over time, but the growth over time did not have a negative effect on the catalyst -- Ideal
 * Spot assays showed toxic results between 1.8V-2.1V (water-splitting conditions)
 * ROS species are the cause of toxicity because it prevents the reactions necessary, AKA it reacts with hydrogen to form hydrogen peroxide(an ROS) rendering the process inefficient
 * This is why carbon dioxide is forced into the system - rids excess oxygen
 * Bovine liver catalase decomposes hydrogen peroxide, so it was introduced into the cathode department where high H2O2 levels were detected
 * When it was deactivated via heating, it did not decompose the h2o2, and toxicity occurred
 * These 2 observations prove that h2o2 is detrimental to the system
 * The HER cathode was replaced with an SS electrode because the SS electrode produces less ROS species, leading to a more efficient conversion to biomass from starting materials.
 * R. Eutropha H16 was experimentally replaced with another strain, Re2133-pEG12, because it performs better when nutrients are more scarce
 * Re2133-pEG12 does not undergo to the side reaction to produce the storage polymer polyhydroxybuterate (PHB).
 * This strain designed an engineered pathway. After Acetyl CoA is produced, the PHB pathway is disrupted and 4 different genes are expressed to produce isopropanol.
 * Kethothialase phA from R. Eutropha
 * acetoacetyle CoA transferase (ctf) from R. Eutropha
 * acetoacetate decarboxylase (adc) from Clostridium
 * Alcohol dehydrogenase (adh) from Clostridium
 * Re2133-pEG12 produced the highest yield of isopropanol ever recorded for a bioelectrochemical system

Notes on Discussion Conclusion Notes
 * high cell potentials are linked to a higher rate of production of ROS species at the cathode end in the electrochemical cell, decreasing viability.
 * Toxic conditions are also a result of hypochlorite at cell potential 1.36V (at the anode region)
 * Chloride has been known as a toxic agent
 * As a result, multichamber designs to protect the acode region from similar species has been implemented
 * At low potentials, ROS species are produced at the cathode rather than the anode under pH 7 conditions
 * The production of radicals, superoxide and hyroxyl, along with h2o2 are thermodynamically favored when paired with the HER cathode.
 * lower potentials increase the faradaic efficiency of ROS species
 * This is how onset potential of 2.3V - 2.7V was determined --- finding out the potential at which H2 production at the cathode overpowers the ROS production
 * The output biomass was calculated as follows:
 * mass = total energy input[kJ] x 52 [mgDCW/kJ] x faradaic efficiency x 1.23V / Ecell x efficiency of biomass production from Hydrogen
 * Ecell efficiency is what is really affected by the cobalt phosphate catalyst that causes the increase in biomass production
 * Systems with high cell potentials prefer cathodic current for HER
 * Ratio of HER to ROS at these potentials overpowers the toxicity of ROS
 * Low potentials favor cathodic ROS production

Torelli/Gagliardi Cite Notes on Minty,Singer,Scholz,Bae,Ahn,Foster,Liao, and Lin

Abstract and Intro
 * Fuel and energy production from bacteria and other microbes has been a desired area of study within metabolic engineering in recent years.
 * This journal reviews the biosynthesis of isobutanol from fungal bacteria Trichoderma Reesei and E. Coli
 * T. Reesei hydrolyzes lignocellulosic enzymes into soluble saccharides, then E. Coli metabolizes the products into the desired fuel alcohol
 * Lignocellulosic biomass
 * High processing costs and inefficient biocatalysts prevent any useful utilization of biosynthesis for fuel sources in industry.
 * Consolidated Bioprocessing (CBP): incorporating all biologically mediated transformations in a single step
 * Engineering microbes that meet the needs of all steps in a pathway is difficult
 * Most successful pathway has been ethanol production
 * Consortia : a community or collective
 * Many microbial consortia exist in nature, but synthetic microbial consortia are more desired because they are overall more useful.
 * Examples : E. Coli for simultaneous fermentation of hexose and pentose sugars
 * Synthetic consortiums are often unreliable because they lack stability, thus limiting their commercial use.
 * This article studies a system where a fungal bacteria that has cellulolytic properties works with E. Coli, which is very useful in fermentation, to metabolize lignocellulose cells into a fusal alcohol
 * Conversion of microcrystalline cellulose (MCC) and ammonia fiber expansion(AFEX) pre-treated from corn stover (CS) to isobutanol using the consortia mentioned above
 * This study focuses on T. reesei, E. Coli, and isobutanol but the concepts can be used for a wide variety of consortia and biofuels

