To meet the greenhouse gases emission reduction targets the demand for renewable biofuels has increased. For this, efforts must be made to increase industrial productivity, and for that, better control of microbiological contamination is highly necessary.
The yeast cells reuse during the season countless times can influence the contamination level of the must by bacteria and wild yeasts (Lopes et al. 2016, Brexó and Sant’Ana 2017). The development and predominance of wild strains of yeasts is undesirable for the process due to the lower productivity, flocculation, foaming, and biofilm formation produced by these microorganisms (Beckner et al. 2011, Della-Bianca and Gombert 2013, Della-Bianca et al. 2013). These drawbacks increase the use of antifoam, acids and antibiotics in the industry plant (Brexó and Sant’Ana 2017).
The samples of wort submitted to the treatments T0, T1, T2, T3, T4 and T5 presented 7.00, 3.72, 3.31, 2.41, 0.00 and 0.00 Log (CFU +1) ml-1 (p<0.05) of total mesophiles, respectively (Fig. 1). These results correspond to an efficiency control of the microorganism of 99.94%, 99.97%, >99.99%, >99.99%, 100% and 100% (p<0.05) for T1, T2, T3, T4 and T5, respectively.
For total bacteria, T0, T1, T2, T3, T4 and T5 presented 5.00, 2.33, 3.22, 2.33, 0.00 and 0.00 Log (CFU +1) ml-1, respectively. These results correspond to an efficiency of control of 99.99%, 99.98%, >99.99%, 100% and 100% for T1, T2, T3, T4 and T5, respectively.
Therefore, the microbial contamination of the worst decreased as the irradiation dose increased, in accordance with Sampa et al. (2007) study.
Only treatments T4 (80 kGy) and T5 (steam - positive control) were able to sterilise the must. However, T3 (40 kGy) was sufficient for 4.59-log reduction of total mesophiles and 2.67-log reduction of total bacteria, corresponding to inactivation of more than 99.99% of the microorganisms present in the must.
In a study carried out by Nobre et al. (2007) when submitting sugarcane juice to treatment with ionizing radiation (g - Co60), the dose of 15 kGy was not enough to fully inactivate the Bacillus subtillis culture, but achieved a reduction of more than 99.9% for these bacteria. In the present study, inactivation of total bacteria higher than 99.9% was achieved in the dose of 10 kGy. Similar results were observed for total mesophiles (Fig. 1). Furthermore, the study conducted by Nobre et al ( 2007) used pure cultures of bacteria while the present study used the total microbiota from a sugarcane field.
Most of the literature about microbial radioresistance is based on reports of experiments typically involving pure cultures grown under near-optimal conditions (Shuryak 2019). In this study, we used the microbiota from the soil of a sugarcane field since it is known that present many microorganisms groups in different levels. Also, other authors have reported that bacterial contamination in alcoholic fermentation is mostly from the sugarcane field soil (Gallo 1989, Figueiredo et al. 2008, Costa et al. 2015).
A study carried out by Costa et al. (2015) assessing microbial diversity at different stages of sugarcane ethanol production identified 22 archaeal groups, 203 fungi groups and 355 bacterial groups. The authors also mentioned that the microbial contamination increases through the processes in the ethanol plant and are mostly from the feedstock and soil impurities.
Many microorganisms in soil are organic matter decomposers and also opportunistic plant/animals pathogens (Diezmann and Dietrich 2009, Sykes et al. 2014). This way of living requires being able to tolerate and possibly exploit the oxidizing compounds used as a defence mechanism by their hosts (Heller and Tudzynski 2011) this may justify the high radiotolerance of some soil microorganisms.
Also, some microorganisms can synthesize antioxidant compounds and pigments that aid in radioprotection (Kim et al. 2007), such as vitamin C (Mao et al. 2006), carotenoids (Parvathy 1983, Jain et al. 2015) and flavonoids (Molins 2001, Shuryak et al. 2017, Shuryak 2019). These compounds are commonly found in sugarcane juice (Abbas et al. 2014) and also present in the sugarcane molasses, the raw material used in this study (Table 1).
