decrease in ethanol production (data not shown). This is reasonable,
since lactic acid is a stronger acid (pKa = 3.86) than acetic acid
(pKa = 4.75). Results suggest that acetic acid may be a more useful
indicator of harmful bacterial contamination than lactic acid or
reduced pH.
Contaminants were further analyzed based on their glucose
fermentation patterns. Facultatively heterofermentive isolates
produced lower levels of acetic acid than did obligately heterofermentative
isolates, and acetic acid production was correlated with
lactic acid production only in obligately heterofermentative strains
(Fig. 5A). Only 3% of total contaminants were obligately homofermentative
species, and as expected, none of these produced significant
levels of acetic acid. Obligately heterofermentative isolates
were most inhibitory to ethanol production, and this inhibition
was moderately correlated with acetic acid production (R2 = 0.50,
Fig. 5B).
This pattern was consistent among the four major contaminant
strains. Isolates of the facultatively heterofermentative species
L. casei and L. plantarum isolates generally produced lower levels
of acetic acid and did not strongly inhibit ethanol production
(Fig. 6A and D). In contrast, isolates of the obligately heterofermentative
species L. fermentum and L. mucosae produced higher levels
of acetic acid (Fig. 6B and C). Inhibition of ethanol production
was strongly correlated with acetic acid production by
L. fermentum (R2 = 0.77) and moderately correlated with acetic acid
production by L. mucosae (R2 = 0.50).
Although production facilities routinely monitor lactic and
acetic acid concentrations as a measure of microbial contamination,
the role of organic acids in ethanol inhibition is unclear.
Studies have shown that added lactic or acetic acid can inhibit
ethanol production under certain conditions (Narendranath et al.,
2001; Graves et al., 2006). Basso et al. (2014) reported that heterofermentative
lactobacilli were more deleterious to yeast than
homofermentative strains. Graves et al. (2007) reported that lactic
and acetic acids act synergistically to inhibit ethanol production.
Among the most abundant contaminants in the current study,
the facultatively heterofermentative species L. plantarum and
L. casei generally produced lower amounts of acetic acid and were
less inhibitory to ethanol production, while the obligately heterofermentative
species L. fermentum and L. mucosae produced higher
levels of acetic acid and were more inhibitory to ethanol production.
Since the biofuel production facility was considered healthy
and productive throughout the study period, this suggests that
potentially inhibitory contaminants are always present in a normal,
healthy microflora. It is possible that stuck fermentations
occur when the balance of this microflora is upset by unknown
factors. Parallels exist in animal and human pathology, and in this
sense the fermentation should perhaps be viewed as a living
organism rather than as a chemical factory.
In terms of practical control of contaminants, results suggest
that it may be important to maintain a desirable balance of
innocuous lactic acid bacteria, and particularly of facultatively