PHOTO 1
Pilot-scale airlift reactor with external recycling: Pilot-scale airlift fungal cultivation of the soluble-thin stillage fraction (24 L) with 0.8 L air/L thin stillage/min on the day of spore inoculation (day 0). Note the recycle in external tubes to promote oxygen transfer and fungal pellet formation and recovery. Fungal pellets filled the reactor by day 3. The reactors were designed by van Leeuwen and custom built.

PHOTO 2
Bench-top reactor: Airlift bench-top reactor with draft-tube design and aseptic addition of 4.5 L thin stillage. Reactor built in our labs by members of our team.

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Introduction

Corn dry-grind ethanol plants generate ~6 gallons leftovers/gallon ethanol after distillation - stillage. Creating value-added byproducts from stillage and recycling water is important as the annual ethanol production in the U.S. approaches eight billion gallons.

Stillage from fermentation, followed by distillation, contains fiber, yeasts, and dissolved organics in water, measured as total chemical oxygen demand (COD) of nearly 100 g/L. Most solids are removed by centrifugation and dried to distillers dried grain (DDG). The centrate, thin stillage is partially recycled directly to the fermentation process, but limited to 50% to prevent build-up of total and dissolved solids, especially lactic acid, acetic acid and glycerol. The remaining thin stillage is currently concentrated by flash evaporation - an energy-intensive process - and blended with DDG, producing DDG with solubles (DDGS). DDGS is used for livestock feed, but is low in essential amino acids, e.g., lysine, limiting its usage, particularly for hogs and chickens. Fortunately, thin stillage contains biodegradable organic compounds, sufficient micronutrients, at pH 4.5, which makes thin stillage an ideal fungal cultivation feedstock.

A fungal treatment process for thin stillage has the following merits

1. Energy savings by avoiding stillage evaporation,
   
2. Recovery of protein-rich fungal biomass, and
   
3. Potential for water recycling of treated effluent
   

Solids separation and removal of organic materials are important for recycling the effluent as process water. This research investigated the cultivation of the food-grade fungus Rhizopus microsporus on thin stillage and the potential for water recycling.

Methodology

The research team designed and operated stirred and airlift fungal bioreactors. Fungal inoculum was prepared from spores in shake flasks using sterile thin stillage as growth medium to reduce the culture lag phase and reduce costs.

Phase 1 used lab- and pilot-scale stirred bioreactors (1.25-, 5-, and 50-L working volumes) for batch fungal treatment of thin stillage.

Phase 2 investigated the design and operation of lab- and pilot-scale airlift bioreactors (5- and 24-L working volumes) to improve oxygen transfer and improve fungal pellet formation and recovery. The team constructed a 5-L airlift bioreactor; diffused air lifts the liquid and fungus inside a concentric draft tube with continuous liquid recycle in the annulus between the draft tube and reactor wall. An improved 24-L airlift reactor was developed with external recycle pipes.

Aeration rates were varied from 0.2 to 1.0 L air/L reactor/min (vvm). Feed stillage and reactor samples were analyzed for total and soluble COD, total and volatile suspended solids (TSS, VSS), glycerol, and lactic and acetic acids, all of which are critical for recycling the effluent as process water.

Results

1. Batch experiments in all bioreactors showed prolific fungal growth under non-sterile conditions.
   
2. COD, suspended solids, glycerol, and organic acid removals, critical for stillage recycling, reached 80 -100% with 5-day fungal treatment in stirred bioreactors.
   
3. The fungi grew in large spherical pellets in the airlift bioreactor in 2-3 days and were harvested with course screens (1/4” openings) - easily dewatered to 20-30% solids.
   
4. Fungal yields reached 0.45 g biomass/g soluble COD removed.
   
5. Amylase activity in the water was roughly equivalent to about 25% of the enzymes required prior to fermentation in the corn-to-ethanol production process.
   

The fungal biomass is high in lysine and nutraceuticals chitin/chitosan, enhancing its potential as a nutritionally-beneficial livestock feed. The fungus could be co-fed with DDG to mono- gastrics: swine and poultry, to avert anticipated corn shortfall created by the booming ethanol industry.

Discussion

Originality and innovation

A novel, low-energy fungal process was developed that remediates dry-grind corn ethanol thin stillage with simultaneous generation of nutritious fungal biomass and useful enzymes

Complexity

Thin stillage is rich in a variety of organic substances with a COD of 100 g /L including 30 g/L suspended solids. The main undesirable components in yeast fermentation are the solids and the fermentation byproducts - glycerol, lactic acid, and acetic acid - which all occur in appreciable concentrations. The complexity of thin stillage requires evaporation of the water to recover the organics as an alternative to disposal of the stillage.

R. microsporus was able to utilize most stillage organic substances, was not inhibited and it enmeshed suspended solids. The fungal growth occurred as large spherical pellets in air-lift reactors, easy to separate by settling or course screening (1/4” openings), and readily dewaterable by mild pressure, e.g. hand squeezing, simulating a pressure filter. These are important properties in the economical operation of a full-scale fungal process.

Socio-economic impacts

The US ethanol industry consumes 35 billion gallons water per year to produce 8 billion gallons ethanol. Water consumption could be reduced by at least 10 billion gallons if all dry- grind ethanol plants recovered water by this process. The current evaporation process costs about $0.13 per gallon ethanol produced at current natural gas prices. Energy savings from eliminating stillage evaporation could save $800 million/year nationwide. Enzymes recycled with fungal-treated water from stillage could further save $60 million in value per year. The potential revenue from high-quality livestock feed production, along with expanding the DDG market, is expected to be worth another $400 million/year. Preliminary cost estimates of implementing a fungal stillage treatment process indicate that the amortization and operational costs of the fungal fermentors and separation equipment amount to about 50% of the savings and additional income. The value of the energy savings, fungal biomass as livestock feed, and enzymes produced is estimated at 20 ¢/gal ethanol, with half of that required in alternative processing costs. Considering all these cost figures, it is estimated that the value added for the ethanol industry would be $0.6 billion per year. The fungal biomass is also an ideal source of the nutraceuticals chitin/chitosan, constituting 5-9% of the biomass, traditionally obtained from crustaceans at a cost of about $8,000 per ton. These have been demonstrated to im- prove animal growth and health and eliminate the need for antibiotics. This would lead to healthier meat products. Industrial implementation of fungal treatment of stillage will lead to job creation and improved rural prosperity.

Research quality

The concept has a patent pending and publications were submitted to top journals in the field.