The D10 (The required dose to destroy 90% of the population) for total mesophiles was 3.06 kGy, whereas for total bacteria it was 4.81 kGy. Bacteria (prokaryotic) are more radioresistant than other microorganisms such as fungi and viruses, so it is justified that the total bacteria D10 is higher than the total mesophiles. In addition, the values found are in accordance with the literature, which states that fungi and bacterial spores present D10 values between 1 and 10 kGy (Confalonieri and Sommer 2011, Jung et al. 2017, Shuryak et al. 2017)
Other studies report radioresistant microorganisms like fungi that present chronic and acute radioresistance (D10 from 0.1 to 6.5 kGy) (Shuryak et al. 2017), bacteria, such as Deinococcus radiodurans capable of withstanding high doses of radiation (D10 of 16 kGy) (Omelchenko et al. 2005) and ability to reconstruction the functional genome (Confalonieri and Sommer 2011), and also archaea, such as Thermococcus gammatolerans sp. nov., which was isolated after exposure of 30 kGy (g- radiation)(Jolivet et al. 2003).
Moreover, Lactobacillus plantarum, one of the major contaminants of alcoholic fermentation (Dong et al. 2015, Dellias et al. 2018), is described as a chronic and acute radioresistant microorganism (Daly et al. 2004, Shuryak et al. 2017).
Most bacterial contaminants are found in the Lactobacillus genera (Bonatelli et al. 2017), especially lactic acid bacteria (LAB), like L. plantarum, which are responsible for reducing yeast cell viability due to the competition for nutrients and the production of toxic compounds, such as lactic and acetic acids during the fermentation (Narendranath et al. 1997, Costa et al. 2008).
In general, ethanol plants use antibiotics in order to control bacterial contamination. However, in some cases, does not prevent Lactobacilli infections recurrence, since these microorganisms can form biofilm, which is tolerant to the high concentration of the antibiotics and cleaning (Dellias et al. 2018, Saunders et al. 2019).
The large-scale use of antibiotic can induce bacterial resistance (Carvalho et al. 2020). Also, antibiotic residues such as virginiamycin can be found in distillers dried grain (DDG), from bioethanol fermentation of corn, which is utilized for animals feed (Bischoff et al. 2016). Regarding sugarcane bioethanol, there is a concern about antibiotic resistance in microorganisms that may be discharged into the environment through the fertigation using vinasse, the liquid waste obtained from the distillation of the wine (Mendonça et al. 2016). Furthermore, the presence of antibiotic in the vinasse can negatively affect its anaerobic digestion for the production of biogas through the inhibition of acetogenic bacteria and methanogenic archaea (Sanz et al. 1996) and reduce the potential to use vinasse to produce other products.
Therefore, a more efficient disinfection process is needed, such as ionizing radiation (IR). However, the use of IR may promote the formation of inhibitors by-products from sugar degradation (Molins 2001).
In our study, it was not observed formation and alteration in the concentration of the inhibitors flavonoids, furfural and 5-HMF (p>0.05) in any condition of treatment evaluated (Table 1). Such compounds are generally produced from sugar degradation, especially in thermal conditions (Molins 2001, Eggleston and Amorim 2006, Chi et al. 2019). However, it did not occur in this study, including in the steam treatment (T5).
The aldehydes like furfural and HMF may inhibit key enzymes intervening in the rate of protein synthesis of the central metabolism of the yeasts affecting negatively the growth and fermentation (Cabañas et al. 2019). Because of that, the presence of these compounds is highly unwanted in the fermentation substrate.
On the other hand, it was observed a gradual increase of phenolic compounds levels (6%, 9.4%, 17.8% and 19.8%, for T1, T2, T3 and T4, respectively) according to the radiation dose applied (Fig. 2). The steam treatment (T5) presented a significantly lower (p<0.05) concentration of phenolic compounds than 40 and 80 kGy, but it was statistically equal to treatments 10 and 20 kGy.
According to Rasmussen et al. (2014), the degradation of carbohydrates, especially D-glucose, D-xylose and L-arabinose can be related to the production of compounds such as phenolics. These compounds have been considered biocatalyst inhibitors (Chi et al. 2019), and their gradual production according to e-beam dose increase was also observed in Lima et al. (2016) study. But, in our work, the presence of phenolics did not inhibit yeast cell viability and biomass production (p>0.05) in any treatment during the fermentation (Table 3).
