Optimization of bioprocesses with Brewers' spent grain and Cellulomonas uda.

Brewers' spent grain (BSG) is a low-value by-product of the brewing process, which is produced in large quantities every year. In this study, the lignocellulosic feedstock (solid BSG) was used to optimize fermentations with Cellulomonas uda. Under aerobic conditions, maximum cellulase activities of 0.98 nkat∙mL-1, maximum xylanase activities of 5.00 nkat∙mL-1 and cell yields of 0.22 gCells∙gBSG -1 were achieved. Under anaerobic conditions, enzyme activities and cell yields were lower, but valuable liquid products (organic acids, ethanol) were produced with a yield of 0.41 gProd∙gBSG -1. The growth phase of the organisms was monitored by measuring extracellular concentrations of two fluorophores pyridoxin (aerobic) and tryptophan (anaerobic) and by cell count. By combining reductive with anaerobic conditions, the ratio of ethanol to acetate was increased from 1.08 to 1.59 molEtOH∙molAc -1. This ratio was further improved to 9.2 molEtOH∙molAc -1 by lowering the pH from 7.4 to 5.0 without decreasing the final ethanol concentration. A fermentation in a bioreactor with 15 w% BSG instead of 5 w% BSG quadrupled the acetate concentration, whilst ethanol was removed by gas stripping. This study provides various ideas for optimizing and monitoring fermentations with solid substrates, which can support feasibility and incorporation into holistic biorefining approaches in the future.

Brewers' spent grain as carbon source for itaconate production with engineered Ustilago maydis.

Brewers' spent grain (BSG) is produced worldwide in millions of tons during the beer brewing process. Due to its high content of structural carbohydrates, BSG is a promising material for being valorised in biorefineries. In this study three process routes for producing itaconate from BSG hydrolysates are presented using the previously engineered smut fungi UstilagomaydisMB215Δcyp3ΔPria1::Petef as whole-cell biocatalyst. Using a fermentation medium based on BSG hydrolysate a yield of 0.38gIta/gSugar and a productivity of 0.11gIta/(L·h) were achieved. The addition of detoxified hydrothermal supernatant to the fermentation medium did not result in improved performance parameters but resulted in a decreased yield (0.29gIta/gSugar) and productivity (0.053 gIta/(L·h)). Simultaneous saccharification and fermentation with hydrothermal pretreated BSG is possible, although at lower rate. In summary, the valorisation of BSG in fungal fermentations might complement the end-of-life options of this industrial side product.

Diffusion profiles in L. lactis biofilms under different conditions.

Despite being an important topic in biofilm research, we still know little about diffusion in biofilms. Emerging biofilms of Lactococcus lactis growing in custom-made flow-cells were monitored and diffusion constants across the height of the biofilms recorded. The biofilms showed different diffusional behavior with regard to flow rate and pH variations, despite growing to similar thickness. At a higher flow rate, the biofilm exhibits slower diffusion compared to the reference cultivation at lower flow rate. By increasing pH, the biofilm exhibited fast growth and little difference in diffusion compared to the reference cultivation. Furthermore, the diffusion inside of the biofilms differed depending on the position in the flow-cell. The present study reveals new insights in how external factors can affect structure and density of biofilms. The method can be reliably used for L. lactis biofilms with a thickness up to 120 µm.

Improved FRAP Measurements on Biofilms.

We expand the standard fluorescence recovery after photobleaching (FRAP) model introduced by Axelrod et al. in 1976. Our goal is to capture some of the following common artifacts observed in the fluorescence measurements obtained with a confocal laser scanning microscope in biofilms: 1) linear drift, 2) exponential decrease (due to bleaching during the measurements), 3) stochastic Gaussian noise, and 4) uncertainty in the exact time point of the onset of fluorescence recovery. To fit the resulting stochastic model to data from FRAP measurements and to estimate all unknown model parameters, we apply a suitably adapted Metropolis-Hastings algorithm. In this way, a more accurate estimation of the diffusion coefficient of the fluorophore is achieved. The method was tested on data obtained from FRAP measurements on a cultivated biofilm.

Addition of Riboflavin-Coupled Magnetic Beads Increases Current Production in Bioelectrochemical Systems via the Increased Formation of Anode-Biofilms.

Shewanella oneidensis is one of the best-understood model organisms for extracellular electron transfer. Endogenously produced and exported flavin molecules seem to play an important role in this process and mediate the connection between respiratory enzymes on the cell surface and the insoluble substrate by acting as electron shuttle and cytochrome-bound cofactor. Consequently, the addition of riboflavin to a bioelectrochemical system (BES) containing S. oneidensis cells as biocatalyst leads to a strong current increase. Still, an external application of riboflavin to increase current production in continuously operating BESs does not seem to be applicable due to the constant washout of the soluble flavin compound. In this study, we developed a recyclable electron shuttle to overcome the limitation of mediator addition to BES. Riboflavin was coupled to magnetic beads that can easily be recycled from the medium. The effect on current production and cell distribution in a BES as well as the recovery rate and the stability of the beads was investigated. The addition of synthesized beads leads to a more than twofold higher current production, which was likely caused by increased biofilm production. Moreover, 90% of the flavin-coupled beads could be recovered from the BESs using a magnetic separator.

