The natural environment is a good source of industrially useful strains. In our previous work we isolated from the environmental probe the new bacteria strains able to 1,3-PD from glycerol (Leja et al. 2011), which have a variety of industrial applications, such as chemical intermediates used in the manufacture of polymers, cosmetics, medicines and heterocyclic compounds (Kośmider et al. 2011). During that experiment we obtained Cl. bifermentans strains which were not known as 1,3-PD producers yet. It occurred that these isolates are also able to produce another industrially useful metabolite from glycerol, lactic acid (Myszka et al. 2012), which is widely used in the food, cosmetic, pharmaceutical, and chemical industries and has received increased attention for potential use as a monomer in the production of biodegradable poly (lactic acid) (Wee et al. 2006). In the existing literature there is no information that the species of Cl. bifermentans is able to lactic acid synthesis. Generally, the metabolite profile of the species of Cl. bifermentans is not investigated sufficiently as yet. Thus the present authors decided to investigate into lactic acid production by these species. Our work’s aim was to check whether or not a medium pH and a carbon source exert an influence on lactic acid production by Cl. bifermentans strains. Some scientists argued that Cl. bifermentans exhibit adaptability in extreme environmental conditions (Lauro et al. 2004) and that they are able to survive in extreme pH levels (Sengupta et al. 2011). Moreover, Gibbs (1964) stated that even incubation at pH 10.0 or pH 3.0 has no significant effects on the ability of spores of Cl. bifermentans to germinate and that the vegetative cells are able to survive in these extreme conditions. We decided thus to investigate changes in metabolite profiles depending on the pH level of a fermentative medium which includes radical values such as 3 or 13. It occurred that the decreasing of pH value to 8.0, 7.0, and 6.0 results in the increased yield of lactic acid production. The data presented in the existing literature confirms this observation: lactic acid production requires strict control of the pH, mostly at values between 6 and 8 (Kascak et al. 1996; Litchfield 1996; Hofvendahl & Hahn-Hägerdal 2000). For example, the optimal pH for lactic acid synthesis for Lactobacillus bulgaricus is 6.0 (Venkatesh et al. 1993) and for Lactobacillus casei is 6.5 (Panesar et al. 2010). Our isolates prefer pH 7 (KM 371 and KM 374) and 6 (KM 376).
We selected glycerol for our research on microbiological production of industrially useful metabolites because a significant increase in biodiesel production was observed within the last decade (Kośmider et al. 2011). Presently, the most often used biodiesel fuels are vegetable oil fatty acid methyl or ethyl esters produced by transesterification. For every three molls of ethyl esters one mol of crude glycerol is produced, which is an equivalent to approximately 10% of total biodiesel production (Kośmider et al. 2011; Rahman et al. 2002). It is estimated that by 2016 the world biodiesel market will achieve the quantity of 37 billion gallons, which means that much more than 4 billion gallons of crude glycerol will be produced every year (Kośmider et al. 2011). Accordingly, it is necessary to find a new effective method to utilized this amount of crude glycerol. The research on production 1,3-PD from crude glycerol by microbiological way is extensively described worldwide e.g., (Hiremath et al. 2011; Mendes et al. 2011; Vaidyanathan et al. 2011; Chatzifragkou et al. 2011; Wilkens et al. 2012; Ringel et al. 2012), but only a few papers concern the production of lactic acid from this by-product, and moreover, publications concentrate mainly on genetic engineered strains (Posada et al. 2012; Ruhal & Choudhury 2012); at the same time only a few papers discuss lactic acid production from other renewable resources (Hofvendahl & Hahn-Hägerdal 2000; Yadav et al. 2011). During our work it occurred that Cl. bifermentans strains are indeed able to synthesize lactic acid from glycerol. The yields of lactic acid for KM 371, KM 374, and KM 376 were, respectively, YLA=0.16, YLA=0.17, and YLA=0.17. These values are lower than the ones quoted in the work by (Ruhal & Choudhury 2012) on the mutant of Propionibacterium freudenreichii subspp. shermanii in which they obtained YLA=0.3. However, in our work the bacteria utilized more glycerol (68.22%, 79.08%, and 80.22%, respectively) than in the above mentioned work, in which only 25.00% was consumed. Our results in the yield of lactic acid obtained by isolates of Cl. bifermentans were comparable with the results obtained in other investigations in which some kinds of variable renewable resources were used, such as a carbon source; e.g., in the case of lactic acid from whey permeate by Lactobacillus lactis sp. lactis 2432 YLA=0.21, from solid waste by Lb. lactis sp. lactis NRRL B-4449 YLA=0.16, and from wheat flour hydrolyzed by Lb. delbrueckii sp. bulgaricus ATCC 11842 YLA=0.11 (Hofvendahl & Hahn-Hägerdal 2000).
