Nitrogen-Fixing Fermentation for L-Glutamic Acid Without Haber-Bosch
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Solution Overview
Problem
Existing methods for producing L-glutamic acid rely on the Haber-Bosch process, which is energy-intensive, and there is a need for a more efficient method that maximizes the nitrogen fixing ability of nitrogen-fixing bacteria to convert atmospheric nitrogen into ammonia for L-glutamic acid production.
Innovation Solution
Culturing nitrogen-fixing bacteria with introduced genes for citrate synthase, 2-methylcitrate synthase, and citrate transporter in a nitrogen-limited medium containing organic acids like citric acid, allowing them to produce L-glutamic acid or its salt from atmospheric nitrogen molecules.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If nitrogen sources (ammonium salts, nitrates, peptones) are present in the environment, then nitrogen-fixing bacteria can grow rapidly, but nitrogen fixing ability is suppressed
Solution Approach 1:
The patent applies preliminary action by pre-culturing nitrogen-fixing bacteria in a nitrogen-free medium to deplete endogenous nitrogen reserves and activate nitrogenase expression before transferring to production medium. This preliminary cultivation step ensures the bacteria are in a nitrogen-starved state, maximizing their nitrogen fixing ability for subsequent L-glutamic acid production.
Solution Approach 2:
The patent utilizes parameter changes by precisely controlling nitrogen concentration in the culture medium. The nitrogen content is maintained at specific low levels (0-50 mg/L as N) to suppress assimilation pathways while allowing nitrogen fixation. This parameter optimization resolves the contradiction by creating conditions where bacteria must fix atmospheric nitrogen rather than assimilating readily available nitrogen sources.
2Object-generated harmful factors
If nitrogen-free medium is used to maximize nitrogen fixing ability, then atmospheric nitrogen can be converted to ammonia, but bacterial growth is limited
Solution Approach 1:
The patent applies partial action by providing trace amounts of nitrogen (0-50 mg/L) in the culture medium - enough to support minimal bacterial growth and metabolism, but insufficient to trigger nitrogen assimilation pathways. This partial nitrogen supplementation allows simultaneous achievement of bacterial proliferation and nitrogen fixing ability activation.
Solution Approach 2:
The patent uses preliminary action through a two-stage cultivation process: first growing bacteria in nitrogen-free medium to activate nitrogenase, then transferring to production medium with controlled nitrogen levels. This preliminary adaptation ensures bacteria maintain nitrogen fixing capability while achieving sufficient biomass for productive L-glutamic acid synthesis.
3Quantity of substance
If conventional Haber-Bosch process is used to produce ammonia, then sufficient nitrogen source is available for L-glutamic acid production, but energy consumption is high
Solution Approach 1:
The patent applies self-service by enabling the bacteria to produce their own ammonia through nitrogen fixation using atmospheric nitrogen as the sole nitrogen source. The nitrogen-fixing bacteria autonomously convert N2 to ammonia in situ, eliminating the need for external ammonia addition and the energy-intensive Haber-Bosch process. This self-sufficient approach simultaneously provides sufficient nitrogen and reduces energy consumption.
Solution Approach 2:
The patent uses nitrogen-fixing bacteria as an intermediary system that bridges atmospheric nitrogen and L-glutamic acid production. Instead of directly producing ammonia via Haber-Bosch, the bacteria serve as a biological mediator that converts atmospheric nitrogen to bioavailable ammonia, which is then assimilated into L-glutamic acid. This intermediary approach reduces energy consumption while ensuring adequate nitrogen supply.
4Ease of operation
If nitrogen-fixing bacteria are cultured in static conditions, then simple cultivation is possible, but nitrogen fixing ability is not maximally exhibited
Solution Approach 1:
The patent applies parameter changes by optimizing specific cultivation parameters including nitrogen concentration (0-50 mg/L), pH (6.5-7.5), temperature (25-37°C), and dissolved oxygen levels. These parameter optimizations enable nitrogen-fixing bacteria to exhibit maximum nitrogen fixing ability under controlled yet operationally simple conditions, resolving the contradiction between ease of operation and nitrogen fixing performance.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This method enables the production of L-glutamic acid without relying on the Haber-Bosch process, enhancing nitrogen fixing ability and increasing L-glutamic acid yield, while utilizing nitrogen molecules from the atmosphere.
Implementation Method 1
Certain microorganisms have the ability to convert nitrogen molecules in the atmosphere to ammonia. Such ability is referred to as nitrogen fixing ability.
Implementation Method 2
The enzymes involved in nitrogen fixiation are nitrogenases, which catalyze the following reaction under ideal reacting conditions in vitro.
Implementation Method 3
Method for fermentatively producing l-glutamic acid derived from nitrogen molecule
Data Source
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AI summary
The purpose of the present invention is to develop a method for fermentatively producing L-glutamic acid based on a nitrogen molecule in air using nitrogen-fixing bacteria. A culture method, by which a state in which the nitrogen-fixing bacteria have enhanced nitrogen fixation ability is maintained, has been developed. The culture method is characterized by containing an organic acid such as citric acid in a nitrogen-limited medium put in an non-hermetic vessel. A method for fermentatively producing L-glutamic acid dependent on nitrogen fixation could be developed by culturing genetically modified strains, which highly express a citric acid synthase gene and a citric acid transporter gene, by means of the culture method. A method for fermentatively producing L-glutamic acid dependent on nitrogen fixation could be developed by using modified strains, which highly express a citric acid synthase gene or a 2-methylcitric acid synthase gene, in the process to reach the aforementioned step. The present invention pertains to a production method and a culture method for such microorganisms.