Fermentation

Being Selective and Specific

Our Fermentation Equipment

From Fermentable Sugars to Value-Added Precursors

Fermentation is a vital unit operation in the biomass conversion process offering the potential for complete utilization of biomass into multiple bioproducts.  Of special interest in biorefining are platform intermediate chemicals from fermentation that can be converted into numerous consumer and industrial products, including succinic acid and butanol.

At ABPDU our focus is on biological fermentation as it offers the most selectivity and specificity when it comes to producing a product or intermediate. Microorganisms and recombinant organisms are used to metabolize lignocellulosic sugars and other intermediates to form a wide array of alcohols, acids, and enzymes—precursors to biofuels, biochemicals, and biomaterials.

High Yields Depend on Extensive Process Development

While fermentation is a very well understood unit operation in first-generation biofuels and bioproducts, it is a relatively new process area in the conversion of lignocellulosic sugars. Achieving high yields and economic viability requires extensive process development.

We apply process intensification and integration concepts to the traditionally removed fermentation and product recovery steps. Our intricate fermentation systems integrate either into deconstruction or recovery to assess the viability and economics of the integrated processes at a relevant scale.

Concept Development and Scale Up

Some companies design their pathway and select the organism to obtain a particular molecule. Based on your specifications, we can develop the fermentation process to a point where it is stable and ready for scale up.

Process Optimization and Validation

If you have a completely optimized bench scale fermentation process, we can control and optimize the process and then demonstrate it at a larger scale.

Achieving End Product Specifications

Feedstock can represent >40% of all process costs in a biomass-to-bioproduct process. Therefore, it is critical to rapidly and efficiently metabolize sugars and other molecules from biomass.

In designing a successful fermentation process, the required end product or intermediate drives our decision-making. Analytical chemistry is an essential part of our fermentation process development, enabling, among other things:

  •  Tight control of process parameters such as temperature, pH balance, nutrients, etc.
  • Optimum selection of microorganisms and fermentation methods
  • Improved product concentration
  • Control of inhibitory effects for better process integration

Our Equipment

Fermentation Process Options

 

Environments
Aerobic and anaerobic fermentations in batch, fed-batch, and continuous modes.
Simultaneous Sachcharification
In simultaneous saccharification (SSF), the fermenting microorganism simultaneously consumes the released sugars from enzymatic hydrolysis, avoiding product inhibition of enzymes and also decreasing the probability of contamination.
Co-Fermentation
There are substantially greater savings in capital and operating costs to be gained by applying a co-fermentation process in which the hexose and pentose sugars derived from both the cellulose and hemicellulose fractions are simultaneously fermented to bio-product in a single operation. Using genetically modified organisms or a community of organisms, this method metabolizes more than one molecule at the same time, e.g glucose-6 carbon, xylose-5 carbon, and lignin.
Simultaneous Sachcharification and Co-Fermentation
Enzymatic hydrolysis can be performed simultaneously with the co-fermentation of glucose and xylose in a process referred to as simultaneous saccharification and co-fermentation (SScF). Besides reduced capital cost, the SScF process offers several advantages, which include continuous removal of end-products of enzymatic hydrolysis that inhibit cellulases or β-glucosidases and higher ethanol productivity and yield than separate hydrolysis and fermentation.
Consolidated Bio Processing
Consolidated bioprocessing (CBP) approaches combine enzyme production, substrate hydrolysis, and fermentation into one process. CBP has potential to lower the cost of biomass processing compared to process configurations featuring a dedicated step for cellulase production due to the elimination of operating and capital costs associated with dedicated enzyme production and more effective biomass solubilization.

Related Papers and Publications

Streptomyces Venezualae as a Platform Organism for Biofuels

Biosynthesis of highly branched short and long-chain hydrocarbons would enable production of biofuels with desirable and tunable properties, including compression ignition fuels with low freezing points. Polyketide synthase (PKS) pathways are a promising route towards production of such compounds; engineering of PKS pathways favors a native host- this work therefore focuses on development of Streptomyces venezualae ATCC 10712 as a platform organism. While S. venezualae is well characterized for industrial production of antibiotics, currently available protocols for high density fermentation make use of rich media and high-purity dextrose. The viability of Streptomyces as a platform organism for large-scale cellulosic biofuel production is therefore currently unknown.

This study focuses on the development of protocols for high density fed-batch fermentation of S. venezualae with cellulosic sugar feed in minimal medium to evaluate the viability of this strain as a production organism for fuels and commodity chemicals.

Reducing Nutrient Supply in Mixed Feedstock Fermentation

Mixed feedstocks can help reduce the risk associated with feedstock availability for bio-based production of fuels and chemicals. This study was performed to evaluate cellulosic hydrolysates for fermentation to biofuels and also probe the possibility of reducing nutrient concentration in the broth media.

The study demonstrated that mixed feedstocks can release 80 -100% of the sugar that is obtained from corn stover alone. A hundred percent of the released sugars from mixed feedstocks can be converted to ethanol. The study also showed that alkali pretreated mixed feedstock has higher ethanol yield but lower glucose yield compared to IL pretreated mixed feedstock due to inhibition of microbial growth by residual EmimAcetate. The same ethanol yield can be achieved with lower nutrient supplied but with longer fermentation time.

