New insights into the influence of pre-culture on robust solvent production of C. acetobutylicum.

Clostridia are known for their solvent production, especially the production of butanol. Concerning the projected depletion of fossil fuels, this is of great interest. The cultivation of clostridia is known to be challenging, and it is difficult to achieve reproducible results and robust processes. However, existing publications usually concentrate on the cultivation conditions of the main culture. In this paper, the influence of cryo-conservation and pre-culture on growth and solvent production in the resulting main cultivation are examined. A protocol was developed that leads to reproducible cultivations of Clostridium acetobutylicum. Detailed investigation of the cell conservation in cryo-cultures ensured reliable cell growth in the pre-culture. Moreover, a reason for the acid crash in the main culture was found, based on the cultivation conditions of the pre-culture. The critical parameter to avoid the acid crash and accomplish the shift to the solventogenesis of clostridia is the metabolic phase in which the cells of the pre-culture were at the time of inoculation of the main culture; this depends on the cultivation time of the pre-culture. Using cells from the exponential growth phase to inoculate the main culture leads to an acid crash. To achieve the solventogenic phase with butanol production, the inoculum should consist of older cells which are in the stationary growth phase. Considering these parameters, which affect the entire cultivation process, reproducible results and reliable solvent production are ensured. KEY POINTS: • Both cryo- and pre-culture strongly impact the cultivation of C. acetobutylicum • Cultivation conditions of the pre-culture are a reason for the acid crash • Inoculum from cells in stationary growth phase ensures shift to solventogenesis.

Municipal green waste as substrate for the microbial production of platform chemicals.

In Germany alone, more than 5·106 tons of municipal green waste is produced each year. So far, this material is not used in an economically worthwhile way. In this work, grass clippings and tree pruning as examples of municipal green waste were utilized as feedstock for the microbial production of platform chemicals. A pretreatment procedure depending on the moisture and lignin content of the biomass was developed. The suitability of grass press juice and enzymatic hydrolysate of lignocellulosic biomass pretreated with an organosolv process as fermentation medium or medium supplement for the cultivation of Saccharomyces cerevisiae, Lactobacillus delbrueckii subsp. lactis, Ustilago maydis, and Clostridium acetobutylicum was demonstrated. Product concentrations of 9.4 gethanol L-1, 16.9 glactic acid L-1, 20.0 gitaconic acid L-1, and 15.5 gsolvents L-1 were achieved in the different processes. Yields were in the same range as or higher than those of reference processes grown in established standard media. By reducing the waste arising in cities and using municipal green waste as feedstock to produce platform chemicals, this work contributes to the UN sustainability goals and supports the transition toward a circular bioeconomy.

Fermentative α-Humulene Production from Homogenized Grass Clippings as a Growth Medium.

Green waste, e.g., grass clippings, is currently insufficiently recycled and has untapped potential as a valuable resource. Our aim was to use juice from grass clippings as a growth medium for microorganisms. Herein, we demonstrate the production of the sesquiterpene α-humulene with the versatile organism Cupriavidus necator pKR-hum on a growth medium from grass clippings. The medium was compared with established media in terms of microbial growth and terpene production. C. necator pKR-hum shows a maximum growth rate of 0.43 h-1 in the grass medium and 0.50 h-1 in a lysogeny broth (LB) medium. With the grass medium, 2 mg/L of α-humulene were produced compared to 10 mg/L with the LB medium. By concentrating the grass medium and using a controlled bioreactor in combination with an optimized in situ product removal, comparable product concentrations could likely be achieved. To the best of our knowledge, this is the first time that juice from grass clippings has been used as a growth medium without any further additives for microbial product synthesis. This use of green waste as a material represents a new bioeconomic utilization option of waste materials and could contribute to improving the economics of grass biorefineries.

Characterization of an Aerosol-Based Photobioreactor for Cultivation of Phototrophic Biofilms.

Phototrophic biofilms, in particular terrestrial cyanobacteria, offer a variety of biotechnologically interesting products such as natural dyes, antibiotics or dietary supplements. However, phototrophic biofilms are difficult to cultivate in submerged bioreactors. A new generation of biofilm photobioreactors imitates the natural habitat resulting in higher productivity. In this work, an aerosol-based photobioreactor is presented that was characterized for the cultivation of phototrophic biofilms. Experiments and simulation of aerosol distribution showed a uniform aerosol supply to biofilms. Compared to previous prototypes, the growth of the terrestrial cyanobacterium Nostoc sp. could be almost tripled. Different surfaces for biofilm growth were investigated regarding hydrophobicity, contact angle, light- and temperature distribution. Further, the results were successfully simulated. Finally, the growth of Nostoc sp. was investigated on different surfaces and the biofilm thickness was measured noninvasively using optical coherence tomography. It could be shown that the cultivation surface had no influence on biomass production, but did affect biofilm thickness.

Production of ß-carotene with Dunaliella salina CCAP19/18 at physically simulated outdoor conditions.

Batch growth and ß-carotene production of Dunaliella salina CCAP19/18 was investigated in flat-plate gas-lift photobioreactors with a light path of 2 cm, operated in physically simulated outdoor conditions. Dunaliella salina CCAP19/18 showed robust growth with respect to pH 8.0-9.0 and 15-35°C at increasing salinity, simulating the evaporation of open photobioreactors. The highest ß-carotene concentration of 25 mg L-1 (3 mg gCDW -1) was observed in batch processes at pH 8.5, 15-30°C and increasing salinity up to 110 g L-1, simulating a typical Mediterranean summer climate. Intracellular ß-carotene accumulation of D. salina CCAP19/18 was shown to be independent of light availability, although nutrient limitation (K2HPO4, MgSO4, and/or ammonium ferric citrate) seems to enable stable ß-carotene content in the algal cells despite increasing cell densities in the photobioreactor. Fully controlled, lab-scale photobioreactors simulating typical climate conditions of any region of interest are valuable tools for enabling a realistic characterization of microalgae on a laboratory scale, for production processes projected in open photobioreactor systems (e.g. thin-layer cascade photobioreactors).

Material utilization of green waste: a review on potential valorization methods.

Considering global developments like climate change and the depletion of fossil resources, the use of new and sustainable feedstocks such as lignocellulosic biomass becomes inevitable. Green waste comprises heterogeneous lignocellulosic biomass with low lignin content, which does not stem from agricultural processes or purposeful cultivation and therefore mainly arises in urban areas. So far, the majority of green waste is being composted or serves as feedstock for energy production. Here, the hitherto untapped potential of green waste for material utilization instead of conventional recycling is reviewed. Green waste is a promising starting material for the direct extraction of valuable compounds, the chemical and fermentative conversion into basic chemicals as well as the manufacturing of functional materials like electrodes for electro-biotechnological applications through carbonization. This review serves as a solid foundation for further work on the valorization of green waste.