Rhizobium biomass production in batch and continuous culture with a malt-sprouts medium

SummaryThe growth of nine strains of four different species ofRhizobium (R. leguminosarum, R. meliloti, R. phaseoli andR. japonicum) was studied with media containing malt sprouts extract (MSE), in place of yeast extract (YE), as source of nitrogen and growth factors. The results obtained in batch cultures indicated that all the strains grew well in MSE medium. In the case of fast-growing strains the biomass increased with increase in the concentration of MSE in the medium, but with the slow-growing strains, likeR. japonicum, growth was inhibited completely by using a concentration of MSE (containing 3.75% w/v of solids) of 40% v/v. With all the strains the concentration of cells attained was greater than 5×109 cells/ml, which clearly indicates that MSE is a suitable component of media, and one which can be used instead of YE. The results of continuous culture experiments showed that a high productivity of cells (15.3×108 cells/ml/h) could be obtained using MSE medium.RésuméLa croissance de neuf souches appartenant à quatre éspèces différentes deRhizobium (R. leguminosarum, R. meliloti, R. phaseoli etR. japonicum) a été étudiée avec des milieux contenant, comme source d'azote et de facteurs de croissance, un extrait de germes de malt (MSE) au lieu d'extrait de levure (YE). Les résultats obtenus en culture discontinue (batch) montrent que toutes les souches poussent bien en milieu MSE. Dans la cas des souches à croissance rapide, la biomasse augmente proportionnellement à la concentration de MSE dans le milieu. Par contre, pour les souches à croissance lente, commeR. japonicum, la croissance est complètement inhibée lorsque la concentration en MSE (contenant 3.75% w/v de solides) atteint 40% (v/v). Avec toutes les souches, on obtient une concentration en cellules supérieure à 5×109/ml, ce qui indique clairement que le MSE convient bien et peut être utilisé à la place de YE. Les expériences réalisées en culture continue montrent qu'on peut obtenir avec le milieu MSE une productivité en cellules très élevée (15.3×108 cellules/ml/h.).ResumenSe estudió el crecimiento de nueve cepas de cuatro especies diferentes deRhizobium (R. leguminosarum, R. meliloti, R. phaseoli y R. japonicum) en medios de cultivo conteniendo extracto de raíz de malta (ERM), en reemplazo de extracto de levaduras (EL), como fuente de nitrógeno y factores de crecimiento. Los resultados obtenidos en cultivos en batch indicaron que todas las cepas crecieron bien en los medios con ERM. En el caso de las cepas de crecimiento rápido la biomasa se incrementó con la concentración de ERM en el medio de cultivo, pero con las cepas de crecimiento lento(R. japonicum) el desarrollo celular se inhibió completamente para concentraciones de ERM (conteniendo 3,75% p/v de sólidos) de 40% v/v. Con todas las cepas empleadas la concentración celular alcanzada fue superior a 5×109 cél/ml, lo que indica claramente que el ERM es un adecuado componente para los medios de cultivo que puede reemplazar al EL. Los resultados de los experimentos realizados en sistema continuo mostraron que se puede alcanzar una alta productividad (15,3×108 cél/ml/h) utilizando un medio de cultivo suplementado con ERM.


Introduction
Several media have been proposed for growing cultures of Rhizobium spp.(Burton 1979;Subba Rao 1982).In general, the majority include yeast extract (YE) or yeast water as a source of nitrogen and growth factors, mannitol, sucrose or glycerol as carbon source, and mineral salts.
According to the recommendations of a workshop on Priorities in Biotechnology (1982) the use of other components cheaper than YE, and which can replace it, is highly desirable.In this respect some byproducts have been used, such as corn-steep (Burton 1979) and proteolyzed pea husk (Gulati 1979), but in general YE has only partially been replaced.Malt sprouts, a byproduct of the malt industry, which contain growth factors and nitrogen sources, and which have already been used by us in other fermentation processes (Cuevas & Ertola 1973;Arcas et al. 1984), is an interesting and cheap product which could be an adequate component of media for rhizobia.The suitability of malt sprouts extract (MSE) for this purpose has been investigated.
The strains were maintained on medium 1 (Table 1).Inocula for the experiments were prepared in 250-ml Erlenmeyer flasks each containing 50 ml of medium 2. The flasks were seeded with 72-or 120-h agar-slant cultures, for fast and slow growing strains, respectively, and located on a rotary shaker operated at 200 rev/min.The temperature was 30 ~ After 40 or 70 h for the fast-or slow-growing strains respectively, a sufficient amount of each culture was used to inoculate the flasks to give an initial concentration between 1 X 108 and 3 • 108 cells/ml, according to the strain.

Media
Media used are indicated in Table 1.Medium 1 was employed for maintaining the strains and medium 2 for inocula preparation for all the strains.Medium 3 was used as control   with YE for R. japonicum, R. meliloti and R. leguminosarum and medium 4 for R. phaseoli (Boiardi 1984).Media 5, 6, 7, and 8 contained 10, 20, 40 and 80% v/v of MSE, respectively.Media 9 and 10 contained 4 g/1 of YE and 20% v/v of MSE respectively, both supplemented with KNO3 as additional nitrogen source.
The MSE was prepared by heating a mixture of 1 part of malt sprouts plus 9 parts of water (w/w at 100 ~ for 0.5 h and centrifuging after cooling.A yellow liquid was obtained containing 3.75% w/v of solids, 0.18% w/v of nitrogen and 0.6% w/v of total reducing sugars.

