Figs. 1-3 show the relative lactic acid (LA) production, cell growth and glucose consumption of a fermentation broth composed of a mixture of fresh culture media and saturated culture media with the specific solvent at proportions of 25, 50, 75 and 100 vol%, (referred to the saturated culture media). Figs. 1-3 are based on a culture media that were not in contact with the studied organic compounds (control fermentation) and for which the LA production, cell growth, and glucose consumptions were also measured. Taking into account the Marták criteria [29], dodecane was non-toxic for the four tested proportions. Cell growth and LA production were still 16% lower than the control fermentation at the four proportions, while glucose consumption was still 6% higher. For dodecane molecular toxicity test, the glucose concentration in the culture media increases after the contact stage due to the transport of water and some nutrients from the culture media to the organic phase. Thus, the glucose concentration of the saturated culture media was between 1.4% and 5.3 % (SD = 1.5 - 7.6%), higher than the control fermentation, and in consequence, the glucose consumption was promoted achieving values of glucose consumption slightly above the 100 %. Taking into account a higher glucose consumption is expected an increase in biomass and LA production. However, they were till 10% lower but within the standard deviation (maximum SD = 11.2%). The toxicity of dodecane on the bacteria was low enough that it does not have an important effect on the measured fermentation variables.

Figure 1 Source: The authors

Figure 2 Source: The authors

Figure 3 Source: The authors

Oleyl alcohol was non-toxic for volumetric proportions from 25 to 75 % considering LA production, cell growth, and glucose consumption (with a minimum and maximum standard deviation of ±0.9 and ±19.6). However, it can be observed that for cell growth (Fig. 2) at proportions from 25 to 75 %, alcohol can be classified as medium toxic if the standard deviations are taking into account. Oleyl alcohol was medium toxic for a proportion of 100 % based on LA production, cell growth, and glucose consumption (with a minimum and maximum standard deviation of ±5.6 and ±18). The glucose consumption (Fig. 3) was reduced by 20 % for 25, 50 and 75 % proportions and it was reduced by 30 % for a proportion of 100 %. On the other hand, both cell growth and LA production decrease around 20 % for proportions of 25, 50 and 75 % and around 30 % for a proportion of 100 %. These results seem to indicate that the bacteria use glucose equitably to produce LA and biomass when oleyl alcohol is present.

Dodecanol has a medium toxicity for a proportion of 25 %, and it was toxic for the other proportions based on LA production and cell growth (Fig. 1). However, glucose consumption was reduced around 40 % for a proportion of 25 % and around 60 % for proportions of 50, 75 % and 100 %. Also, cell growth rate was reduced by 60 % for a proportion of 25 % and around 90 % for proportions of 50, 75 % and 100 %. LA production was reduced around 50 % for a proportion of 25 % and around 80 % for the other proportions. The aforementioned results show that the bacteria consumes more glucose to survive than the biomass and LA produced. It uses this glucose for maintenance and this behavior is more intense at proportions above 50 % where there are a lower cell growth and LA production as compared to the glucose consumption.

Aliquat 336 (a quaternary ammonium salt mixed with tertiary amines C8-C10-alkyl, octanol, and decanol) was toxic for all tested proportions, taking into account both LA produced and cell growth. LA produced was reduced by 95 % as compared with control fermentation. The consumed glucose was reduced around 95 % for volumetric proportions of 25, 75 and 100 %, but it was reduced by 86 % for a proportion of 50 %. The cell growth was reduced around 95 % for proportions of 25 and 100 % and it was reduced by 88 % for a proportion of 50 %. The aforementioned results show that the bacteria use the consumed glucose to produce biomass but reduce its LA production to survive and for maintenance. For a proportion of 75 % the cell growth was reduced by 82 %. At this point, the bacteria produced an amount of LA in accordance with the consumed glucose but the relative fraction of biomass produced was higher than the consumed glucose. Aliquat 336 was the most toxic amine among tested extractants. Aliquat 336 contains trioctylmethylammonium chloride (TOMAC) that has a chloride ion in its structure and can interact with some substances from the MRS, such as sodium acetate and triammonium citrate to produce sodium chloride and ammonium chloride. Ammonium chloride is soluble in water and increases the medium acidity, whereas low pH can inhibit the lactic acid bacteria [11,16,31].

Trioctylamine (TOA) is toxic for the four volumetric proportions tested. Based on the cell growth, it is medium toxicity for a proportion of 25 % in terms of LA production and it is toxic for the other proportions. However, this value of medium toxicity could be read as toxic considering the standard deviation. The consumed glucose is used to equitably produce LA and biomass for proportions of 75 and 100 %, and as well for a proportion of 50 % if the standard deviations of glucose consumption and cell growth are taking into account. For a proportion of 25 %, the glucose consumption and LA production were reduced by 75 % but cell growth was reduced by 85 %, showing that the bacteria reduces its biomass production for maintenance.

