Sustaining biogenic methane release from Illinois coal in a fermentor for one year
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Zhang, Ji↗
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The Complete Genome Sequence of Hyperthermophile Dictyoglomus turgidum DSM 6724™ Reveals a Specialized Carbohydrate Fermentor
In this study we report the complete genome sequence of the chemoorganotrophic, extremely thermophilic bacterium, Dictyoglomus turgidum , which is a Gram negative, strictly anaerobic bacterium. D. turgidum and D. thermophilum together form the Dictyoglomi phylum. The two Dictyoglomus genomes are highly syntenic, and both are distantly related to Caldicellulosiruptor spp. D. turgidum is able to grow on a wide variety of polysaccharide substrates due to significant genomic commitment to glycosyl hydrolases, 16 of which were cloned and expressed in our study. The GH5, GH10, and GH42 enzymes characterized in this study suggest that D. turgidum can utilize most plant-based polysaccharides except crystalline cellulose. The DNA polymerase I enzyme was also expressed and characterized. The pure enzyme showed improved amplification of long PCR targets compared to Taq polymerase. The genome contains a full complement of DNA modifying enzymes, and an unusually high copy number (4) of a new, ancestral family of polB type nucleotidyltransferases designated as MNT (minimal nucleotidyltransferases). Considering its optimal growth at 72°C, D. turgidum has an anomalously low G+C content of 39.9% that may account for the presence of reverse gyrase, usually associated with hyperthermophiles.
59 BASIC BIOLOGICAL SCIENCES↗
Off-gas capture: a promising strategy for removal and recovery of toxic bioproducts in aerobic fermentation
In many bioprocesses, maximum achievable titers are limited below economically viable levels by toxic accumulation of the primary end-product. To combat end-product inhibition, a variety of in situ product removal technologies have been developed to selectively remove or partition toxic bioproducts, thereby prolonging fermentation and improving overall process efficiency. Use of an in situ organic overlay to partition toxic hydrophobic products is a commonly employed approach, but this technique occupies valuable space in the fermentor, imposes replacement costs for unrecovered solvent, and increases downstream separation due to formation of stable emulsions. In addition, for many volatile hydrophobic products produced under aerobic conditions—including medium-chain alcohols, esters, monoterpenes, and other aviation fuel precursors—a significant fraction of the product is volatilized to the fermentor off-gas and must be recovered separately to maximize product yield. To address these challenges, we explore the viability of leveraging existing aeration energy to fully strip and recover volatile products from the fermentor off-gas. We compare two strategies of in situ product removal—liquid–liquid extraction and direct recovery from fermentation off-gas—for the production and recovery of intermediates used to generate isoprene and DMCO (1,4-dimethylcyclooctane), a high-performance jet fuel. We evaluate product toxicity, solvent toxicity, solvent partitioning, and the impact of aeration and internal overlay configurations on product volatilization rates. We then optimize product recovery from fermentor off-gas via condensation in chilled solvent, achieving 84% capture efficiency. In addition to greatly simplifying downstream processing, relying on aeration for product volatilization in the absence of an internal overlay enables continuous removal of toxic fermentation products up to maximum isoprenol titers of 20.4 g/L, the highest reported to date.
isoprene↗
Systems and methods for continuously fermenting C5 and C6 saccharides
This invention provides optimized fermentation of cellulosic and hemicellulosic sugars. Biomass-derived hemicellulosic and cellulosic sugars are independently conditioned and separately fermented, utilizing reuse and recycle of microorganisms, metabolic intermediates, and nutrients. Conditioned sugars can be fermented in separate vessels, where excess cells from glucose fermentation are conveyed to hemicellulose sugar fermentation along with raffinate from solvent recovery, to enhance productivity and product yield. Some variations provide a method of fermenting C5 and C6 sugars to fermentation products, the method comprising: fermenting a C6-rich sugar feed to a first fermentation product; fermenting a C5-rich sugar feed to a second fermentation product; removing microorganism cells from the first fermentor, to maintain a cell concentration within a selected range; conveying microorganism cells to a second fermentor; and removing microorganism cells from the second fermentor, to maintain a microorganism cell concentration that is greater than that in the C6-rich fermentor.