Notes on Results Fig. 1 Summary
 * Why isobutanol? "It's a promising next-generation biofuel with superior properties"
 * 2-ketoisovalerate: an endogenous valine biosynthesis intermediate
 * Overall process: Lignocellulose cells are hydrolyzed into soluble saccharides which are then further metabolized into desired biofuels
 * T. reesei is chosen to hydrolyze because it produces sufficient amounts of cellulase, is also physiologically compatible with E. Coli, and does not degrade or attack other bacteria
 * Cellulases producted by T. reesei: cellobiohydrolase I and II and endoglucanase
 * oligosaccharides produced from these cellulases are hydrolyzed even more by beta glucosidase
 * Leads to higher glucose concentration at the T. reesei cell surface
 * These saccharides allow growth of fermentation starting substrates - glucose and cellebiose
 * Partial Rank Correlation Coefficients -- PRCC : measure of strength and direction of a linear relationship between two continuous variables whilst controlling for the effect of one or more other continuous variables
 * One of the most influential parameters in the growth/substrate uptake kinetics is the initial E. Coli mass fraction
 * The fraction of substrate bonds accesible to enzymes and initial cellulose and microbe concentrations have predominant effects on controlling rates of hydrolysis
 * Studies from experiment show that cellulose hydrolysis rate and T. reesei growth rate are very much dependent on each other.
 * There is a trade off between cell hydrolysis rate and isobutanol production.
 * As isobutanol reaches its maximum of 0.41 g i-but / g cellulose, the hydrolysis rate of cellulose approaches 0
 * Feel like you might need to ask Dr. Martinez some questions here

Experimental Validation
 * For study, a monculture of T. reesei RUTC30, and bicultures of T. reesei RUTC30 with E. Coli NV3 and K12 were evaluated
 * Studies show that the trade off between cellulose hydrolysis rate and yield of isobutanol exists.
 * The RUTC30/NV3 biculture has a much lower E. Coli concentration, earlier cease of growth, and higher death rates of both E. Coli and T. reesei.
 * NV3 has a higher biomass yield --> lower cell concentrations
 * Cease of growth early on is probably due to NV3 toxicity
 * oxygen limitation may slow the process too
 * NV3 contains a defective rpoS allele --> decreases stationary stage survival
 * They then studied an NV3 monoculture
 * Isobutanol was produced with a higher selectivity.
 * The lower selectivity in the biculture may be a result of the loss of isobutanol production plasmids
 * Initial conditions variation that increases isobutanol production generally decrease the rate of hydrolysis
 * Ask about the last sentence in the last paragraph of this section because it's probably pretty important

Isobutanol Production from Corn Stover(CS) notes


 * Another demonstration of isobutanol produced from lignocellulosic biomass
 * Demonstration: RUTC30/NV3 bicultures over a wide range of NV3/RUTC30 innoculation ratios on AFEX treated Corn Stover
 * AFEX pretreated CS contains hemmicellulose(a mix of pentose and hexose) and crystalline cellulose
 * Ask what ammonia fiber expansion is
 * What is innoculation ratio
 * Highest isobutanol yield and titer with an NV3/RUTC30 ratio of 1
 * 1.88 g/L abd 62% yield from the theoretical
 * Higher relative amounts of isobutanol and succinate in the AFEX system compared to the MCC