Fermentation procedure
Growth experiments using different concentrations of MSE were carried out in 1000-ml Erlenmeyer flasks each containing 200 ml of the appropriate medium.The conditions used in these experiments were the same as those used to prepare the corresponding inocula.
The continuous culture experiments were carried out in an LKB 1601 Ultroferm Fermentation Unit (LKB Produkter, BOX 305, S-16126, Bromma, Sweden).A working volume of 3 1 was used.The temperature was 30 ~ the agitation 300 rev/min and the aeration rate 0.3 v/v/min.Different values of dilution rates were employed and the parameters were determined under steady state conditions which were defined by the pressure of dissolved O2 (Biotech galvanic probe, LKB), percentage of 02 in the outcoming gas (Beckman 02 analyser OM 14, Beckman Instruments Inc., 2500 Harbor Bv.Fullerton, California 92634, USA), cellular concentration and pH.

Analytical techniques
The biomass was determined by optical density at 625 nm and the number of total cells in a Petroff-Hausser chamber.Glycerol was evaluated by the phloroglucin method (Lambert & Neish 1950) and the reducing sugars by the o-toluidine method (Hyvarinnen & Nikkila 1962).
The oxygen uptake rate was calculated by measuring the flow rate and oxygen content of the incoming and outgoing air of the fermenter, according to Cooney et al. (1977).

Results and discussion
The results obtained in Erlenmeyer flasks with different media are shown in Fig. 1 and Table 2.The results of the experiments carried out with the strain of R. meliloti B 323 are presented in Fig. 2 and those for continuous culture in Fig. 3.
The results show that all the strains grew well with MSE and in several cases the concentration of cells attained was higher than that achieved by using 4 g/1 of YE.Therefore, it is clear that MSE contains all the nutritional requirements for nitrogen and vitamins of the Rhizobium strains used.It is known for instance that R. leguminosarum and R. phaseoli need several vitamins (Vincent 1977) which are supplied by the MSE.In particular, the strain of R. phaseoli F-45 has an absolute requirement for biotin as was previously reported (Boiardi 1984).The inhibition observed when 40% v/v, or higher concentration, of MSE are used for R. japonicum may reflect the presence of high contents of certain components probably similar to those occurring in YE (Sherwood 1972) such as glycine, alanine and certain D-amino acids, since it is known that the protein fraction of MS contains a similar ratio of amino acids to that found in YE (Pomeranz & Robbins 1971).This inhibition effect of MSE is not evident for the fast growing strains although in the case of the strain F-45 very large and deformed cells were observed when the concentration of MSE used was 40% v/v.Growth curves of R. meliloti (B 323) in MSE media compared with YE medium.Media: e, control (medium 3); A, MSE 10% v/v (medium 5); ~, MSE 20% v/v (medium 6); o, MSE 40% v/v (medium 7); ~, MSE 80% v/v (medium 8); *, Control + KNO 3 (medium 9); x, MSE 20% v/v + KNO 3 (medium 10).
For the strain of R. meliloti B 323 a partial inhibition was observed with 80% v/v of MSE.This, again, could be the result of a similar effect to that mentioned above.
With all the strains used, the concentration of cells attained was higher than 5 X 109 cells/ml, which is the normal limit for industrial production (Subba Rao 1982).In addition, it is interesting to point out that these concentrations were obtained in 40 h or less for the fast growing strains and in 80 h or less for the slow ones.Figure 2 also shows that no differences in the growth curve were observed between medium 3 (control), medium 9 (which contained KNO3) and medium 6, which contained 20% v/v of MSE.The same result occurred when medium 6 was supplemented with KNO3 (mediuml0) which means that nitrogen was not a limiting factor in either medium 6 or 9.
Figure 3 shows the results of continuous culture experiments with R. meliloti B 323.According to Wang et al. (1977) these should be considered non-chemostat continuous cultures because the medium used was complex and the limiting component was unknown.
For these reasons results are difficult to interpret.The 02 uptake rates are in agreement with previous observations (Ertola et al. 1969) related to the requirements of rhizobia.The uptake of 02 found was 340 ml/1/h for the maximum productivity.When comparing O2 uptake rates it is important to define the condition under which this parameter is measured (which is not commonly done) because the requirement for 02 varies according to the conditions of growth.
According to Fig. 3 it seems that even at the lowest dilution rates (0.025 h -i) the glycerol was not completely used, which may indicate the presence of another carbon source in the MSE which was used instead.As the reducing sugars were not also utilized at a dilution rate of 0.075 h -1, it is clear that the medium used for continuous culture can be simplified.
The growth curve shows a continuously decreasing cell concentration which is normally observed when complex substrates are used in continuous culture.The curve observed in the case of OD may be ascribed to the increase of polysaccharide production with the dilution rate, as has been indicated by De Hollander et al. (1979).
The results shown in Fig. 3 also indicate that the productivity was very high, a fact which could be important from the industrial point of view because high productivity can lead to a smaller investment in plant.
It can be concluded that MSE is a suitable component of media for rhizobia cultures and particularly for legume inoculant production, considering its low cost and ease of preparation from malt sprouts.

Summary
The growth of nine strains of four different species of Rhizobium (R. leguminosarum, R. meliloti, R. phaseoli and R. ]aponicum) was studied with media containing malt sprouts extract (MSE), in place of yeast extract (YE), as source of nitrogen and growth factors.The results obtained in batch cultures indicated that all the strains grew well in MSE medium.In the case of fast-growing strains the biomass increased with increase in the concentration of MSE in the medium, but with the slow-growing strains, like R. japonicum, growth was inhibited completely by using a concentration of MSE (containing 3.75% w/v of solids) of 40% v/v.With all the strains the concentration of cells attained was greater than 5 • 109 cells/ml, which clearly indicates that MSE is a suitable component of media, and one which can be used instead of YE.The results of continuous culture experiments showed that a high productivity of cells (15.3 • 108 cells/ml/h) could be obtained using MSE medium.

Table 1
Composition of media