Survase, Shrikant↗
Consultancy on Large-Scale Submerged Aerobic Cultivation Process Design - Final Technical Report: February 1, 2016 -- June 30, 2016
NREL is developing an advanced aerobic bubble column model using Aspen Custom Modeler (ACM). The objective of this work is to integrate the new fermentor model with existing techno-economic models in Aspen Plus and Excel to establish a new methodology for guiding process design. To assist this effort, NREL has contracted Genomatica to critique and make recommendations for improving NREL's bioreactor model and large scale aerobic bioreactor design for biologically producing lipids at commercial scale. Genomatica has highlighted a few areas for improving the functionality and effectiveness of the model. Genomatica recommends using a compartment model approach with an integrated black-box kinetic model of the production microbe. We also suggest including calculations for stirred tank reactors to extend the models functionality and adaptability for future process designs. Genomatica also suggests making several modifications to NREL's large-scale lipid production process design. The recommended process modifications are based on Genomatica's internal techno-economic assessment experience and are focused primarily on minimizing capital and operating costs. These recommendations include selecting/engineering a thermotolerant yeast strain with lipid excretion; using bubble column fermentors; increasing the size of production fermentors; reducing the number of vessels; employing semi-continuous operation; and recycling cell mass.
09 BIOMASS FUELS↗
Bioprocess and microbe engineering for total carbon utilization in biofuel production
Some aspects of this invention provide methods and bioreactors for converting a carbon source into a lipid. In some embodiments, lipid production is carried out in an aerobic fermentor and carbon dioxide generated during lipid production is converted into a carbon substrate by CO2 fixation in an anaerobic fermentor. In some embodiments, the carbon substrate generated by CO2 fixation is used as the carbon source for lipid production, thus achieving total carbon utilization in lipid production.
Stephanopoulos, Gregory↗
Membrane assisted continuous production of solvents with integrated solvent removal using liquid-liquid extraction
The aim was to demonstrate an industrially relevant pilot scale integrated process to produce butanol and other solvents from lignocellulosic sugars produced with AVAP® biomass fractionation technology from southern pine wood. The concentrated sugars were concurrently fermented using genetically engineered Clostridium acetobutylicum to n-butanol, acetone, isopropanol and ethanol in continuous membrane assisted cell recycle fermentation with steady-state solvent productivity exceeding 10 g/L/h. The solvents from the cell free permeate were recovered with extractant of butyl butyrate in a continuous liquid-liquid extraction column and aqueous product (raffinate) along with unused sugars, nutrients, and metabolic intermediates were recycled back to the fermentors. The total solvent yield approached 0.30 g/g sugars.
09 BIOMASS FUELS↗
Sweet sorghum bagasse and corn stover serving as substrates for producing sophorolipids
To make the process of producing sophorolipids by Candida bombicola truly sustainable, we investigated production of these biosurfactants on biomass hydrolysates. This study revealed: (1) yield of sophorolipds on bagasse hydrolysate decreased from 0.56 to 0.54 and to 0.37 g/g carbon source when yellow grease was dosed at 10, 40 and 60 g/L, respectively. In the same order, concentration of sophorolipids was 35.9, 41.9, and 39.3 g/L; (2) under similar conditions, sophorolipid yield was 0.12, 0.05 and 0.04 g/g carbon source when corn stover hydrolysate was mixed with soybean oil at 10, 20 and 40 g/L. Sophorolipid concentration was 11.6, 4.9, and 3.9 g/L for the three oil doses from low to high; and (3) when corn stover hydrolysate and yellow grease served as the substrates for cultivating the yeast in a fermentor, sophorolipid concentration reached 52.1 g/L. Upon further optimization, sophorolipids production from ligocellulose will be indeed sustainable.
09 BIOMASS FUELS↗
Efficient Production of Polymalic Acid by a Novel Isolated Aureobasidium pullulans Using Metabolic Intermediates and Inhibitors
Polymalic acid (PMA) is a linear anionic polyester composed of L-malic acid monomers, which have potential applications as drug carriers, surgical suture, and biodegradable plastics. In this study, a novel strain of Aureobasidium pullulans var. melanogenum GXZ-6 was isolated and identified according to the morphological observation and deoxyribonucleic acid internal-transcribed spacer sequence analysis, and the product of PMA was characterized by FT-IR, {sup 13}C-NMR, and {sup 1}H-NMR spectra. The PMA titer of GXZ-6 reached 62.56 ± 1.18 g L{sup −1} with productivity of 0.35 g L{sup −1} h{sup −1} using optimized medium with addition of metabolic intermediates (citrate and malate) and inhibitor (malonate) by batch fermentation in a 10-L fermentor. Besides that the malate for PMA synthesis in GXZ-6 might mainly come from the glyoxylate cycle, based on results, citrate, malate, malonate, and maleate increased while succinate and fumarate inhibited the production of PMA, which was different from that of other A. pullulans. This study provided a potential strain and a simple metabolic control strategy for high-titer production of PMA and shared novel information on the biosynthesis pathway of PMA in A. pullulans.
