There is a growing need of substrate flexibility for biobased production of energy and value-added products that allows the application of variable biodegradable residues within a circular economy. It can be used to balance fluctuating energy provision of other renewable sources. Hydrolysis presents one of the biggest limitations during anaerobic digestion. Methods to improve it will result in broader process applicability and improved integration into regional material cycles. Recently, one focus of anaerobic digestion research has been directed to systems with a separate hydrolysis–acidogenesis stage as it might be promised to improve process performance. Conditions can be adjusted to each class of microorganisms individually without harming methanogenic microorganisms. Extensive research of separate biomass pretreatment via biological, chemical, physical or mixed methods has been conducted. Nevertheless, several methods lack economic efficiency, have a high environmental impact or focus on specific substrates. Pretreatment via a separate hydrolysis stage as cell-driven biotransformation in a suspension might be an alternative that enables high yields, flexible feeding and production, and a better process control. In this review, we summarize existing technologies for microbial hydrolytic biotransformation in a separate reactor stage and the impacts of substrate, operational parameters, combined methods and process design as well as remaining challenges.

Background: Single-use rocking-motion-type bag bioreactors provide advantages compared to standard stirred tank bioreactors by decreased contamination risks, reduction of cleaning and sterilization time, lower investment costs, and simple and cheaper validation. Currently, they are widely used for cell cultures although their use for small and medium scale production of recombinant proteins with microbial hosts might be very attractive. However, the utilization of rocking- or wave-induced motion-type bioreactors for fast growing aerobic microbes is limited because of their lower oxygen mass transfer rate. A conventional approach to reduce the oxygen demand of a culture is the fed-batch technology. New developments, such as the BIOSTAT^® CultiBag RM system pave the way for applying advanced fed-batch control strategies also in rocking-motion-type bioreactors. Alternatively, internal substrate delivery systems such as EnBase^® Flo provide an opportunity for adopting simple to use fed-batch-type strategies to shaken cultures. Here, we investigate the possibilities which both strategies offer in view of high cell density cultivation of E. coli and recombinant protein production. Results: Cultivation of E. coli in the BIOSTAT^® CultiBag RM system in a conventional batch mode without control yielded an optical density (OD₆₀₀) of 3 to 4 which is comparable to shake flasks. The culture runs into oxygen limitation. In a glucose limited fed-batch culture with an exponential feed and oxygen pulsing, the culture grew fully aerobically to an OD₆₀₀ of 60 (20 g L^-1 cell dry weight). By the use of an internal controlled glucose delivery system, EnBase^® Flo, OD₆₀₀ of 30 (10 g L^-1 cell dry weight) is obtained without the demand of computer controlled external nutrient supply. EnBase^® Flo also worked well in the CultiBag RM system with a recombinant E. coli RB791 strain expressing a heterologous alcohol dehydrogenase (ADH) to very high levels, indicating that the enzyme based feed supply strategy functions well for recombinant protein production also in a rocking-motion-type bioreactor. Conclusions: Rocking-motion-type bioreactors may provide an interesting alternative to standard cultivation in bioreactors for cultivation of bacteria and recombinant protein production. The BIOSTAT^® Cultibag RM system with the single-use sensors and advanced control system paves the way for the fed-batch technology also to rocking-motion-type bioreactors. It is possible to reach cell densities which are far above shake flasks and typical for stirred tank reactors with the improved oxygen transfer rate. For more simple applications the EnBase^® Flo method offers an easy and robust solution for rocking-motion-systems which do not have such advanced control possibilities.

Milk kefir is a traditional fermented milk product whose consumption is becoming increasingly popular. The natural starter for kefir production is kefir grain, which consists of various bacterial and yeast species. At the industrial scale, however, kefir grains are rarely used due to their slow growth, complex application, bad reproducibility and high costs. Instead, mixtures of defined lactic acid bacteria and sometimes yeasts are applied, which alter sensory and functional properties compared to natural grain-based milk kefir. In order to be able to mimic natural starter cultures for authentic kefir production, it is a prerequisite to gain deep knowledge about the nature of kefir grains, its microbial composition, morphologic structure, composition of strains on grains and the impact of environmental parameters on kefir grain characteristics. In addition, it is very important to deeply investigate the numerous multi-dimensional interactions among different species, which play important roles on the formation and the functionality of grains.