Klebsiella pneumoniae – a useful pathogenic strain for biotechnological purposes: 1,3-propanediol biosynthesis CURRENT STATUS: POSTED

Background Despite being a well-known human pathogen, Klebsiella pneumoniae plays a significant role in the biotechnology field, being considered as a microbial cell factory in terms of valuable chemicals biosynthesis. In fermentation processes, K. pneumoniae is mostly used to biosynthesize 1,3-propanediol (PDO), a very important intermediate compound utilized in the biodegradable bioplastics industry. This strain owns the ability to metabolize glycerol and to produce PDO under both aerobic and anaerobic circumstances, and under low oscillations of pH. Results In this work, we investigated the K. pneumoniae strain DSMZ 2026 in batch bioreactor cultivation. The bacterial strain was inoculated in 2 L culture broth containing 43.5 g·L- 1 main substrate and the pH was adjusted at 7 at the beginning of the process. After 12 h of batch cultivation, the pH values decreased to 4.77, and we obtained 51 g·L-1 wet biomass, 17 g·L-1 PDO, while the starting substrate reached 15 g·L- 1. The morphology of K. pneumoniae cells was analyzed on both solid and liquid media, by being identified large mucoid colonies on Columbia agar, and individual and grouped cells were observed by methyl blue staining. Conclusion From this study it can be concluded that K. pneumoniae can grow successfully in mineral broth under anaerobic environment and low decrease of pH, and can biosynthesize valuable chemicals like PDO.


Abstract
Background Despite being a well-known human pathogen, Klebsiella pneumoniae plays a significant role in the biotechnology field, being considered as a microbial cell factory in terms of valuable chemicals biosynthesis. In fermentation processes, K. pneumoniae is mostly used to biosynthesize

Background
Among Enterobacteriaceae, Klebsiella spp. are recognized mostly in the medical field as famous opportunistic germs associated with pathogenic infections [1][2][3]. Klebsiella pneumoniae represents a saprophytic pathogen that might affect both plants [4], animals [4,5] and humans [6][7][8], but which can be successfully used for biotechnological applications [9,10]. In medical terms, K. pneumoniae is a pathogenic strain responsible for multiple nosocomial infections including pneumonia, urinary tract and soft tissue infections, and septicemias [6,7,11]. K. pneumoniae cells are able to spread very quickly especially in hospital environment, the main cause being the unclean hands of personnel [11].
The pathogenic potential of the Gram-negative K. pneumoniae is mainly due to the external cell structure [4,9]. The main virulence factor is the outer cell membrane that consist of capsule, lipopolysaccharides (recognized as endotoxins in humans), siderophores, and pili [1,11]. The capsular polysaccharides are attached to the peripheral membrane, and play a major role in capsular antigen synthesis and export [12].
In the biotechnology field, pathogenic bacteria like Klebsiella, Clostridium, E. coli or Bacillus have been studied for more than 100 years to produce important chemical compounds such as propanediols (1,2-propanediol, 1,3-propanediol), organic acids (lactic, acetic, citric, succinic, pyruvic) or alcohols (ethanol, 1-butanol) [13][14][15]. K. pneumoniae is a representative strain used at large scale for the biosynthesis of PDO by means of fermentation processes at neutral pH values, under both aerobic and anaerobic conditions, starting from glycerol as the main nutrient substrate [16][17][18]. K. pneumoniae is mostly used for PDO synthesis at large scale because of its natural ability to produce the B12 co-enzyme, a very important factor for microbial PDO synthesis [19]. The biotechnological production of PDO is extensively studied by multiple research groups because of the major development of market demands worldwide for PDO [20][21][22]. PDO plays an important role in the synthesis of biodegradable plastics, namely for the polytrimethyleneterephtalalte (PTT) production [18,23].
Considering the microbial potential of the pathogenic strains like K. pneumoniae in biotechnology field, in this study was used K. pneumoniae DSMZ 2026 (risk group: level 2) [24, 25] for PDO production in bioreactor batch fermentation, in anaerobic conditions and different values of pH. The biomass evolution, pH oscillation, PDO biosynthesis and substrate consumption were monitored during 12 h of fermentation. The bacterial morphology on both solid and liquid media was analyzed.

Results And Discussions
The biomass evolution, pH decrease, substrate consumption and PDO biosynthesis were monitored during 12 h of batch fermentation, and the results are presented in Table 1: After 12 h of batch cultivation the pH decreased from 7 to 5, while the wet biomass increased from 12 g·L-1 to 51 g·L-1 ( Figure 1). More than a half of the initial concentration of the substrate was   shake-flask level, in anaerobic conditions, and after 12 h they achieved maximum concentrations ranging between 33 g·L-1 and 54 g·L-1 of PDO. For the same study, the pH values were kept at 7 [28]. Higher PDO concentrations in batch trials were obtained by Zhao et al. [29] who used microencapsulated K. pneumoniae type ZJU 5205. They achieved 63 g·L-1 after 11 h of cultivation starting from high initial glycerol concentration of 120 g·L-1 [29].
Placed on solid media and incubated at 37oC for 24 h, Klebsiella cells developed large (>1 mm), 5 opaque, cream-colored and glistening mucoid colonies (Figure 3, left). Under microscope light, individual cells surrounded by a thin halo could be observed (Figure 3, right), structure which constitutes the capsule of the bacteria. According to Evrad et al. [8], the voluminous capsular layer is made of polysaccharides that covers the entire bacterial surface, and its role is to protect de bacteria cell against macrophage phagocytosis in animal and human models [8]. In biotechnological processes instead, large amounts of capsular polysaccharides induce mucoviscosity [13] and obstruct the separation of bacteria cells from fermentation media during the downstream process [9].

Microorganism and culture conditions
In this study, we used K. pneumoniae DSMZ 2026 obtained from the German Collection of Microorganisms and Cell Culture (DSMZ, Braunschweig, Germany); the cultivation conditions and fermentation broth components were similar to those used by Menzel et al. [30].

Batch cultivation at bioreactor level
Initially, 200 mL of pre-culture broth was inoculated with a 24 h colony of 108 CFU·mL-1. The preculture was maintained at 37oC for 24 h and 200 RPM. The experiment was performed using a 5 L bioreactor (B. Braun Biotech International) filled with 2 L of culture broth, where the inoculum was added in sterile conditions. The fermenter was fitted with temperature, pH, and rotation speed control. Temperature was maintained at 37°C and rotations were maintained at 200 RPM. pH was not kept constant, in order to observe its influence on PDO production. The batch trial run for 12 h anaerobically. Samples were collected every 2 h during 12 h for specific tests.

Testing methods
The wet biomass quantity was determined by weighing 10 mL of sample centrifuged at 7000 RPM for 15 min, washed twice with double distilled water, and removing the supernatant.
The glycerol consumption was determined by using the enzymatic test purchased from Sigma Aldrich, Glycerol Assay Kit.

Declarations
Ethical approval and consent to participate Not applicable.

Consent for publication
Not applicable.

Availability of data and materials
Not applicable.

Competing interest
The authors declare that they have no competing interests.  Substrate consumption and PDO production during 12 h of fermentation using K.
14 Figure 3 The K. pneumoniae DSMZ 2026 colony appearance on Columbia solid media (left), and the microscopic examination of bacterial cells after methylene blue staining (right). The red arrow indicates the cell capsule.