60 APPLIED LIFE SCIENCES↗
Microbial Approach to Low-Cost Production of Photovoltaic Nanomaterials
Photovoltaic (PV)-generated electricity can participate in renewable grid parity after meeting conditions of low-cost PV materials and economic manufacturing of solar cells. In this paper, we report low-cost, scalable microbial synthesis of Cu(In,Ga)Se 2 (CIGSe) and Cu(In,Ga)S 2 (CIGS), which are among the promising candidates to serve as light absorbing layers in solar panels. Microbial synthesis uses reducible chalcophiles and empirically stoichiometric metal components to produce CIGSe and CIGS with band gaps and intra- and intercrystallite compositional homogeneity similar to that produced with traditional techniques. Importantly, microbially produced photovoltaic materials described herein use inexpensive precursor materials at moderate temperatures (65 °C). The microbially facilitated processes do not utilize high temperature, vacuum, or toxic organic solvents. The potential to upscale microbial synthesis without loss of material quality is demonstrated here, indicating a high potential for industrial applications of this technology for production of nanomaterials for PV applications. We estimate that a 50 000 gallon fermentor could generate about 100 kg/month of CIGSe nanoparticles, which could be processed into 0.2 MW of PV cells.
stoichiometric homogeneity↗
Scale Up of Malonic Acid Fermentation Process: Cooperative Research and Development Final Report, CRADA Number CRD-16-612
The goal of this work is to use the large fermentation vessels in the National Renewable Energy Laboratory's (NREL) Integrated Biorefinery Research Facility (IBRF) to scale-up Lygos' biological-based process for producing malonic acid and to generate performance data. Initially, work at the 1 L scale validated successful transfer of Lygos' fermentation protocols to NREL using a glucose substrate. Outside of the scope of the CRADA with NREL, Lygos tested their process on lignocellulosic sugars produced by NREL at Lawrence Berkeley National Laboratory's (LBNL) Advanced Biofuels Process Development Unit (ABPDU). NREL produced these cellulosic sugar solutions from corn stover using a separate cellulose/hemicellulose process configuration. Finally, NREL performed fermentations using glucose in large fermentors (1,500- and 9,000-L vessels) to intermediate product and to demonstrate successful performance of Lygos' technology at larger scales.
09 BIOMASS FUELS↗
Bio-Oxo Technology
Easel Biotechnologies, LLC’s Bio-Oxo process has demonstrated that isobutyraldehyde can be biologically produced from corn stover hydrolysate up to 56 g/L in a 14L fermentor. This was accomplished by metabolically engineering bacterial strains to not only produce isobutyraldehyde, but to do so by co-utilizing corn stover hydrolysate sugars, glucose and xylose. Also essential to the success of the Bio-Oxo process was that it utilized gas stripping as a means of product separation, allowing for the continuous removal of isobutyraldehyde. This aided in not only reducing energy costs associated with separation, but also alleviating product toxicity, resulting in higher production. Although we were not able to complete our economic analysis based on pilot scale fermentations, the improvements we have made from strain engineering to product separation, should result in the reduced cost of isobutyraldehyde. Still, as the project has ended prematurely, there is room for additional optimization. Improvements in productivity and sugar utilization would result in a further reduction in capital and recovery costs. As a biological-based process, the utilization of corn stover results in reduced greenhouse gas emissions as compared to petroleum-based chemical synthesis. In addition, as a true replacement chemical “drop in” system, no downstream production units need to be changed. Jobs can also be created as farm waste needs to be collected and transported to the new production facility.
09 BIOMASS FUELS↗
Application of photosynthetic N2-fixing cyanobacteria to the CELSS program
Commercially available air lift fermentors were used to simultaneously monitor biomass production, N2-fixation, photosynthesis, respiration, and sensitivity to oxidative damage during growth under various nutritional and light regimes, to establish a data base for the integration of these organisms into a Closed Ecological Life Support System (CELSS) program. Certain cyanobacterial species have the unique ability to reduce atmospheric N2 to organic nitrogen. These organisms combine the ease of cultivation characteristics of prokaryotes with the fully developed photosynthetic apparatus of higher plants. This, along with their ability to adapt to changes in their environment by modulation of certain biochemical pathways, make them attractive candidates for incorporation into the CELSS program.
