Production of the biocontrol agent Pantoea agglomerans PBC-1 in a stirred tank reactor by batch and fed-batch cultures

Abstract

Concerns about food safety as well as the development of resistance to many fungicides by major postharvest pathogens have increased recently. Biological control, using microorganisms antagonistic to the fungal plant pathogens, appears to be promising as an alternative to fungicides. The microbial biocontrol agent has to be produced on an industrial scale, maintaining its biocontrol efficacy. The purpose of the current study was to optimize the conditions for microbial biomass production of the biocontrol agent Pantoea agglomerans PBC-1 in a 2-l mechanically stirred reactor (STR), defining mixing and mass transfer technological parameters and the growth kinetics for different saccharides. In the batch mode, different impellers and spargers were tested. Despite the oxygen mass transfer improvement achieved with marine propeller combined with porous sparger, the biomass did not increase, if compared with the use of a Rushton turbine and L-sparger, pointing out the relevance of a radial flux for better broth homogenization. Different carbon sources were used: sucrose, glucose and fructose; each of which led to viable populations 3.9 9 10 9 , 1.4 9 10 9 , 3.9 9 10 9 c.f.u/ml, respectively, after 20 h of incubation. Fedbatch technology allows the maintenance of high cell viability for longer periods of time in the stationary growth phase, which can be crucial for the scale-up of biocontrol agent production process that is achieved together with a reduction of 85% on the incidence caused by the pathogens, brought about by fresh microbial biomass preparation on artificially wounded apples or oranges, stored for 7 days at 25°C against Penicillium expansum and Penicillium digitatum.

Carob pulp as raw material for production of the biocontrol agent P. agglomerans PBC-1

“…Similar growth profiles were obtained in previous studies on the production of this biocontrol agent at different sucrose concentrations 5, 10, 15 and 20 g l -1 [26]. In those studies, at the beginning of the stationary phase, 18 h, the viable populations were similar in all sucrose concentrations, 3-4 9 10 9 cfu ml -1 .…”

“…In those studies, at the beginning of the stationary phase, 18 h, the viable populations were similar in all sucrose concentrations, 3-4 9 10 9 cfu ml -1 . Sucrose at 5 g l -1 was chosen for the following scale-up experiments, since it was the concentration that led to achieve the higher biomass yield [26]. In our study the viable populations achieved after 19 h of inoculation was 4.2 9 10 9 , 3 9 10 9 , 2.5 9 10 9 and 7.2 9 10 8 cfu ml -1 , at 5, 10, 15 and 20 g l -1 , respectively.…”

“…Fresh cells of P. agglomerans PBC-1, at an initial concentration of 1 9 10 5 cfu ml -1 , were inoculated in 250-ml conical flasks with 50 ml of culture medium. Flasks were incubated, at 30°C under orbital agitation, at 150 rpm in an orbital incubator (Pentlab, Portugal) and determination of viable cells (cfu ml -1 ), optical density, pH and sugar contents in the broth was carried out following the methodology described by Manso et al [26].…”

“…The temperature, pH and dissolved oxygen were measures by specific probes and the values were constantly registered. Samples were taken, immediately after inoculation and at regular intervals, to determine the OD 640 , viability population counts and sugar concentrations, as previously described [26]. Fresh weight biomass was determinated after centrifugation at 1,920g (Universal 320, Hettich Zentrifugen, Germany), for 15 min.…”

The science, development, and commercialization of postharvest biocontrol products

“…In general, liquid-phase cultures are used for bacteria and yeasts and solid-phase cultures are used for most fungi. Optimized mass production systems have been described for some postharvest biocontrol agents, including bacteria such as P. agglomerans CPA-2 (Costa et al, 2001), P. agglomerans PBC-1 (Manso et al, 2010) and B. subtilis CPA-8 (Yánez-Mendizábal et al, 2012b), yeasts such as C. sake CPA-1 (Abadias et al, 2003a,b), A. pullulans (Mounir et al, 2007), and Rhodotorula minuta (Patiño-Vera et al, 2005), and fungi such as Penicillium frequentans 909 (De Cal et al, 2002), and Epicoccum nigrum (Larena et al, 2004).…”