Cultivation of Mycolicibacterium spp. Mutants in Miniaturized and High-Throughput Format to Characterize Their Growth, Phytosterol Conversion Ability, and Resistance to the Steroid Products.

This chapter describes methods for cultivation and characterization of the growth of Mycolicibacterium spp. mutants in a microbioreactor system in the presence of steroids and/or phytosterols followed by high-throughput mass spectrometry analysis to describe their ability to convert phytosterols into the target steroid androstenedione (AD). We focus on Mycolicibacterium neoaurum NRRL B-3805 ΔkstD which can convert phytosterol into androstenedione (AD) as one of its major steroid products, and mutants thereof with increased tolerance towards this end-product. By using BioLector 48-well plates with optodes at the bottom of each well, bacterial growth can be monitored online despite the turbidity of the growth medium resulting from non-dissolved phytosterol and steroid particles. To cope with the large number of samples that accumulate during growth experiments in microbioreactors and similar formats (e.g., microtiter plates), protocols for extraction and subsequent RapidFire-MS analysis are presented. This reduces the analysis time per sample to 10 s from 10 min required for regular LC-MS analysis.

Bioconversion of Phytosterols into Androstenedione by Mycolicibacterium.

The chapter describes the bioconversion of phytosterols into androstenedione (AD) by Mycolicibacterium spp. in shake flasks and fermenters, as well as LC-MS-based methods for analysis of phytosterols and steroid products. Phytosterols are derived as by-products of vegetable oil refining and manufacture of wood pulp. They contain the same four-ring nucleus as steroids and may be converted to high-value steroids by removing the sidechain at C17 and minor changes at other sites in the ring structure. Many bacteria, including Mycolicibacterium spp., can degrade phytosterols. Mutants of Mycolicibacterium spp. unable of ring cleavage can, when growing on phytosterols, accumulate the steroid intermediates androstenedione (AD) and androstadienedione (ADD). The practical challenge with microbial conversion of phytosterols to steroids is that both the substrate and the product are virtually insoluble in water. In addition, some steroids, notably ADD, may be toxic for the cells. Two main strategies have been employed to overcome this challenge: the use of two-phase systems and the addition of chemically modified cyclodextrins. The latter method is used here. Defined cultivation and bioconversion media for both shake flask and fermenter are given, as well as hints how to minimize the practical problems due to the water-insoluble phytosterol. Sampling, sample extraction, and quantification of substrates and products using LC-MS analysis are described.

Towards a low CO2 emission building material employing bacterial metabolism (1/2): The bacterial system and prototype production.

The production of concrete for construction purposes is a major source of anthropogenic CO2 emissions. One promising avenue towards a more sustainable construction industry is to make use of naturally occurring mineral-microbe interactions, such as microbial-induced carbonate precipitation (MICP), to produce solid materials. In this paper, we present a new process where calcium carbonate in the form of powdered limestone is transformed to a binder material (termed BioZEment) through microbial dissolution and recrystallization. For the dissolution step, a suitable bacterial strain, closely related to Bacillus pumilus, was isolated from soil near a limestone quarry. We show that this strain produces organic acids from glucose, inducing the dissolution of calcium carbonate in an aqueous slurry of powdered limestone. In the second step, the dissolved limestone solution is used as the calcium source for MICP in sand packed syringe moulds. The amounts of acid produced and calcium carbonate dissolved are shown to depend on the amount of available oxygen as well as the degree of mixing. Precipitation is induced through the pH increase caused by the hydrolysis of urea, mediated by the enzyme urease, which is produced in situ by the bacterium Sporosarcina pasteurii DSM33. The degree of successful consolidation of sand by BioZEment was found to depend on both the amount of urea and the amount of glucose available in the dissolution reaction.