Production of acids from a large number of sugars, sugar polymers, and sugar
alcohols (1% w/v each) in otherwise identical media composition was studied with
four Bacillus strains – B. circulans var. alkalophilus (facultative alkaliphile),
B. alkalophilus spp. halodurans, and Bacillus sp.17-1 – all of which were able to
grow also in neutral pH. The fourth B. alcalophilus was an obligate alkaliphile
[5]. The acids were studied with different chromatographic techniques together with
acid-base titrations to assess total acid and basic compounds. In general, all the
alkaliphiles produced acetic acid (4.5–5 g/L at maximum). Among the tested
bacteria, formic acid was produced only by B. circulans var. alkalophilus (up to
2 g/L). Acetoin, butanediol, or ethanol were not detected, which is a difference to
neutrophilic Bacillus species. The studied alkaliphiles differed from each other in
amounts of production of less common branched-chain organic acids. They were
probably generated through amino acid metabolism. Since the amino acid source
was the same, the acid composition reflects differences in the amino acid metabolism. The high pH and high buffering capacity of the medium favored the formation
of acids and oxoacids. At low pH, hydroxy acids seem to be advantageous. At high
pH, the protons formed via NAD(P) from substrates can be easily secreted to
medium, while at low pH, it is easier to transfer protons to form hydroxy acids
(like lactate) to avoid the over-acidification [5]. The keto acids are useful compounds
for biomaterials since they contain the reactive carbonyl groups.
Alkaliphiles have exceptionally effective respiratory chain. Non-alkaliphilic
Bacillus mutants have less cytochromes and cytochrome species than the
corresponding alkaliphilic cells [6, 7]. NADH oxidase activity (nmol/min/mL) was
5–20 times higher than the activities of the key enzymes in the glycolysis pathway
[2]. An interesting feature of alkaliphiles is their ability to fluctuate the medium pH
when grown in carbohydrate-containing media [8]. In standard experiments, the
starting pH of growth was 10.3. During the logarithmic growth, the pH dropped
sharply to 8 and then slowly increased back to 9.5 even with no growth (even a
decrease in turbidity measured at A 600nm ; Fig. 1). This phenomenon has been often
neglected even if it is likely to be typical to alkaliphiles. It is tempting to explain that
the pH drop during the growth in sugar media originates from secretion of organic
acids into medium, while the pH increase could be uptaking the acids (i.e., change
the catabolism from sugars to acids). The first evidence against that explanation was
that TCA cycle was functioning very slowly with the studied alkaliphilic Bacillus
sp. Moreover, the microbe grew very poorly in 1% acetate, glycerol, or citrate.
Because of an obvious conflict against previous conception for the origin of the pH
changes, the growth was studied with accurate sampling and measurement of acids.
To get accurate timing, an automatic sampling device was developed [8, 9]. During
the middle of the pH drop, there occurred a sharp peak in reducing sugars with starch
substrate. Maximum of secreted acetic acid appeared into the medium 3–5 h later
than the pH minimum (Fig. 1). It is possible to speculate that the microbe produced
also other than acetic and formic acids. Back-titration curve of the cultivation
medium to original starting pH, however, showed similar timing profile as the pH
drop curve. It was possible to trace all carbon in acetic acid, formic acid, and CO 2
and protons, i.e., to make credible total inventory of the sugar metabolism. The slow
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E. Khalikova et al.
alcohols (1% w/v each) in otherwise identical media composition was studied with
four Bacillus strains – B. circulans var. alkalophilus (facultative alkaliphile),
B. alkalophilus spp. halodurans, and Bacillus sp.17-1 – all of which were able to
grow also in neutral pH. The fourth B. alcalophilus was an obligate alkaliphile
[5]. The acids were studied with different chromatographic techniques together with
acid-base titrations to assess total acid and basic compounds. In general, all the
alkaliphiles produced acetic acid (4.5–5 g/L at maximum). Among the tested
bacteria, formic acid was produced only by B. circulans var. alkalophilus (up to
2 g/L). Acetoin, butanediol, or ethanol were not detected, which is a difference to
neutrophilic Bacillus species. The studied alkaliphiles differed from each other in
amounts of production of less common branched-chain organic acids. They were
probably generated through amino acid metabolism. Since the amino acid source
was the same, the acid composition reflects differences in the amino acid metabolism. The high pH and high buffering capacity of the medium favored the formation
of acids and oxoacids. At low pH, hydroxy acids seem to be advantageous. At high
pH, the protons formed via NAD(P) from substrates can be easily secreted to
medium, while at low pH, it is easier to transfer protons to form hydroxy acids
(like lactate) to avoid the over-acidification [5]. The keto acids are useful compounds
for biomaterials since they contain the reactive carbonyl groups.
Alkaliphiles have exceptionally effective respiratory chain. Non-alkaliphilic
Bacillus mutants have less cytochromes and cytochrome species than the
corresponding alkaliphilic cells [6, 7]. NADH oxidase activity (nmol/min/mL) was
5–20 times higher than the activities of the key enzymes in the glycolysis pathway
[2]. An interesting feature of alkaliphiles is their ability to fluctuate the medium pH
when grown in carbohydrate-containing media [8]. In standard experiments, the
starting pH of growth was 10.3. During the logarithmic growth, the pH dropped
sharply to 8 and then slowly increased back to 9.5 even with no growth (even a
decrease in turbidity measured at A 600nm ; Fig. 1). This phenomenon has been often
neglected even if it is likely to be typical to alkaliphiles. It is tempting to explain that
the pH drop during the growth in sugar media originates from secretion of organic
acids into medium, while the pH increase could be uptaking the acids (i.e., change
the catabolism from sugars to acids). The first evidence against that explanation was
that TCA cycle was functioning very slowly with the studied alkaliphilic Bacillus
sp. Moreover, the microbe grew very poorly in 1% acetate, glycerol, or citrate.
Because of an obvious conflict against previous conception for the origin of the pH
changes, the growth was studied with accurate sampling and measurement of acids.
To get accurate timing, an automatic sampling device was developed [8, 9]. During
the middle of the pH drop, there occurred a sharp peak in reducing sugars with starch
substrate. Maximum of secreted acetic acid appeared into the medium 3–5 h later
than the pH minimum (Fig. 1). It is possible to speculate that the microbe produced
also other than acetic and formic acids. Back-titration curve of the cultivation
medium to original starting pH, however, showed similar timing profile as the pH
drop curve. It was possible to trace all carbon in acetic acid, formic acid, and CO 2
and protons, i.e., to make credible total inventory of the sugar metabolism. The slow
162
E. Khalikova et al.