Under industrial fermentation conditions, microorganisms face many physiological and non-physiological stresses, including Acid Stress, high-temperature stress, inhibitor stress, hyperosmotic stress and oxidative stress. To keep a production environment good for microbial fermentation, people often make adjustments. They add neutralizers, cool things down, or use other methods. So, during production, a hyperosmotic environment may form or energy use may increase.
For example, making organic acids and amino acids can create an acidic environment. This environment is bad for cell growth and for keeping normal metabolic activity. To keep production conditions stable, people usually use external neutralizers to adjust pH. In industrial production, they add alkaline substances like ammonia water and liquid ammonia. This controls the pH within a suitable range and mitigates Acid Stress.
After fermentation, people want to get amino acid and organic acid products easily. So they add concentrated sulfuric acid or other acids. This lowers the pH of the fermentation broth to 3.5 or even lower. If we can make the acid tolerance of amino acid or organic acid production strains better, fermentation can happen in an acidic environment, reducing Acid Stress. This can cut down the use of acids and alkalis a lot. It works for both fermentation and product separation. Also, because the production strains are more acid-tolerant, the final yield may get higher.
Besides, keeping fermentation pH relatively low helps stop miscellaneous bacteria from contaminating. So, making industrial microorganisms more able to handle environmental stress can keep cells active under stress. This not only makes production more efficient but also uses less energy. It also reduces pollutant emissions. This helps build a resource-saving and environment-friendly industry.
01 Types and Impacts of Acid Stress
Many types of acids move in and out of cells. They use passive transport or active transport. When acid builds up more than a certain limit, acid stress happens. Acid stress affects the concentration of protons and anions inside and outside cells. This damages cell membranes, ribosomal RNA, DNA and active enzymes. So, it disrupts the normal growth and metabolism of microorganisms.
Common acidic substances include acetic acid, formic acid, lactic acid, butyric acid, citric acid, malic acid, fumaric acid, succinic acid, amino acids (like aspartic acid and glutamic acid) and propionic acid. They also include acids added from outside. For example, benzoic acid and sorbic acid are used as preservatives.
At the cellular level, acid stress affects microorganisms in three ways. First, the transmembrane ΔpH decreases. This makes the transmembrane proton motive force (ΔP) go down. So, the cell membrane becomes uncoupled. Second, too many protons build up inside cells. This causes depurination and damages DNA. Genomic DNA from E. coli grown in low pH conditions has more strand breaks. Third, the pH inside cells drops. This badly affects enzymes that only work at normal pH. So, acid stress stops microorganisms from growing and metabolizing normally.
02 Tolerance Mechanisms to Acid Stress
Different microorganisms have developed their own ways to handle acid stress over long evolution. These ways mainly include: using F1F0-ATPase and molecular efflux pumps to push protons or acids out of cells; reducing intracellular protons by using up protons and making ammonia compounds; repairing and preventing damage to biological macromolecules from acids; changing how the cell wall and membrane let protons through; and controlling the whole system at a global level.
Microorganisms also use different tolerance mechanisms for different acids. When they face strong acids, they adjust intracellular pH quickly. They use internal buffering and ion flows. Transcription-level regulation can’t respond fast under strong acid conditions. When microorganisms face weak acids, things are more complex. Weak acids dissociate slower than strong acids. The specific physical and chemical properties of different weak acids, and the surrounding pH, have complex effects on different microorganisms.
For example, to deal with acid stress from amino acids, E. coli usually reduces intracellular proton concentration. It uses decarboxylation or deamination. Lactic acid bacteria use lactate-malate antiporters. These pumps push lactic acid out of cells. Studies on yeast’s acetic acid tolerance mechanism have found related regulatory mechanisms. These include acetic acid transport, cell wall remodeling and transcription factor regulation.
03 Modification Approaches for Microbial Acid Stress Tolerance
Currently, industrial fermentation strains are only a small part of all strains in nature. So, finding strains from nature that can be used in industrial production is still a potential method.
For acid stress tolerance, one way is to look for acidophilic strains that can be used in industry. Another way is to find strains with strong acid resistance from “contaminated” strains. Miscellaneous bacteria around specific fermentation environments are more able to handle environmental stresses.
Traditional strain modification methods include mutagenesis breeding, domestication, hybridization and protoplast fusion. These methods have something in common. They cause random mutations or recombination in the genome. They use acid stress as a screening tool. Finally, they get strains with better acid tolerance.
Using traditional strain modification methods has some downsides. The modification process is quite random. The screening process takes time and effort. But the strains usually become more acid-resistant by improving one of the acid stress tolerance mechanisms.
In recent years, research on the acid stress tolerance mechanisms of various microorganisms has deepened. Modifying industrial microorganisms with genetic engineering, metabolic engineering and other technologies has developed quickly. By understanding microbial acid stress tolerance mechanisms, people can improve acid tolerance of strains to some extent. They do this by overexpressing or knocking out key genes related to these mechanisms.
But simple genetic engineering and metabolic engineering operations are hard to get ideal results for traits controlled by multiple genes. Also, overexpressing or knocking out only a few genes may disrupt the metabolic network.
So, the future direction and focus will be using systems biology and synthetic biology methods. These methods help analyze biological systems systematically. They do quantitative and qualitative analysis at a global level. They use a design idea of assembly based on sequence modularization and componentization. They also combine several acid stress tolerance mechanisms. This allows systematic and programmed modification of industrial microorganisms at a global level.
Currently, many research groups at home and abroad have started to collect and summarize regulatory elements. These elements respond to acid stress. They also collect functional elements that enhance acid resistance from different microorganisms. People design and build these elements into acid-resistant devices artificially. They use systems biology techniques to evaluate acid resistance and chassis adaptability. They test them in model chassis microorganisms and industrial chassis microorganisms. Finally, they will form a standardized library of acid-resistant components. This will provide important guidance for studying strain acid resistance and modification methods.
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