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.