DISCUSSION
Since the fermentation was carried out in unsteady state batch mode, the
rates of production and consumption changed during the runs. In such
cases, the maximum rate or average rate can be calculated and utilized
for analysis. In practice, the instantaneous maximum rate achieved is
impossible to obtain for the entire process duration. Therefore in our
case, average rates were calculated and given in Tables 1 and 2, which
in our opinion, reflect the overall performance better.
The peak amount obtained and duration until that peak time was utilized
to calculate the average production rates.
Tables 1 and 2 also reported yields and sugar recovery. Here, since
products were utilized back by the microorganism, the peak amount
obtained and duration until that peak time was utilized to calculate the
yields, based on the assumption that the batches would be stopped at
those times in an industrial process. Sugar recovery shows the
efficiency of the microorganism to convert the starch available in the
PPW to glucose.
Substrate conversion efficiency is another particularly useful parameter
for characterizing ethanol and lactic acid production reported in Tables
1 and 2.
Figures 1 and 2 show that glucose, DP4+, ethanol, and lactic acid
concentrations peak at 24 hours (8% loading rate slightly retards the
peak). This is interesting because the metabolism of filamentous fungi
is slower compared to bacteria or yeast [8,9].
The fast process is probably due to the species used, Rhizopus
spp . grow faster than other fungi [10]. Process conditions used
also affect the metabolic rate, filamentous fungi were shown to grow
faster in liquid media compared to solid media [11]. Indeed,
according to prior research [12,13] and published data [14–16],R. oryzae reaches the stationary phase after 18-24 hours,
depending on pH, temperature, the working volume of the growth medium,
and the initial spore concentration. In the log phase of growth primary
metabolites and enzymes are synthesized, one of which is amylase,
therefore DP4+ and glucose concentrations peak at this time. Lactic acid
and ethanol production follow closely.
Even though there is no other published study previously in the
literature that explores the use of PPW by R. oryzae as
thoroughly as this study, some comparable works exist that either report
utilization of other agro-wastes by R. oryzae or the utilization
of PPW by other microorganisms.
The only comparable study in the literature involved drying, powdering,
and sieving PPW similar to this work, however only a single particle
size (1.2-1.6 mm) was used. Obtained PPW was pretreated by the steam
explosion for hydrolyzation before fermentation by R. oryzae .
However, the authors reported very limited results for this process
(only the final lactic acid concentration was given as 66.5 g/L)
[17].
Another remotely comparable study again used drying, powdering, and
sieving PPW, again only a single particle size (0.211 mm) was used and
it was for the production of ethanol by yeast fermentation. The authors
concluded that the ethanol yield was not comparable with that of corn
[6].
Notable studies that deal with the utilization of agro-food waste
materials as substrates for lactic acid production by R. oryzaeare listed below.
One such study explores the use of six different types of agro-food
wastes (sapota peel, banana peel, papaya peel, potato peel, corn cob
powder, and carboxymethyl cellulose). Substrates were dried, rounded,
and screened to collect particles of the size between 1.2 and 1.6 mm and
steam explosion treated to obtain hydrolysates, which were then used for
fermentation. All six substrates supported the growth and lactic acid
productions of 66-72 g/L were obtained. The best substrate was sapota
peel with a lactic acid productivity of 1 g/L·h and a yield of 3.6 g/g
[17].
Another comparable study includes eight different agro-food wastes (rice
bran, wheat bran, ragi bran, rice starch water, tea waste, sugar cane
bagasse, groundnut, and coconut oil cakes) that were dried, pulverized,
pretreated (steam explosion, direct and acid hydrolysis) and used as a
carbon source for lactic acid fermentation. Lactic acid production was
in the range of 69-72 g/L for 100 g/L agro-waste [18].
A similar study also targeted to production of lactic acid using dry
grass, coconut husk, sugarcane waste, and wood chips as substrates.