High-Cell Density Fermentation to Produce “Milk Proteins”

In a collaboration with Muufri, a fed-batch process to express milk proteins through Pichia species was optimized by conducting several tests at 2L scale. The optimal process at 2L was scaled to 300L followed by centrifugation for supernatant recovery. The downstream recovery process was developed to concentrate and purify proteins from the supernatant. These proteins are now being tested in several milk formulations.

Process Development of Three Novel High Value Fermentation Products

Lygos developed a biological process for malonic acid production and provided ABPDU with fermentation parameters optimized at the bench scale. At the 300-L scale, we used LabVIEW VI to control external pumps and regulate the progress of fermentation. The successful scale-up of this fermentation pathway demonstrated the ability in replacing traditional petroleum-based malonic acid production process, which requires hazardous cyanide and chloroacetic acid.

FATER ABPDU Waste-to-Bioenergy Process Partnership

ABPDU has been developing and validating an integrated waste-to-energy process under a DOE work-for-others (WFO) agreement with FATER, an Italian JV between Procter & Gamble and the Angelini Industrial Group.

Key outcomes indicate that post-consumer absorbent hygiene products (AHP) can be readily and economically converted — without using harsh or expensive pretreatment routes — to fermentable sugar intermediates as well as biofuel and bio-based chemical products.

Bisabolene Production Scale-up at ABPDU

Bisabolene Production Scale-up at ABPDU

Biosynthetic bisabolene is a precursor to bisabolane – a potential renewable diesel fuel alternative. Bisabolane has attractive physical properties including a much lower cloud point (-78°C) than traditional diesel (-35°C) making bisabolane a potential cold weather additive to current fuels.
Demonstrating Bisabolene Production at 1.8 L Scale

Demonstrating Bisabolene Production at 1.8 L Scale

Bisabolane has been identified as a potential biosynthetic alternative to D2 diesel fuel. Researchers at JBEI have engineered S. Cerevisiae for the production of bisabolene, bisabolane’s immediate precursor, by the introduction of bisabolene synthase from A. Grandis. In order to produce large enough quantities of bisabolane for engine testing and to better understand the challenges that arise at larger production scales, JBEI and the Advanced Biofuels Process Demonstration Unit (ABPDU) have developed a fed-batch fermentation process for bisabolene production for eventual scale-up to production scale. Presented here are preliminary results from 1.8 L fed-batch fermentations conducted at ABPDU with a discussion of challenges in scaling up the process.

Using FT-NIR to Facilitate Dynamic Fed-Batch Fermentations

Using FT-NIR to Facilitate Dynamic Fed-Batch Fermentations

  • Fed-batch fermentations involve controlled feeding of a growth limiting nutrient to a batch culture enabling higher cell densities. Some classic fed-batch strategies employ pH, OUR (oxygen uptake rate), DO (Dissolved oxygen) etc, as control parameters, based on their indirect correlations with growth. This indirect feedback control could lead to an un-optimized process with compromised productivity.
  • FT-NIR spectroscopy is an analytical tool that uses electromagnetic spectrum (~800 – 2500 nm) to cause vibrational energy changes in matter resulting from fluctuations in molecular dipole moment and provides a response that can be used to quantify composition of the material.
  • The FT-NIR probe can penetrate much farther into a medium allowing instant measurements, and little to no sample preparation. However, its meaningful implementation as a standard practice requires vigorous optimization.
  • In this study, we present direct online monitoring of cell density, substrate and product profiles using a single FT-NIR (Fourier Transform Near Infrared) probe during yeast fermentation. This online measurement can then be used to directly control fed batch fermentations, resulting in a precisely optimized process.
Scale-up of Thermophilic Ionic Liquid-tolerant Cellulase Cocktail for Lignocellulosic Biofuel Production

Scale-up of Thermophilic Ionic Liquid-tolerant Cellulase Cocktail for Lignocellulosic Biofuel Production

Ionic liquid pretreatment of lignocellulosic biomass shows great potential in effectively reducing the biomass recalcitrance with less enzyme requirement for saccharification of biomass into sugars. However, commercially available cellulase enzymes are sensitive to residual ionic liquids from pretreatment necessitating extensive and expensive washing steps to remove ionic liquids from pretreated material. As a result, high costs associated with the extensive pretreatment process followed by washing and separate hydrolysis has become one of the main barriers for commercialization. Consolidation of pretreatment and enzymatic saccharification would address the current techno- economic challenges related to high cost of separate pretreatment and saccharification. To accomplish this, researchers at Joint Bioenergy Institute (JBEI) have developed a thermophilic ionic liquid-tolerant cellulase cocktail called “JTherm” for biofuel production. The cocktail can tolerate up to 20-30% ionic liquid over a range of temperatures, 50-70 deg C, when incubated at pH 5.5 for 72 hours.

In this study, we present the results from the scale-up of production of JTherm cocktail in 75L bioreactors at Advanced Biofuel Process Demonstration Unit (ABPDU). Initial results showed excellent scalability of the process with enzyme yields similar to that of shake flasks and further optimization would expand the cellulolytic enzymes landscape to integrate various ionic liquid pretreatment technologies overcoming barriers to production of economically viable lignocellulosic biofuels.