A new easy method for determination of surface adhesion of phototrophic biofilms.

Terrestrial cyanobacteria grow as phototrophic biofilms and offer a wide spectrum of interesting products. For cultivation of phototrophic biofilms different reactor concepts have been developed in the last years. One of the main influencing factors is the surface material and the adhesion strength of the chosen production strain. In this work a flow chamber was developed, in which, in combination with optical coherence tomography and computational fluid dynamics simulation, an easy analysis of adhesion forces between different biofilms and varied surface materials is possible. Hereby, differences between two cyanobacteria strains and two surface materials were shown. With longer cultivation time of biofilms adhesion increased in all experiments. Additionally, the content of extracellular polymeric substances was analyzed and its role in surface adhesion was evaluated. To test the comparability of obtained results from the flow chamber with other methods, analogous experiments were conducted with a rotational rheometer, which proved to be successful. Thus, with the presented flow chamber an easy to implement method for analysis of biofilm adhesion was developed, which can be used in future research for determination of suitable combinations of microorganisms with cultivation surfaces on lab scale in advance of larger processes.

Process Technologies of Cyanobacteria.

Although the handling and exploitation of cyanobacteria is associated with some challenges, these phototrophic bacteria offer great opportunities for innovative biotechnological processes. This chapter covers versatile aspects of working with cyanobacteria, starting with up-to-date in silico and in vitro screening methods for bioactive substances. Subsequently, common conservation techniques and vitality/viability estimation methods are compared and supplemented by own data regarding the non-invasive vitality evaluation via pulse amplitude modulated fluorometry. Moreover, novel findings about the influence the state of the pre-cultures have on main cultures are presented. The following sub-chapters deal with different photobioreactor-designs, with special regard to biofilm photobioreactors, as well as with heterotrophic and mixotrophic cultivation modes. The latter topic provides information from literature on successfully enhanced cyanobacterial production processes, augmented by own data.

Sporosarcina pasteurii can be used to print a layer of calcium carbonate.

When using microbiologically induced calcium carbonate precipitation (MICP) to produce calcium carbonate crystals in the cavities between mineral particles to consolidate them, the inhomogeneous distribution of the precipitated calcium carbonate poses a problem for the production of construction materials with consistent parameters. Various approaches have been investigated in the literature to increase the homogeneity of consolidated samples. One approach can be the targeted application of ureolytic organisms by 3D printing. However, to date, this possibility has been little explored in the literature. In this study, the potential to use MICP to print calcium carbonate layers on mineral particles will be investigated. For this purpose, a dispensing unit was modified to apply both a suspension of Sporosarcina pasteurii and a calcination solution containing urea and calcium chloride onto quartz sand. The study showed that after passing through the nozzle, S. pasteurii preserved consistent cell vitality and therefore its potential of MICP. Applying cell suspension and calcination solution through a printing nozzle resulted in a layer of calcium carbonate crystals on quartz sand. This observation demonstrated the proof of concept of printing calcium carbonate by MICP through the nozzle of a dispensing unit. Furthermore, it was shown that cell suspensions of S. pasteurii can be stored at 4°C for a period of 17 days while maintaining its optical density, urease activity and cell vitality and therefore the potential for MICP. This initial concept could be extended in further research to printing three-dimensional (3D) objects to solve the problem of homogeneity in consolidated mineral particles.

In vivo and in silico screening for antimicrobial compounds from cyanobacteria.

Due to the emerging rise of multi-drug resistant bacteria, the discovery of novel antibiotics is of high scientific interest. Through their high chemodiversity of bioactive secondary metabolites, cyanobacteria have proven to be promising microorganisms for the discovery of antibacterial compounds. These aspects make appropriate antibacterial screening approaches for cyanobacteria crucial. Up to date, screenings are mostly carried out using a phenotypic methodology, consisting of cyanobacterial cultivation, extraction, and inhibitory assays. However, the parameters of these methods highly vary within the literature. Therefore, the common choices of parameters and inhibitory assays are summarized in this review. Nevertheless, less frequently used method variants are highlighted, which lead to hits from antimicrobial compounds. In addition to the considerations of phenotypic methods, this study provides an overview of developments in the genome-based screening area, be it in vivo using PCR technique or in silico using the recent genome-mining method. Though, up to date, these techniques are not applied as much as phenotypic screening.

The Beneficial Effects of Cyanobacterial Co-Culture on Plant Growth.