In a study conducted by (Martín et al. 2007) using sugarcane bagasse hydrolysate a concentration of 2100 µg ml-1 of phenolic compounds was responsible for the yeast (S. cerevisiae) inhibition and consequently, poor fermentability. In the same study using an adapted strain of the same yeast it was observed higher ethanol yield on total sugar after 24 h (0.38 g g-1) than the non-adapted yeast (0.18 g g-1) in wort with 1400 µg ml-1 of phenolic compounds. In our study, although the yeast has not been adapted to inhibitory toxins, the concentration of phenolics was below 1089.38 µg ml-1 (Table 1) in all fermented treatments, which probably reflected the S. cerevisiae tolerance to these compounds.
In addition to the low formation of inhibitors, it was not observed significant inversion of TRS (Total Reducing Sugars) in all treatments (p>0.05) (Table 2). Otherwise, Lima et al. (2016) observed significant (p<0.05) TRS inversion in sugarcane juice irradiate with 20 kGy e-beam dose.
The fact that in our study the reduction in the concentration of TRS was not observed is interesting because low sugar degradation is essential in a decontamination method aiming no decrease in the ethanol yield due to the sugars degradation (Alcarde et al. 2000, 2003).
Regarding sucrose, it was observed a decrease in steam treatment (p<0.05). However, there was no decrease in concentration (p>0.05) in the irradiated treatments (Fig. 3). In a study by Podadera (2007), using electron beam to sterilise invert sugar syrup, it was observed a significant decrease (p<0.05) in sucrose concentrations between the control, 5, 10 and 30 kGy samples. Moreover, glucose and fructose concentrations increased significantly (p<0.05). Which indicated the degradation of the disaccharide with the breakdown of the glycosidic bond and formation of the reducing sugars glucose and fructose.
At the end of the fermentation, sucrose was not detected in any treatment, in addition, the residual sugars glucose and fructose presented low concentrations (<0.05%) in all treatments (p>0.05), evidencing efficient consumption of sugars by yeasts or other microorganisms during the fermentation process (Table 3).
The concentration of glycerol was similar in the wine from all treatments, approximately 15 grams per liters (p>0.05). Bai et al. (2008) indicate that during the fermentation commonly a level of about 1% (w/v, 10g l-1) of glycerol is produced.
The high concentration of glycerol in wine can be an indicator of the yeast response to the adversity. High sugar values lead to high concentrations of glycerol in the must, due to the increase in osmotic pressure (Ponce et al. 2016), as well as the presence of bacterial contamination (Li et al. 2009).
There was bacterial contamination in the wine of all treatments. However, the control treatment presented a higher value (p<0.05) of 5.55 log (CFU ml-1+1). The presence of bacteria in all the treatments may be due to the contamination during the experiment sampling and poor asepsis.
As well as the high bacterial contamination, the control treatment (T0) presented a higher concentration of mannitol (0.41g l-1) when compared with other treatments (p<0.05). Mannitol is a sensitive indicator of contamination and its presence is an indication of the enzymatic dehydrogenation of fructose carried out exclusively by bacteria (Eggleston et al. 2007).
The T3 wine exhibit the lowest concentration of mannitol (0.33 g l-1) when compared with the other treatments (p<0.05). This was expected because among the treatments submitted to fermentation T3 presented the greatest contamination control. This result is remarkably interesting because according to Eggleston et al. (2007) high concentrations of mannitol may promote yeast flocculation and reduce the efficiency and productivity of the fermentation. The authors also described that a concentration around 6 g L-1 of mannitol can cause a decrease of 4% in ethanol yield.
The control (T0) showed the lowest yield (88%) of the treatments. It is justified due to high bacterial contamination in wine (5.55 log) and conversion of sugars into metabolites such as glycerol and mannitol (Table 3). The highest fermentation yield was reached in the steam treatment (T5) with 95% (p<0.05). Right below, the treatments 20 kGy (T2) and 40 kGy (T3) presented 93% yield (p>0.05). Higher than usually fed-batch industrial fermentations with 87% average of fermentation using molasses as raw material (Andrietta and Maugeri 1994, Viegas et al. 2002) and also greater than the yield described by Alcarde et al. (2001) which achieved 90.56% in the fermentation of sugarcane juice treated with 10 kGy (g radiation).