Tri-iso-octylamine (TiOA) was the only extractant that exhibited a medium toxicity. It was medium toxic for a proportion of 25 %, the other proportions and the other amines were toxic based on both, cell growth and LA production. TOA and TiOA are tertiary amines with the same structural formula thus it can be expected the same toxicity level. However, TiOA was less toxic than TOA. The iso structure into the alkyl group for TiOA can produce steric effects that probably reduces its toxic effects.

Although it is known that toxicity of organic solvents depend on the type of microorganism or even of its strain [10,29,34], generally the toxicity is related to the solvent polarity (related to log P_ow values - Table 1) [1,19,24], and its water solubility [10,25]. Therefore, it is expected that dodecanol and oleyl alcohol, which are polar, were more toxic than dodecane (non-polar). The toxicity with the alkane is similar to other studies where alkanes were non-toxic or low toxic on bacteria [29,35]. For alcohols, it is known that its toxicity decreases as its long-chain increases [1,29,35,36]. Also, this study shows that oleyl alcohol (C18) is less toxic than dodecanol (C12) on the bacteria Lactobacillus casei ATCC 393 (Fig. 2).

Table 1

Physical properties were calculated with ProPed-ICAS 14 software (CAPEC-DTU) using Marrero and Gani, Constantinou and Gani methods [32,33]. Water solubility was taken from MSDS. ^aPhysical property at 293.15 K. ^bPhysical property calculated for TOMAC using connectivity index neglecting ionic bond.

The logarithm of P_ow (Table 1) is a common parameter to study toxic effects on microorganisms [24,37]. A Log P_ow higher than 4, apparently does not have any effect on the biocatalytic activity of the cell [37]. However, a type of Lactobacillus Delbruekii is sensitive to organic solvents with Log P_ow between 1 to 4 [24]. Taking into account Log P_ow as a parameter of toxicity [24,37], the tested solvents can be considered to be low toxic, except dodecanol that it can be considered as non-toxic due to its Log P_ow value, nevertheless, another scientific literature have shown that Log P_ow is an unreliable parameter [1] to infer the toxicity level. However, other parameters as solubility and surface tension can affect the microbial growth (biomass and LA production, and glucose consumption).

Surface tension and water solubility have also been used to predict the toxic effect of the solvent on the microorganism. A high surface tension involves a rapid loss of cellular activity [19,37] where the cell membrane fluidity increases [19]. Also, the lower the solvent solubility in water is, the lower the probability of contact between the solvent and the microorganism will be. Thus, microorganisms will be less affected for the solvent presence into the culture media. For these reasons oleyl alcohol is less toxic than dodecanol (water solubility in Table 1) in spite of having a similar surface tension. And dodecane has a low toxicity because it has a lower surface tension and lower water solubility than dodecanol and Oleyl alcohol.

In extractants, the higher the surface tension is the higher the toxicity on the bacteria will be, resulting Aliquat 336 with the highest toxicity in spite of being the extractant with the lowest water solubility. The lower toxicity of TiOA as compared with TOA is due to TOA has a higher surface tension and in consequence, it disturbs the cell membrane function as a barrier, as a matrix for enzymes and as energy transducer [23].

Figs. 4-6 shows lactic acid production, cell growth and glucose consumption (respectively) for the culture media after contact with mixes of TiOA or TOA (less toxic amines) in dodecane (less toxic diluent) at volumetric proportions of 10, 20 and 30 % of the respective tertiary amine in dodecane.

Figure 4 Source: The authors

Figure 5 Source: The authors

Figure 6 Source: The authors

TiOA mixed in n-dodecane was non-toxic at the three volumetric proportions for both, LA production and cell growth. For cell growth, the toxicity effect on the bacteria rises as the TiOA concentration increases, being non-toxic for volumetric proportions of 10 and 20 % and medium toxic at 30 %. There is a LA production according to the glucose consumption for three proportions of TiOA. However, as TiOA concentration rises, the cell growth decreases, perhaps in order to survive due to a high energy consumption of the bacteria trying to tolerate the organic solvent. These microorganisms have developed several mechanisms to increase its tolerance for organics solvents, one of them involves the action of efflux pumps to expel the organic solvent cumulated into the membrane and another one involves to change the membrane rigidity in order to do it less permeable to the organic solvents [21]. Both mechanisms require an additional energy consumption.

TOA was non-toxic at a volumetric proportion of 10 % for LA production and it was medium toxic for both cell growth and glucose consumption. It was medium toxicity at 20 % of TOA and toxic at 30 %. It means that the higher the TOA concentration is the higher the toxicity effect on the bacteria will be. For both proportions 20 and 30 % of TOA, the consumed glucose was used, in higher proportion, to produce LA than biomass. LA production is proportional to the glucose consumed but the biomass production is reduced. This means that for every single cell the LA production is increased as compared to the control fermentation. The metabolic changes inside the cell do not clearly explain this behavior.