Cyanobacteria are ubiquitous phototrophic prokaryotes that find a wide range of applications in industry due to their broad product spectrum. In this context, the application of cyanobacteria as biofertilizers and thus as an alternative to artificial fertilizers has emerged in recent decades. The benefit is mostly based on the ability of cyanobacteria to fix elemental nitrogen and make it available to the plants in a usable form. However, the positive effects of co-cultivating plants with cyanobacteria are not limited to the provision of nitrogen. Cyanobacteria produce numerous secondary metabolites that can be useful for plants, for example, they can have growth-promoting effects or increase resistance to plant diseases. The effects of biotic and abiotic stress can as well be reduced by many secondary metabolites. Furthermore, the biofilms formed by the cyanobacteria can lead to improved soil conditions, such as increased water retention capacity. To exchange the substances mentioned, cyanobacteria form symbioses with plants, whereby the strength of the symbiosis depends on both partners, and not every plant can form symbiosis with every cyanobacterium. Not only the plants in symbiosis benefit from the cyanobacteria, but also vice versa. This review summarizes the beneficial effects of cyanobacterial co-cultivation on plants, highlighting the substances exchanged and the strength of cyanobacterial symbioses with plants. A detailed explanation of the mechanism of nitrogen fixation in cyanobacterial heterocysts is given. Finally, a summary of possible applications of co-cultivation in the (agrar-)industry is given.

Passively immobilized cyanobacteria Nostoc species BB 92.2 in a moving bed photobioreactor (MBPBR): Design, cultivation, and characterization.

The cyanobacterium Nostoc sp. BB 92.3. had shown antibacterial activity. A cultivation as biofilm, a self-forming matrix of cells and extracellular polymeric substances, increased the antibacterial effect. A new photobioreactor system was developed that allows a surface-associated cultivation of Nostoc sp. as biofilm. High-density polyethylene carriers operated as a moving bed were selected as surface for biomass immobilization. This system, well established in heterotrophic wastewater treatment, was for the first time used for phototrophic biofilms. The aim was a cultivation on a large scale without inhibiting growth while maximizing immobilization. Cultivation in a small photobioreactor (1.5 L) with different volumetric filling degrees of carriers (13.4%-53.8%) in a batch process achieved immobilization rates of 70%-85% and growth was similar to a no-carrier-control. In a larger photobioreactor (65 L) essentially all of the biomass was immobilized on the carriers and the space-time yield of biomass (0.018 gcell dry weight L-1 day- ​​​​​​​1 ) was competitive compared to phototrophic biofilm cultivations from literature. The use of carriers increased the gas exchange in the reactor by a factor of 2.5-3 but doubled the mixing time. Enriched gassing with carbon dioxide resulted in a short-term increase in growth rate, but unexpectedly it also adversely changed the growth morphology.

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.

Insights into the Development of Phototrophic Biofilms in a Bioreactor by a Combination of X-ray Microtomography and Optical Coherence Tomography.

As productive biofilms are increasingly gaining interest in research, the quantitative monitoring of biofilm formation on- or offline for the process remains a challenge. Optical coherence tomography (OCT) is a fast and often used method for scanning biofilms, but it has difficulty scanning through more dense optical materials. X-ray microtomography (µCT) can measure biofilms in most geometries but is very time-consuming. By combining both methods for the first time, the weaknesses of both methods could be compensated. The phototrophic cyanobacterium Tolypothrix distorta was cultured in a moving bed photobioreactor inside a biocarrier with a semi-enclosed geometry. An automated workflow was developed to process µCT scans of the biocarriers. This allowed quantification of biomass volume and biofilm-coverage on the biocarrier, both globally and spatially resolved. At the beginning of the cultivation, a growth limitation was detected in the outer region of the carrier, presumably due to shear stress. In the later phase, light limitations could be found inside the biocarrier. µCT data and biofilm thicknesses measured by OCT displayed good correlation. The latter could therefore be used to rapidly measure the biofilm formation in a process. The methods presented here can help gain a deeper understanding of biofilms inside a process and detect any limitations.

Co-cultivation of diazotrophic terrestrial cyanobacteria and Arabidopsis thaliana.

Diazotrophic cyanobacteria are able to fix N2 from the atmosphere and release it as bioavailable nitrogen what other organisms can utilize. Thus, they could be used as living nitrogen supplier whereby the use of fertilizer could be reduced in agricultural industry what results in a decrease of laughing gas released during fertilizer production. The diazotroph cyanobacterium Desmonostoc muscorum (D. muscorum) was characterized in shake flasks cultivated in nitrogen-free and nitrogen-containing medium. Similar growth rates were reached in both cultivations and the release of ammonium by D. muscorum was detected under nitrogen depletion. Subsequently, D. muscorum was co-cultivated with Arabidopsis thaliana (A. thaliana) in nitrogen-free medium. Additionally, the plant was cultivated in nitrogen containing and nitrogen-free medium without D. muscorum as reference. A co-cultivation led to higher growth rates of the cyanobacterium and similar growth of A. thaliana with similar maximum photochemical efficiency of photosystem II compared to the growth of nitrogen containing medium. Further, accumulation of cyanobacterial cells around the roots of A. thaliana was detected, indicating a successfully induced artificial symbiosis. Based on these results, D. muscorum could be a promising cyanobacterium as living nitrogen supplier for plants.

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.

New procedure for separation and analysis of the main components of cyanobacterial EPS.