There are great importance and interest in increasing the yield of industrial fermentation. A yield of 93% could be responsible for a significant increase in ethanol production and, consequently, in the revenue of the industrial plant.
Regarding ethanol productivity, it decreased with the contamination whereas the control treatment showed the lowest value of 0.85 g l-1h-1 (Table 3). The highest productivity has been achieved in steam treatment (0.91 g l-1h-1) followed by 10 and 20 kGy treatments with 0.89 g l-1h-1 in both (p>0.05) and 40 kGy treatment with 0.87 g l-1h-1 (p<0.05). Thus, the treatment of 20 kGy showed better productivity (p<0.05) and the same yield as treatment 40kGy, which requires more energy.
Due to that, the dose of 20 kGy is the most recommended as it requires less energy consumption to allow the results with very positive response in the control of the contamination and on the fermentation process.
It is believed that greater changes in the fermentative behaviour of the irradiated wort could have been observed if consecutive fermentative recycles and acid treatment of yeast from the control treatment had been carried out. Since the microbial contamination tends to increase throughout the fermentative recycles during the harvest season, as described in the literature (Ceccato-Antonini 2018).
It is also necessary to study more the application of this technology, as well as the increase of the scale and the economic viability.
In our work, the electrical energy consumption to operate the electron accelerator at full power in one hour of use was approximately 150 kWh. Only the electron beam is responsible for 25% (37.5 kWh) of this total. The cooling system, vacuum system, compressed air and other devices consumed the remaining 75% (112.5 kWh). In this case, the cost was US$ 6.43 per hour.
For the present case, the electron beam was not used at its maximum power. Due to this, for each hour of use of the accelerator, the electrical energy consumption was 122 kWh, of which 113.58 kWh were consumed by the devices of each system mentioned above. Only 8.42 kWh was consumed by the electron beam. The cost was about US$ 5.23 per hour of operation.
Table 4 presents the operating cost, taking into account only the energy consumption of the electron accelerator for each treatment. Δt is the processing time (or sterilisation) of the samples by e-beam. Their values were obtained considering the conveyor speed of 0.112 m s-1 and the linear length of two aligned trays equal to 0.40 m.
Therefore, operating at a dose of 20 kGy, the energy consumption by the electron accelerator is estimated at 146.18 kWh (33.68 kWh) consumed by the electron beam and the rest by the peripheral equipment. The cost of each hour of operation of the accelerator is estimated at US$ 6.27. Considering that in the ethanol-producing plants in Brazil an average of 450 m3 of wort is produced per hour, the estimated cost of processing 1 m3 of this material is US$0.014.
It is important to note that the sugarcane mills can process 1 m3 of the wort in a short time. Furthermore, the e-beam technology is very fast, and, in a few seconds, the desired result in microbial control can be achieved. This allows the treatment of large volumes of wort in a short time, which facilitates this process implementation in large industries.
It should be mentioned that apart from sterilisation of the wort, to have success in the contaminating control in the ethanol industry it is necessary an adequate system to cleaning fermenters, pipeline, centrifuges, valves and other compartments used to transport or store wine, yeast cream and wort.
With that, the use of e-beam for sterilisation of the substrate could also make it possible to use more productive yeast strains in the fermentation, such as the thermotolerant strains of S. cerevisiae described by Pattanakittivorakul et al. (2019), which show extremely high ethanol production at 40 ˚C, in addition to tolerating high gravity fermentation and high concentrations of furfural, HMF and acetic acid.
It should be noted that the e-beam could also be applied in other processes within the industry, as in the pretreatment of biomass for the production of second-generation ethanol (Postek et al. 2018). The biomass irradiation can facilitate enzymatic hydrolysis with lower temperatures and minimal formation of inhibitory by-products when compared to conventional pretreatments (Singh et al. 2016).