2. Effect of Associated Bacteria on the Growth and 2-KGA Production of K. vulgare
2.1. Fermentation Process
In the second step of the two-step fermentation method,
K. vulgare and associated bacteria are usually prepared into mixed bacterial seeds through cocultivation and then inoculated into the fermentation medium
[24][51]. It has been shown that the centrifugation supernatant and cytoplasmic matrix of
B. megaterium can promote the growth of
K. vulgare, and the presence of
B. megaterium significantly shortens the lag time of
K. vulgare [25][52]. At this stage, the associated bacteria grow rapidly, and the biomass of
K. vulgare increases slowly, producing only a small amount of 2-KGA
[24][26][48,51].
The coculture fermentation medium usually includes L-sorbose, corn steep liquor, urea, potassium dihydrogen phosphate, magnesium sulfate, and calcium carbonate
[10][27][10,53]. Given that the associated bacteria cannot use L-sorbose, corn steep liquor is the only energy source for the associated bacteria, besides providing carbon sources, nitrogen sources, and growth factors. With the progress of fermentation, the nutrient components in the medium are rapidly consumed, leading to a lack of nutrition and especially energy. Poor nutritional conditions lead to the formation of spores by
Bacillus [26][28][48,54]. Subsequently,
Bacillus releases spores, which are accompanied by cell lysis, and the associated bacteria that cannot form spores undergo autolysis. As a result, peptides, proteins, purines, pyrimidines, and small molecules from the cytoplasm are released, which provides new nutrients for
K. vulgare. From this moment on,
K. vulgare enters the stage of rapid growth and rapid transformation of L-sorbose to 2-KGA
[24][51].
The associated bacteria quickly absorb nutrients from the culture medium, except for L-sorbose, and synthesize the nutrients required for their own growth. Subsequently, the associated bacteria release new components and ratios of nutrients through secretion, release of spores, or cell lysis, which are utilized by
K. vulgare. This phenomenon of sequential conversion and reutilization of substrates is common in cocultivation systems, such as the process of cellulose-to-ethanol transformation in the cocultivation of cellulose-decomposing microorganisms and ethanol-producing microorganisms
[17]. Nutrients with new compositions and proportions are released by associated bacteria, stimulating the growth of
K. vulgare and the production of 2-KGA.
2.2. Supplementation of Key Substance
Although
K. vulgare has defects in amino acid, vitamin, coenzyme, and sulfur metabolism, it has a strong resource utilization and transportation system. In the GSMM of
K. vulgare WSH-001, 103 genes were found responsible for the transport of exogenous polypeptides, and about 58 genes encoded aminopeptidases or peptidases that could hydrolyze polypeptides into amino acids
[29][35]. Maximizing the use of nutrient elements in the environment is the way for many auxotrophic strains to survive in nature, and this is why associated bacteria can promote the growth and 2-KGA production of
K. vulgare.
On the soft agar medium plate, the increase in amino acid concentration in the medium around
K. vulgare colonies attracted
B. megaterium to move and aggregate to
K. vulgare; erythrose, erythritol, avian purines, and inositol were depleted by
K. vulgare; and 2-KGA content in agar increased dramatically
[28][54]. The same movement phenomenon was found when
K. vulgare was cocultured with
B. thuringiensis. Erythrose, erythritol, guanine, and inositol accumulated around
B. thuringiensis were consumed by
K. vulgare, and the production of 2-KGA increased sharply
[30][55]. Additionally,
B. megaterium WSH002 can secrete pantothenic acid and L-cysteine, which may be two potential growth promoters of
K. vulgare because they are precursors of CoA
[31][50]. Other studies have also shown that the adenine, guanine, and hypoxanthine required for
K. vulgare in the middle stage of fermentation probably come from the decomposition of the associated bacteria
[32][56].
In addition to the small-molecule metabolites, the associated bacteria also secrete proteins that can promote the growth and 2-KGA production of
K. vulgare.
B. megaterium secretes two different proteins, with molecular weights of 30–50 kDa and > 100 kDa, which can promote the growth of
K. vulgare and increase the production of 2-KGA
[33][37]. However, the specific protein and the mechanism by which it promotes
K. vulgare growth and 2-KGA production have not yet been isolated and studied.
Reasonable utilization of material exchange between microorganisms can improve the biomass and product yield of coculture systems. For example, in the nitrogen- and fatty acid-limiting environment of water kefir, the presence of
Saccharomyces cerevisiae improves the growth of
Lactobacillus hordei by providing gluconate, fructose, amino acids, fatty acids, etc.
[34][57]. Yeast utilizes oxygen respiration to produce carbon dioxide, while microalgae convert carbon dioxide into lipids and oxygen through photosynthesis
[35][58]. Coculture of
Rhodotorula glutinis and
Scenedesmus obliquus in a photobioreactor increases the fermentation biomass by 40–50% and total lipids by 60–70%.