Hydrolysates of these materials were obtained by steam and acid
hydrolysis and fed as a substrate to R. oryzae . After
fermentation, lactic acid concentrations between 34-70 g/L were obtained
[19].
Lactic acid production is not always that efficient, however.
In a study that reported bioconversion of waste office paper to lactic
acid by R. oryzae a fter enzymatic hydrolysate, the lactic acid
yield and production rate were only 0.59 g/g and 16.3 g/L·d,
respectively, which were less than glucose medium. The authors suggested
that the production rate may be inhibited by xylose derived from
hemicellulose, and the yield may be inhibited by unknown compounds
derived from paper pulp [20].
Likewise, in another study where rice straw was used to obtain lactic
acid by R. oryzae , only a small amount of lactic acid is produced
even though R. oryzae can consume glucose in rice straw-derived
hydrolysates. The inhibitory effect of polyphenols in rice straw-derived
dissolved organic matter was found to be responsible for ineffective
lactic acid production [21].
On the other hand, yield, rate, and concentrations reported in the work
by Zain et al. (2021) are comparable to this study. They used solid
pineapple waste as substrate for fermentation by R. oryzae and
under optimized conditions, calculated maximum lactic acid concentration
as 103.7 mg/g, ethanol concentrations as 0.14 mg/g, lactic acid yield as
0.45 mg/g, and average lactic acid production rate as 10.3 mg/g·day
[22].
It is interesting that usually only lactic acid production was reported,
even though R. oryzae possesses lactate dehydrogenase, pyruvate
decarboxylase, and alcohol dehydrogenase enzymes for the co-production
of ethanol and lactic acid. It is previously documented that both
metabolites are produced in defined media by R. oryzae , high
spore inoculation shifts production from lactic acid to ethanol, and
their yields increase as the initial glucose amounts increase
[23,24].
Notable examples of the utilization of PPW or other potato waste for
lactic acid and ethanol production by microorganisms other than R.
oryzae that are available in the literature are listed below.
One such study reported using PPW for ethanol production bySaccharomyces cerevisiae var. Bayanus after enzymatic hydrolysis
to obtain 7.6 g/L ethanol with a yield of 0.46 g ethanol / g sugar
[3].
Another work used undefined mixed cultures inoculated from wastewater
treatment plant sludge to ferment PPW. Lactic acid was the major
product, followed by acetic acid and ethanol. The maximum yields of
lactic acid, acetic acid, and ethanol were 0.22 g/g, 0.06 g/g, and 0.05
g/g, respectively. The highest lactic acid concentration of 14.7 g/L was
obtained from a bioreactor with an initial solids loading of 60 g/L
[7].
Another study reported lactic acid production of 1.2 g/L, acetic acid
production of 0.9 g/L, and lactic acid yield of 0.042 g/g from PPW using
undefined mixed culture, those values were increased up to 5.1, 0.4,
0.18 when PPW was pretreated
(hydrothermal+cellulase+CaCO3) [25].
Future research directions and recommendations drawn from the key
findings of this study are listed below.
Gelling was observed in the nutrient media prepared with PPW particle
sizes smaller than 0.125 mm, and increased media viscosity resulted in
long lag times for production.
R. oryzae can utilize PPW as a substrate to produce DP4+,
glucose, lactic acid, and ethanol. However it consumes those metabolites
back during the fermentation, glucose, and DP4+ were utilized first,
then lactic acid and ethanol were used. Lactic acid and ethanol
concentrations should be monitored as the bioprocess parameters and the
batch should be stopped when their concentrations peak.
Recommended parameters are PPW particle size of 1-2 mm and loading rate
of 8%; the highest maximum amounts of ethanol (18.83 g/L) and lactic
acid (3.14 g/L), highest rates of ethanol (9.41 mg/L·d) and lactic acid
production (1.89 g/L·d), the highest yield of ethanol (0.235 mg/g PPW)
and second highest yield of lactic acid (0.039 mg/g PPW) were obtained
under these conditions.