Monitoring of biofilms grown on differentially structured metallic surfaces using confocal laser scanning microscopy.

Imaging of biofilms on opaque surfaces is a challenge presented to researchers especially considering pathogenic bacteria, as those typically grow on living tissue, such as mucosa and bone. However, they can also grow on surfaces used in industrial applications such as food production, acting as a hindrance to the process. Thus, it is important to understand bacteria better in the environment they actually have relevance in. Stainless steel and titanium substrata were line structured and dotted surface topographies for titanium substrata were prepared to analyze their effects on biofilm formation of a constitutively green fluorescent protein (GFP)-expressing Escherichia coli strain. The strain was batch cultivated in a custom built flow cell initially for 18 h, followed by continuous cultivation for 6 h. Confocal laser scanning microscopy (CLSM) was used to determine the biofilm topography. Biofilm growth of E. coli GFPmut2 was not affected by the type of metal substrate used; rather, attachment and growth were influenced by variable shapes of the microstructured titanium surfaces. In this work, biofilm cultivation in flow cells was coupled with the most widely used biofilm analytical technique (CLSM) to study the time course of growth of a GFP-expressing biofilm on metallic surfaces without intermittent sampling or disturbing the natural development of the biofilm.

Adhesion forces of the sea-water bacterium Paracoccus seriniphilus on titanium: Influence of microstructures and environmental conditions.

The bacterial attachment to surfaces is the first step of biofilm formation. This attachment is governed by adhesion forces which act between the bacterium and the substrate. Such forces can be measured by single cell force spectroscopy, where a single bacterium is attached to a cantilever of a scanning force microscope, and force-distance curves are measured. For the productive sea-water bacterium Paracoccus seriniphilus, pH dependent measurements reveal the highest adhesion forces at pH 4. Adhesion forces measured at salinities between 0% and 4.5% NaCl are in general higher for higher salinity. However, there is an exception for 0.9% where a higher adhesion force was measured than expected. These results are in line with zeta potential measurements of the bacterium, which also show an exceptionally low zeta potential at 0.9% NaCl. In the absence of macromolecular interactions, the adhesion forces are thus governed by (unspecific) electrostatic interactions, which can be adjusted by pH and ionic strength. It is further shown that microstructures on the titanium surface increase the adhesion force. Growth medium reduces the interaction forces dramatically, most probably through macromolecular bridging.

Removing biofilms from stainless steel without changing surface properties relevant for bacterial attachment.

The influence of oxygen (and argon) plasma cleaning and a base-acid cleaning procedure on stainless steel surfaces was studied. The main aim was to clean stainless steel samples from Paracoccus seriniphilus biofilms without changing the surface properties which are relevant for bacterial attachment to allow reuse in a biofilm reactor. It is shown that oxygen plasma cleaning, which very successfully removes the same kind of biofilm from titanium surfaces, is not suitable for stainless steel. It largely influences the surface chemistry by producing thick metal oxide layers of varying compositions and changing phenomenological surface properties such as wettability. A promising method without changing surface properties while cleaning satisfactorily is a combination of base and acid reagents at elevated temperature.

Analyzing the influence of microstructured surfaces on the lactic acid production of Lactobacillus delbrueckii lactis in a flow-through cell system.

Microorganisms growing in biofilms might be possible biocatalysts for future biotechnological production processes. Attached to a surface and embedded in an extracellular polymeric matrix, they create their preferred environment and form robust cultures for continuous systems. With the objective of implementing highly efficient processes, productive biofilms need to be understood comprehensively. In this study, the influence of microstructured metallic surfaces on biofilm productivity was researched. To conduct this study, titanium and stainless steel sheets were polished, micromilled, as well as coated with particles. Subsequently, the metal sheets were exposed to the lactic acid producing Lactobacillus delbrueckii subsp. lactis under laminar and homogeneous flow conditions in a custom-built flow cell. A proof-of-concept showed that biofilm formation in the system only occurred on the designated substratum. Following a 24-h batch cultivation for primary biofilm development, the culture was continuously provided with glucose containing medium. As different experimental series have indicated, the process resulted to be stable for up to eleven days. Primary metabolite productivity averaged around 6-7 g/(L h). Interestingly, the productivity was shown to be affected neither by the type of metal, nor by the applied microstructures. Nevertheless, a higher dry biomass weight determined on micro-milled substratum indicates a complementary differentiation of biofilm components in future experiments.