Cooperator-cheater dynamics for Modifying TrEc consortia
 * Stability: permits the use of continuous reactors or repeated batch fermentation
 * Tunability: allow population composition to be optimized for desired performance
 * Researchers are able to predict results based off of ecology theory
 * Also used to design biological mechanisms
 * Monod equation
 * Game Theory
 * Cheaters and cooperators.
 * In the case of this study, T. reesei is the cooperator - it does a lot of work to give up its enzymes and receives little in result
 * E. Coli is the cheater, it isn't as physiologically burdened as T. reesei; it uses the hydrolyzed products
 * As long as the cooperator reaps the fitness benefits that are concave and have access to its own products
 * Testing the theory:
 * Simplified version of the TrEc model in a system such that the parameters may be adjusted. Example:
 * E.Coli cheating benefits are quantified by the ratio of the maximum growth rate of E. Coli to the max growth rate of T. reesei
 * T. reesei cooperator access is quantified by the increase in glucose concentration relative to the bulk medium at the T. reesei cell surface due to cell wall localized cellobiose hydrolysis
 * Wide range of product compositions can be formed by altering the max growth rate of E. Coli and/or the increase in glucose concentration, and other parameters
 * Equibrium, in the experiment, was attained in 12-60 doubling times. The initial conditions had very little effect, leading one to believe that the equilibrium states are stable and easily attainable
 * To investigate the population and equilibrium composition, cellobiose was used instead of cellulose.
 * Cellobiose is a glucose disaccharide
 * Cellobiose enhances the cooperator-cheater dynamics of the system - concave fitness benefits, cooperator access to appropriate substrates
 * Max growth rate of E.Coli was tuned using the different strains NV3 and K12 because they have different rates, and also by altering pH
 * Results:
 * bi-cultures inoculated at different initial E.coli concentrations
 * steady state e.coli concentration increased with maximum growth rate of e.coli AND time to reach equlibrium decreases
 * Bi-cultures WITH the toxicity effects still display cooperator-cheater dynamics, BUT plasmid maintenance and isobutanol production rendered unreliable and varied with inoculation fractions

Discussion Notes
 * TrEc bicultures were constructed and analyzed as model consortia systems for consolidated bio-processing of lignocellulosic cells into usable fusal bio-compounds. Isobutanol was the model fuel in this study.
 * They demonstrated not just the principles that are the foundation of this model, but also methods to optimize the consortia via initial conditions and parameters.
 * i.e. the cooperator-cheater system that is proved and designed through manipulation of cooperator/cheater benefits
 * The cooperator-cheater model can be used as a general tool for modeling other similar consortia
 * Altering culture conditions and programming cellular behavior are some of the ways that the consortia can be 'tuned'
 * Analysis suggests that the hydrolysis rate of cellulose approaches 0 as the isobutanol yield reaches its maximum of 0.41
 * Some parameters were identified to improve the titer and rate of product formation relative to reactants.
 * Increasing competitiveness of E.Coli compated to T. reesei for glucose
 * Other parameters such as growth rates of E.coli or t.reesei can affect the volumetric or spacial product
 * RUTC30/NV3 consortia yields more isobutanol when glucose availability is high, but this is not favorable in industry because of the expenses.
 * Even manipulating initial conditions and parameters, the biculture process yields 50% lower than the monoculture process
 * Proof-of-concept consolidated bio-processing application:
 * Microcrystalline cellulose(MCC) and ammonia fiber expansion pre-treated corn stover directly converts to isobutanol
 * highest yield/titer to date for CBP production of biofuels
 * Further analysis needs to be conducted before it can be implemented commercially
 * It's an aerobic process which generally is more expensive than anaerobic processes because of oxygen agitation
 * Future Research: why? --> Isobutanol production is clearly reduced in biculture vs. monoculture, what factors may have an effect on this?
 * Further study of E.Coli metabolism under similar biculture conditions
 * Chromosomal integration of plasmid genes
 * Further study of isobutanol production from pentoses and hemicellulose
 * Attempts to improve tolerance of Tr and Ec to isobutanol as an inhibitor
 * Even though the TrEc consortia is technically an aerobic modeled system, it can still perform in anaerobic conditions
 * E.Coli is versatile - it can produce anaerobic metabolites to accomplish the same tasks if oxygen were present.
 * This is one reason why this biculture consortia is more desired in industry - anaerobic processes are easier and much cheaper to regulate
 * Further study can also be done with different cellulolytic components





Other source:

http://www.igem.org.uk/media/405523/biofuels%20final%20report.pdf