In the literature there is a lot of information about lactic acid production from other carbon sources such as saccharides e.g., (Wee et al. 2006; Hujanen et al. 2001; Liu 2003; Jun et al. 2003). Thus we wanted to check if the change of carbon source from glycerol to pure saccharides increases the level of lactic acid synthesis by Cl. bifermentans isolates. It occurred that the highest productions of lactic acid were obtained when mannitol was used – the yield of production increased more than three times: YLA=0.62, YLA=0.78, and YLA=0.76 in the case of KM 371, KM 374, and KM 376, respectively. Moreover, some lactic acid bacteria, as it turned out, are able to ferment mannitol into lactic acid. For instance, Lactobacillus casei utilizes mannitol through the following pathway: mannitol->mannitol-1-phosphate->fructose-6-phosphate->2 pyruvate->2 lactate (Liu 2003). Under aerobic conditions, Lb. casei converts mannitol primarily to lactate only. However, under anaerobic conditions mannitol is fermented to lactate, acetate, formate, and ethanol (Liu 2003). The effect of variable saccharides on the lactic acid production by Rhizopus oryzae was investigated in the work by Yin et al. (1997). These authors tested the efficiency of lactic acid production from glucose, mannose, fructose, sucrose, raffinose, inulin, maltose, rhamnose, xylose, galactose, and corn starch. It occurred that mannitol is a good carbon source also in lactic acid production by Rhizopus oryzae and the YLA=0.70 which is comparable with the results obtained in the present work. This step of our experiment also shows that all the saccharides used (except of xylose, raffinose and, additionally, sorbitol in the case of KM 374) are a preferable carbon source for lactic acid synthesis. The main aim of this work, however, was to investigate into how utilize glycerol as a by-product from biodiesel production. Thus it was checked if the addition of small amount of saccharide to glycerol used as a main carbon source can result in an increase of the level of lactic acid synthetized. Generally, the levels of lactic acid obtained from a mixed carbon source were comparable with the results from our tests with glycerol only. Only in the case of addition of mannitol for all strains, and mannose for KM 376, the yields of lactic acid increased. When some saccharides were used as a carbon source – no 1,3-PD was synthetized, in the situation when the saccharides were added to glycerol, 1,3-PD was synthetized. Moreover, the amount of utilized glycerol was lower and the saccharides were completely consumed. Biebl and Marten (1995) made similar observations. In their experiments, glucose was applied in half the concentration of glycerol for a mixed-substrate culture. It occurred that the addition of glycerol to medium with glucose increased the rate of glucose utilization by Cl. butyricum (up to 8 h). Moreover, product formation changed markedly in comparison with glycerol fermentation as 90% of the glycerol was converted to 1,3-PD and only 10% was used for acids. Additionally, mixed fermentation (glycerol plus glucose) shifted from butyrate to acetate production. Because the addition of the saccharides did not increase the efficiency of lactic acid production, a better solution from the environmental point of view is to optimize the production of metabolites using only glycerol as a carbon source. Growing prices of crude oil and fuels for the transportation sectors have resulted in a rapid growth in biodiesel production worldwide. An increase of biodiesel production leads thus to an increased quantity of its primary co-product, glycerol. Since the existing glycerol supply and demand market was tight, the recent increase in glycerol production from biodiesel refining has created a glut in the glycerol market. This situation made the price of glycerol fall significantly and biodiesel refiners are faced with limited options for managing the glycerol by-product (Johnson & Taconi 2007). One of the solutions of this problem is to use crude glycerol in the production of industrially useful metabolites such as lactic acid and 1,3-PD (Kubiak et al. 2012).