[36][59]. However, in the continuous transformation and reutilization process of substrates, microorganisms that first utilize these substances will consume a portion for growth and metabolism; reducing these losses can improve the conversion efficiency of substrates. Similarly, in the coculture process of
Bacillus and
K. vulgare, a considerable part of the limited nutrients in the medium is locked in the spores. It has been shown that using lysozyme to lyse
B. megaterium releases more intracellular substances than spore formation; the growth rate of
K. vulgare increases by 27.4%, and the productivity of 2-KGA increases by 28.2%.
[25][52]. Second, in the fermentation stage after the formation of spores, most of the nutrients for
K. vulgare growth are provided by the lysate produced during the process of spore release by
Bacillus, which leads to the problem that the substances released after spore decomposition may not completely meet the needs of
K. vulgare. For example, in the coculture system with
B. megaterium as associated bacteria,
B. megaterium forms spores within 7–22 h, but the extracellular contents of L-proline, L-cysteine, L-valine, L-phenylalanine, and L-arginine remain 0 during 10–70 h of fermentation
[37][60]. This indicates that these nutrients are leaked at low concentrations or that they are quickly absorbed and utilized by
K. vulgare during spore release, and nutrients such as L-cysteine and L-proline, which cannot be synthesized by
K. vulgare itself, may be factors limiting
K. vulgare’s growth and 2-KGA production at this time.
2.3. Alleviation of Oxidative Pressure in Fermentation Systems
Autoxidation of flavin dehydrogenase
[38][39][61,62] and non-respiratory flavoproteins in some metabolic pathways
[40][41][42][63,64,65] can generate reactive oxygen species (ROS), including superoxide anion, hydrogen peroxide, hydroxyl radicals, and nitric oxide. ROS are natural products of oxygen metabolism in aerobic organisms. Excessive levels of ROS in cells cause oxidative stress, which results in DNA damage, cell membrane peroxidation, inactivation of enzymes and cofactors, and eventually cell death
[43][66]. The presence of cytochrome c in
K. vulgare couples the production of 2-KGA with the respiratory chain
[44][27], and ROS inhibits the growth of
K. vulgare and the production of 2-KGA
[45][67]. Except for the conversion process of L-sorbose to 2-KGA and other metabolic activities that produce a lot of ROS, 2-KGA as the target product also causes oxidative stress in
K. vulgare [46][47][68,69].
To combat the ROS-induced damage to cells, biological organisms have developed a series of enzymes that can eliminate ROS, including superoxide dismutase, catalase, and peroxidase. The associated bacteria can induce the upregulation of these ROS-scavenging enzymes. For example,
S. cerevisiae induces upregulation of superoxide dismutase, catalase, and oxidative stress–related genes (
sod,
cat, and
gpd), thereby leading to increased 2-KGA production by
K. vulgare [13]. Proteomic studies have shown that the enzymes in the microbial consortium that maintain the metabolic reduction environment of cells, including superoxide dismutase, glutathione S-transferase, NADPH: quinone oxidoreductase, and glucose-6-phosphate dehydrogenase, reach higher levels of expression during 18–23 h of fermentation
[32][56].
Some small-molecular substances released by the associated bacteria may also help
K. vulgare resist ROS. Proline can directly eliminate ROS and may also protect the stability of a variety of antioxidant enzymes
[48][70]. L-cysteine can be used to synthesize GSH—the most important antioxidant in cells. Pyridoxine can quench ROS
[49][71]. Vitamin C can alleviate oxidative stress in the environment and promote the growth and 2-KGA production of
K. vulgare. Hence, using
Saccharomyces cerevicae VTC2, which is genetically engineered to produce vitamin C from D-glucose, as an associated bacteria could increase 2-KGA production by 25% compared to the original strain
[13]. Adenine, guanine, xanthine, and hypoxanthine produced by the associated bacteria may contribute to
K. vulgare’s resistance to ROS
[32][56]. Furthermore, the effect of
B. megaterium–engineered bacteria that cannot form spores in the coculture system is weakened, which may be due to the absence or reduction of antioxidant substances called sporulenes, produced during the sporulation stage of
B. megaterium [50][51][72,73].
Siderophores are secreted by microorganisms such as bacteria and fungi to obtain iron from the environment, and they can efficiently combine with low-molecular-weight substances of iron in the surrounding environment
[52][53][74,75]. Most aerobic and facultative anaerobic microorganisms can synthesize at least one siderophore, and microorganisms growing under aerobic conditions require iron for a variety of functions, including the reduction of oxygen for ATP synthesis, the reduction of nucleoside precursors of DNA, heme formation, and other basic purposes
[54][76]. It has been shown that
K. vulgare cannot synthesize siderophores but has a siderophore-absorption system, and the addition of 500 µg/L
Bacillus pumilus SY-A9 siderophores increases the titer of 2-KGA by 71.45%
[12]. Further research has shown that siderophores cause overexpression of iron absorption system–related genes, electron transfer chain–related genes, ATP synthase–related genes, antioxidant enzyme–related genes, and 2-KGA production enzyme–related factors in
K. vulgare 25B-1, thereby reducing oxidative stress and ensuring energy metabolism
[12].
Despite the fact that the associated bacteria play a significant role in helping
K. vulgare resist oxidation, the addition of antioxidant substances can further alleviate the oxidative stress of the fermentation system and improve the yield of 2-KGA. The addition of glutathione and oxidized glutathione with a concentration ratio of 50:1 in the fermentation system of
B. endophyticus ST-1 as the associated bacteria increases the activities of total antioxidant capacity (t-AOC), total superoxide dismutase (T-SOD), and catalase (CAT) in the fermentation system and upregulates the expression of genes related to superoxide dismutase, such as
sod,
gst,
gr,
zwf, and
gp, thereby eliminating oxidative stress, improving 2-KGA production, and shortening fermentation time
[45][67]. Moreover, during the period when
K. vulgare produces ROS due to the rapid dehydrogenation of L-sorbose to 2-KGA, the associated bacteria usually form spores or cell lysates. The antioxidant substances provided by associated bacteria lack a sustained supply during the high-speed production period of 2-KGA, so manual intervention to alleviate the oxidative pressure of
K. vulgare in the later stage of fermentation may further improve the fermentation intensity.