Packer, L.↗
BIOASPEN: System for technology development
The public version of ASPEN was installed in the VAX 11/750 computer. To examine the idea of BIOASPEN, a test example (the manufacture of acetone, butanol, and ethanol through a biological route) was chosen for simulation. Previous reports on the BIOASPEN project revealed the limitations of ASPEN in modeling this process. To overcome some of the difficulties, modules were written for the acid and enzyme hydrolyzers, the fermentor, and a sterilizer. Information required for these modules was obtained from the literature whenever possible. Additional support modules necessary for interfacing with ASPEN were also written. Some of ASPEN subroutines were themselves altered in order to ensure the correct running of the simulation program. After testing of these additions and charges was completed, the Acetone-Butanol-Ethanol (ABE) process was simulated. A release of ASPEN (which contained the Economic Subsystem) was obtained and installed. This subsection was tested and numerous charges were made in the FORTRAN code. Capital investment and operating cost studies were performed on the ABE process. Some alternatives in certain steps of the ABE simulation were investigated in order to elucidate their effects on the overall economics of the process.
Source record↗
Rejuvenation of Spent Media via Supported Emulsion Liquid Membranes
The overall goal of this project is to maximize the reuseability of spent fermentation media. Supported emulsion liquid membrane separation, a highly efficient extraction technique, is used to remove inhibitory byproducts during fermentation; thus, improving the yield while reducing the need for fresh water. The key objectives of this study are: Develop an emulsion liquid membrane system targeting low molecular weight organic acids which has minimal toxicity on a variety of microbial systems; Conduct mass transfer studies to allow proper modeling and design of a supported emulsion liquid membrane system; Investigate the effect of gravity on emulsion coalescence within the membrane unit; Access the effect of water re-use on fermentation yields in a model microbial system; Develop a perfusion-type fermentor utilizing a supported emulsion liquid membrane system to control inhibitory fermentation byproducts; Work for the coming year will focus on the determination of toxicity of various solvents, selection of the emulsifying agents, as well as characterizing the mass transfer of hollow-fiber contactors.
Wiencek, John M.↗
Rejuvenation of Spent Media via Supported Emulsion Liquid Membranes
The overall goal of this project was to maximize the reuseability of spent fermentation media. Supported emulsion liquid membrane separation, a highly efficient extraction technique, was used to remove inhibitory byproducts during fermentation; thus, improve the yield while reducing the need for fresh water. The key objectives of this study were: (1) Develop an emulsion liquid membrane system targeting low molecular weight organic acids which has minimal toxicity on a variety of microbial systems. (2) Conduct mass transfer studies to allow proper modeling and design of a supported emulsion liquid membrane system. (3) Investigate the effect of gravity on emulsion coalescence within the membrane unit. (4) Access the effect of water re-use on fermentation yields in a model microbial system. and (5) Develop a perfusion-type fermentor utilizing a supported emulsion liquid membrane system to control inhibitory fermentation byproducts (not completed due to lack of funds)
Wiencek, John M.↗
Bioprocess utilizing carbon dioxide and electrodeionization
A method for producing succinic acid is provided, which comprises circulating a fermentation broth containing succinate ion through an electrodeionization apparatus. The apparatus comprises an anode; a cathode; a stacked ion-exchange assembly comprising a one or more oriented ion-exchange units between the anode and the cathode. Each ion-exchange unit comprises a porous ion-exchange resin wafer including an inlet and an outlet together are adapted to circulate a liquid reaction stream containing a carboxylate anion from a fermentor through the resin wafer; a flow-distributing gasket for circulating a product stream through a product reservoir; an anion exchange membrane for transporting at least a portion of the carboxylate anion to the product stream; and a bipolar ion-exchange membrane to direct protons toward the cathode and into the product stream and to direct hydroxyl ions toward the anode and into the reaction stream in the resin wafer of an adjacent ion-exchange unit.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Bench-Scale Fermentation Laboratory (Fact Sheet)
This fact sheet provides information about Bench-Scale Fermentation Laboratory capabilities and applications at NREL's National Bioenergy Center.
09 BIOMASS FUELS↗