Pigments production and cell growth at pH 6.5 with
different nitrogen sources At pH 6.5 both cell growth and
pigments production were significantly inhibited independently to
the nitrogen source employed, especially when nitrate was used.
Because of the extremely low DCW and pigments yield for nitrate,
the data were not shown. The maximum DCW for ammonium and
peptone were approximately 4.5 g/L in both cases (Fig. 3A1 and
B1), corresponding to less than half of the values observed at pH
2.5 and 4.0. The yield of intracellular pigments was also much
lower, around 1/5 to 1/4 of that at pH 2.5 and 4.0 (according to
the absorbance at 410 nm). However, extracellular pigments
secretion was greatly increased and the ratios of intracellular to
extracellular pigments concentration at 132 h (according to the
absorbance at 410 nm) were approximately 5.51 and 0.36 for
ammonium and peptone, respectively (Fig. 3A2 and B2). Unlike
other fermentation conditions, for peptone at pH 6.5 the
concentrations of extracellular pigments were higher than the
intracellular pigments and the absorbance of extracellular
pigments at 510 nm reached 68.11 AU. It was noteworthy that
the absorbance spectra of intracellular and extracellular pigments
(Fig. 3A3 and B3) exhibited large differences from those recorded
at pH 2.5 and pH 4.0. When ammonium was the nitrogen source,
the absorbance spectrum of the extracellular broth presented a
maximum at 475 nm and a shoulder at about 410 nm, whereas
the intracellular extract showed two maxima at 527 nm and
413 nm, with the absorbance at 413 nm slightly higher than that
at 527 nm. The spectrum of the intracellular extract match the spectra of two red pigments previously published by Zheng et al.
(34) showing two maxima with similar absorbance at 414 nm
and 530 nm. In the same work it is reported the spectra of four
different yellow pigments with maxima centered at
386e389 nm. Therefore our data indicate that the intracellular
pigments consisted of red pigments with a relatively low amount
of yellow pigments contributing to the absorbance at 410 nm.
When peptone was the nitrogen source, the spectrum of the
extracellular broth exhibited two maxima at 419 nm and 491 nm
(Fig. 3B3), indicating the presence of different types of red
pigments derivatives. In fact it was previously reported that red
pigments derivatives conjugated with different amino acids have
similar absorbance spectra characterized by two maxima at
about 417e427 nm and 498e525 nm (12). The spectrum of the
intracellular extract, with its relatively intense maximum at
398 nm and a relatively small shoulder at 500 nm (Fig. 3A3),
suggested that intracellular pigments consist mainly of yellow
pigments and a low amount of red pigments derivatives. In fact
the maximum at 398 nm is likely the combination of the
maximum at 386e389 nm of yellow pigments observed by
Zheng et al. (34) with the maximum at 417e427 nm of red
pigments derivatives while the maximum at 498e525 nm of red
pigments derivatives contributed to the shoulder observed at
500 nm.
Interestingly, although the growth and pigments production
were greatly inhibited when nitrate was used as nitrogen source at
pH 6.5, the spectrum of the intracellular extract, with its maximum
at 471 nm, was almost identical to the spectra observed at pH 2.5
and 4.0, suggesting that, differently from what observed for
ammonium and peptone, the intracellular extract consisted mainly
of orange pigments. The spectrum of the extracellular broth presented
two peaks at 407 nm and 492 nm, suggesting that they
might mainly consist of red pigments derivatives.