Phototrophic biofilms produce a matrix of extracellular polymeric substances (EPS), which holds the cells together and functions inter alia as nutrient storage and protection layer. EPS mainly consist of water, polysaccharides, proteins, lipids and nucleic acids as well as lysis and hydrolysis products which makes the composition very complex. Thus, rough simplifications are used and commonly one or at most two components of the EPS are examined. In this work a new procedure for separation and analysis of EPS in the main components (i) polysaccharides, (ii) proteins and (iii) lipids is presented with recovery rates of nearly 100 %. The method was established with synthetic EPS, which based on the composition of real EPS described in literature. Afterwards, the method was transferred to real EPS samples allowing a deeper insight in the composition of EPS from only one sample. The composition of EPS-extracts from Nostoc spec, cultivated under heterotrophic and mixotrophic batch and fed-batch conditions, was analysed during a cultivation period of 14 days. It was observed that mixotrophic cultivation led to higher amounts of carbohydrates, lipids and proteins than heterotrophic cultivation respectively, regardless of batch or fed-batch culture. While the amount of proteins in the EPS increased during the cultivation period, carbohydrates and lipids were dominant in the beginning and decreased afterwards.

Novel method enabling a rapid vitality determination of cyanobacteria.

Cyanobacteria represent a large group of bacteria with underestimated scientific potential. Recent studies indicate them as a great reservoir of secondary metabolites with antifungal, antiviral or antibacterial activity. However, common, well established research techniques cannot be easily adapted to these organisms. Slow growth rates and irregular cell aggregates constitute challenges for researchers dealing with cyanobacteria. In this work, we present an innovative new method enabling a quick, easy and economical vitality determination of cyanobacterial strains, as, e.g. required for the finding of optimal cryopreservation conditions. We were able to measure the vitality of previously cryopreserved and defrosted Trichocoleus sociatus samples within 45 min by means of their O2-production. For each run, a cell wet mass of only 0.5 g was required. By application of this method, we could find DMSO (5% v/v) and glycerin (15% v/v) to be the most promising cryoprotectants for the conservation of T. sociatus cells. DMSO and glycerin guaranteed a vitality rate of 80-90% and 60-70% after up to four weeks of cryopreservation, compared to fresh cell material.

Development of a lightweight multi-skin sheet photobioreactor for future cultivation of phototrophic biofilms on facades.

This article covers the development of a novel emerse photobioreactor (ePBR), using a polycarbonate multi-skin sheet (MSS), to cultivate terrestrial cyanobacteria as surface-associated phototrophic biofilms in an aerosol-based cultivation process. The aerosol, generated by ultrasonic transduction, moistens and nourishes the biofilm inside the multi-skin sheet emerse photobioreactor (MSSePBR). Advantages of the MSSePBR, such as its low weight design and reduced water consumption due to the usage of aerosol, simplify the development for future facade bioreactors. To develop the MSSePBR, surface roughness, static contact angle and luminous transmittance were investigated to characterize the properties of the cultivation surface for phototrophic cultivation. The polymeric MSS showed good luminous transmittance and proofed its optical suitability for the cultivation of terrestrial cyanobacteria. Using the MSSePBR, the terrestrial cyanobacteria Coleofasciculus chthonoplastes and Trichocoleus sociatus were cultivated with either ambient air, air with increased CO2 content or flue gas. The cultivation of terrestrial cyanobacteria showed higher productivities for biomass in the MSSePBR than in suspended systems. Cultivation with increased CO2 contents and flue gas was possible, thus a combination with flue gas treatment is feasible. An up-scaled prototype of the MSSePBR was introduced to show the possibilities for future industrial-sized and facade applications.

A semi-continuous process based on an ePBR for the production of EPS using Trichocoleus sociatus.

Biodiversity forms the basis for a large pool of potential products and productive organisms offered by terrestrial cyanobacteria. They are stuck together by EPS (extracellular polymeric substances) that can obtain antiviral, antibacterial or anti-inflammatory substances. Most stress conditions, e.g. drought, induce the production of protective EPS or biotechnological-products for pharmaceutical application. However, the growth of a phototrophic biofilm is limited under submerged conditions. Therefore, a semi-continuous process to produce EPS by cyanobacteria was developed in an aerosol-based ePBR (emerse photobioreactor) that imitates the natural habitat of terrestrial cyanobacteria. The process consists of a growth-phase (biomass production), followed by a dry-phase (EPS-production) and a consecutive extraction. The EPS-productivities of Trichocoleus sociatus (ranging from 0.03 to 0.04gL-1d-1) were 32 times higher than described in topic-related literature. In comparison to submerge cultivations in shaking flasks, the EPS-productivities were sevenfold higher. To ensure that the extraction solvent has no influence on cell viability, a cell-vitality-test was performed. However, no statistically significant difference between the amount of living and dead cells before and after the extraction was detected. A bioactivity assay was then performed to identify antimicrobial activity within EPS extracts from emerse and submerge cultivations. The EPS revealed an antibacterial effect against gram-negative bacteria (E. coli) which was two times higher than EPS from a submerged cultivation.