Revealing nutritional requirements of MICP-relevant Sporosarcina pasteurii DSM33 for growth improvement in chemically defined and complex media

Abstract

Microbial induced calcite precipitation (MICP) based on ureolysis has a high potential for many applications, e.g. restoration of construction materials. The gram-positive bacterium Sporosarcina pasteurii is the most commonly used microorganism for MICP due to its high ureolytic activity. However, Sporosarcina pasteurii is so far cultivated almost exclusively in complex media, which only results in moderate biomass concentrations at the best. Cultivation of Sporosarcina pasteurii must be strongly improved in order to make technological application of MICP economically feasible. The growth of Sporosarcina pasteurii DSM 33 was boosted by detecting auxotrophic deficiencies (L-methionine, L-cysteine, thiamine, nicotinic acid), nutritional requirements (phosphate, trace elements) and useful carbon sources (glucose, maltose, lactose, fructose, sucrose, acetate, L-proline, L-alanine). These were determined by microplate cultivations with online monitoring of biomass in a chemically defined medium and systematically omitting or substituting medium components. Persisting growth limitations were also detected, allowing further improvement of the chemically defined medium by the addition of glutamate group amino acids. Common complex media based on peptone and yeast extract were supplemented based on these findings. Optical density at the end of each cultivation of the improved peptone and yeast extract media roughly increased fivefold respectively. A maximum OD600 of 26.6 ± 0.7 (CDW: 17.1 ± 0.5 g/L) was reached with the improved yeast extract medium. Finally, culture performance and media improvement was analysed by measuring the oxygen transfer rate as well as the backscatter during shake flask cultivation.

Figure 1

Backscatter data from a microplate cultivation of S. pasteurii in modified chemically defined media to determine nutritional requirements and auxotrophic deficiencies. Illustrated are backscatter data from different growth curves after leaving out grouped or single components from the chemically defined medium (Table 1). Culture conditions: 48-well Flower Well Plate, filling volume 800 $\mathrm{\mu }$L, shaking frequency 1200 rpm, shaking diameter 3 mm and temperature 30 ${}^{\circ }$C. Here, arithmetic mean values derived from biological replicates (N = 2, N = 1 for w/o vitamins) are shown. The error bands depict the standard deviation.

Figure 2

Backscatter data from three different microplate cultivation experiments of S. pasteurii in supplemented chemically defined media to determine substrate growth limitations. Illustrated are backscatter data from different growth curves after adding grouped or single components. The reference data corresponds to cultivation in chemically defined medium (Table 1). The cultivation experiments a, b and c were stopped during stationary phase resulting in different total cultivation durations. Average pH of all cultures: 9.29 ± 0.12. Culture conditions: 48-well Flower Well Plate, filling volume 800 $\mathrm{\mu }$L, shaking frequency 1200 rpm, shaking diameter 3 mm and temperature 30 ${}^{\circ }$C. Here, arithmetic mean values derived from biological replicates (N = 3) are shown. The error bands depict the standard deviation.

Figure 3

Specific growth rates from microplate cultivation of S. pasteurii with substituted main carbon source. All cultivation experiments were performed in improved chemically defined media with modified carbonsource (Table 1, tripled components marked with asterisk). Culture conditions: 48-well Flower Well Plate, filling volume 800 $\mathrm{\mu }$L, shaking frequency 1200 rpm, shaking diameter 3 mm and temperature 30 ${}^{\circ }$C. Here, arithmetic mean values derived from biological replicates (N = 3, N = 6 for the references with and without glucose) are shown. The error bars depict the standard deviation.

Figure 4

Backscatter data from microplate cultivation of S. pasteurii in supplemented complex media. (a) Data from casein and soy peptone (CaSo) medium supplementation. (b) Data from yeast extract (YE) medium supplementation. Dissolved oxygen (DO) data indicates oxygen limitation between 10 and 14 h for the cultivation with the three supplemented YE media showing the highest improvement (see Supplementary Figure S7 online). This explains slightly linear growth during this period. Components are reported as follow: glucose Gluc, K${}_2$HPO${}_4$ PO4, trace elements Trace, thiamine-HCl and nicotinic acid Vitamins, L-methionine and L-cysteine S-amino, triple glutamate group Glutamate. Average pH of all cultures: 9.15 ± 0.60. Culture conditions: 48-well Flower Well Plate, filling volume 800 $\mathrm{\mu }$L, shaking frequency 1200 rpm, shaking diameter 3 mm and temperature 30 ${}^{\circ }$C. Here, arithmetic mean values derived from biological replicates (N = 2) are shown. The error bands depict the standard deviation.

Figure 5

Oxygen transfer rate (OTR) and backscatter of shake flask cultivations of S. pasteurii in different media. Backscatter data was generated using the Cell Growth Quantifier system and cannot be compared to backscatter data from microplate cultivation. Cultivation was carried out in (a) chemically defined medium, (b) improved chemically defined medium (c) improved YE medium. Cells were harvested when the OTR decreased to near 0 mmol/L/h. Average pH of all cultures: 9.26 ± 0.09. Culture conditions: 250 mL shake flasks, filling volume 10 mL, shaking frequency 300 rpm, shaking diameter 50 mm and temperature 30 ${}^{\circ }$C. Due to the experimental setup, no biological replicates were obtained simultaneously. However, the experiment was repeated with the same results (see Supplementary Figure S10).

Figure 6

OD600, CDW, urease activity and specific urease activity of shake flask cultivations of S. pasteurii in different media, corresponding to the OTR and backscatter data in Fig. 5. CDW was calculated with a predetermined correlation from OD600 data. The chemically defined medium is described in Table 1. The improved chemically defined medium is supplemented with glutamate group amino acids. The complex medium corresponds to the YE medium. OTR and backscatter data from cultivation with the simple YE medium can be found in Supplementary Figure S11. The improved complex medium is supplemented YE medium with glucose, phosphate, trace elements, thiamine-HCl, nicotinic acid, L-methionine, L-cysteine and glutamate group amino acids, according to the concentrations of Table 1, based on the results of Fig. 4b (purple line). Here, arithmetic mean values derived from technical replicates (N = 3) are shown. The error bars depict the standard deviation.