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Bio-based Product Research

Filamentous Fungal Capabilities

Filamentous Fungal Discovery Research

We have assembled a collection of filamentous fungi from the three major groups of true fungi: Basidiomycota, Ascomycota and Zygomycota. Almost all of the strains in the collection have been screened for organic acid production using a novel high-throughput-screening technique based on the 96 well format. This new technique greatly accelerated the screening of filamentous fungi for organic acid production and shows that many fungi exude organic acids of interest in bioproduct research. Several fungal strains from the PNNL collection produced organic acids of interest at promising concentrations in the absence of contaminating organic acid co-products. These strains are being assessed as candidates for bioprocesses. The fungal culture collection will be queried again for hemicellulase enzyme activity; this second screening will take advantage of new information about the substrate utilization characteristics of the fungi in the collection resulting from the organic acid screening to refine the query for the hemicellulases. Thus, each query of the collection adds valuable information that can be used to increase the speed of subsequent screenings and the likelihood of their success.

In addition, a survey of the diversity of fungi in composts and soils has resulted in the discovery of novel fungi with novel traits, as well as contributing to the scientific community at large through an estimate of the diversity of this large but understudied kingdom. The diversity of fungi in soils was even greater than expected and many of these fungi are "unculturable" or fastidious. The research indicates that the percentage of "unculturable" fungi is about 70-90%, confirming the hypothesis that the fungal kingdom represents an untapped wealth of microbial diversity. Details: Fungal Discovery (.pdf, 135Kb)

Fungal Bioprocess Research

We have established methods to evaluate and manipulate parameters necessary to create novel fermentation processes utilizing filamentous fungi and to ensure that laboratory-developed techniques are relevant to industrial processes. This integrated capability is necessary to develop novel bioprocessing techniques utilizing fungal organisms to convert biomass into industrial and energy products.

Our hypothesis is that discovery and bioprocess research using microorganisms to convert large amounts of biomass derived carbon into valuable products is most efficiently accomplished by initial screening in milliliter volumes in micro-plates, confirmation in shake flasks and then evaluation in laboratory bioreactors. Basic protocols have been developed for using the "bioreactor instruments" that are available for the further study of fungi identified as having potentially useful properties. Fungi are extremely diverse, but the major taxonomic groups have similarities so that basic techniques for inoculation, biomass estimation, agitation, pH control, foaming and aeration control are generally applicable.

The two general types of bioreactors commonly used for commercial bioprocesses, the airlift fermentor and the aerated stirred tank fermentor, are being employed. Fungal organisms from each of the three major taxonomic groups have been evaluated in the bioreactors. A citric acid producing strain of Aspergillus niger was studied. The design research for a process to evaluate engineered strains of A. niger has been completed. Preliminary research on use of the stirred tank bioreactors has been performed using a Zygomycete (Rhizopus), a Basidiomycete (Phanerochaete) and Ascomycetes/Deuteromycetes (Trichoderma and Aspergillus).

Significant modification or reconfiguration of bioreactor vessels and associated hardware is often required to grow filamentous fungi and considerable effort is often required to define the exact process parameters necessary to achieve the optimal production conditions for these organisms. Once defined, these processes have significant advantages relative to those using common single-cell organisms. New fungi capable of synthesizing desired products and potentially useful at a manufacturing scale can be evaluated in lab-scale bioreactors that simulate the airlift and/or the stirred tank designs. Commercial implementation of a new fungus and new bioprocesses can proceed rapidly because we are using equipment and procedures that model typical conditions in the production plant. Details: Bioprocess Development (.pdf, 162Kb)

The establishment of an Applied Biotechnology Laboratory is an ongoing process; including completing the establishment of standard methods and performing experiments in the 1.25L and 2.5L New Brunswick fermentors. To model airlift processes, a bubble column was acquired and validated, and the initial process design established for an A. niger citric process. The predicted difficulty of obtaining data and adequately controlling all parameters in small vessels using filamentous fungi was confirmed. The installation of additional capacity is ongoing, including a 30 Liter B.Braun fermentor, an important instrument in the Bioprocess laboratory for work with filamentous fungi.

In bioprocess research laboratories, iterative experiments in shake flasks, small fermentors, and process prototype fermentors are employed to establish a new process. One of the significant bottlenecks in this standard approach to bioprocess research is the requirement for large numbers of repetitive experiments using batch fermentation technology. Usually only one variable can be examined in each experiment and large numbers of shake flask experiments are often performed in parallel with the batch fermentor experiments and the time limiting factor is labor and availability of equipment. An alternative to this standard approach is to use continuous culture technology, often referred to as chemostat or auxostat technology. At PNNL we are establishing chemostat technology to complete the set of tools required to perform state-of-the-art fungal research.

For rapid progress toward novel processes and determination of optimum operating conditions, we hypothesize that performing optimization investigations using continuous culture (chemostat technology) will reduce new product/process development time. Support for this hypothesis is being sought by applying it to novel organisms found in a previous screening project. It is further hypothesized that proteomics research will be greatly improved using chemostat technology. A fungus grown in a chemostat can be examined under conditions of balanced growth. Having achieved a steady state, critical factors hypothesized to affect the proteome can be assessed by sampling the steady state, changing the parameter and sampling again. Finally, chemostat technology is further hypothesized to be useful in applying the principles of quantitative genetics to improve desired characteristics of the target fungus using selective pressure.

This work includes preparation of detailed process descriptions. These include the research data and a preliminary process design documenting optimal fermentation conditions, media composition, inoculation strategies and initial separation of the final products.

Filamentous Fungal Genetics Research

The morphology of filamentous fungi in manufacturing processes is critical to optimal product output. Current commercial fungal fermentation processes for citric acid, glucoamylase, microbial rennet, penicillin, and the statins produce tens to hundreds of grams per liter of product. Often the highest productivity is observed when the fungus exhibits a specific morphology. When filamentous fungi are growing in a mycelial mat (i.e., webs of hyphae, or filaments) it is the hyphal tips that extend out into richer nutrient conditions. These actively growing hyphal tips are where the majority of the metabolic activity occurs. It is our hypothesis that under a specific set of physical and chemical conditions that the optimal morphology is observed (e.g., agitation, high oxygen and carbon source concentration and proper concentrations of other critical nutrients). It is logical to suppose that there are genes responsible for regulating and maintaining this optimal morphology. If these genes can be identified, they would have tremendous potential for engineering fungi to generate large amounts of product. For example, a novel fungal strain selected from our screening program for the ability to produce small amounts of a desired product (mg/liter) may be converted to a production organism capable of producing grams per liter of the desired product without the investment of decades of research required in typical strain improvement strategies.

Bio-based Product Research

PNNL Information