Spatial organization and proteome of a dual-species cyanobacterial biofilm alter among N2-fixing and non-fixing conditions.

Many disciplines have become increasingly interested in cyanobacteria, due to their ability to fix CO2 while using water and sunlight as electron and energy sources. Further, several species of cyanobacteria are also capable of fixing molecular nitrogen, making them independent of the addition of nitrate or ammonia. Thereby they hold huge potential as sustainable biocatalysts. Here, we look into a dual-species biofilm consisting of filamentous diazotrophic cyanobacteria Tolypothrix sp. PCC 7712 and heterotrophic bacteria Pseudomonas taiwanensis VLB 120 growing in a capillary biofilm reactor. Such systems have been reported to enable high cell densities continuous process operation. By combining confocal laser scanning and helium-ion microscopy with a proteomics approach, we examined these organisms' interactions under two nitrogen-feeding strategies: N2-fixing and nitrate assimilation. Not only did Pseudomonas facilitate the biofilm formation by forming a carpet layer on the surface area but also did N2-fixing biofilms show greater attachment to the surface. Pseudomonas proteins related to surface and cell attachments were observed in N2-fixing biofilms in particular. Furthermore, co-localized biofilm cells displayed a resilient response to extra shear forces induced by segmented media/air flows. This study highlights the role of Pseudomonas in the initial attachment process, as well as the effects of different nitrogen-feeding strategies and operation regimes on biofilm composition and growth. IMPORTANCE Cyanobacteria are highly interesting microorganisms due to their ability to synthesize sugars from CO2 while using water and sunlight as electron and energy sources. Further, many species are also capable of utilizing molecular nitrogen, making them independent of artificial fertilizers. In this study, such organisms are cultivated in a technical system, which enables them to attach to the reactor surface, and form three-dimensional structures termed biofilms. Biofilms achieve extraordinarily high cell densities. Furthermore, this growth format allows for continuous processing, both being essential features in biotechnological process development. Understanding biofilm growth and the influence technical settings and media composition have on biofilm maturation and stability are crucial for reaction and reactor design. These findings will help to open up these fascinating organisms for applications as sustainable, resource-efficient industrial workhorses.

Whole-genome sequence of the filamentous diazotrophic cyanobacterium Tolypothrix sp. PCC 7712 and its comparison with non-diazotrophic Tolypothrix sp. PCC 7601.

Cyanobacteria are highly promising microorganisms in forthcoming biotechnologies. Besides the systematic development of molecular tools for genetic engineering, the design of chassis strains and novel reactor concepts are in focus. The latter includes capillary biofilm reactors (CBR), which offer a high surface area-to-volume ratio and very high cell densities. In this context, Tolypothrix sp. PCC 7712 was found to be highly suited for this reactor system due to maximal surface coverage, extraordinarily strong biofilm attachment, and high biomass formation. Here, we provide the genome sequence of Tolypothrix sp. PCC 7712 to potentially allow targeted strain engineering. Surprisingly, it was almost identical to an available incomplete genome draft of Tolypothrix sp. PCC 7601. Thus, we completely sequenced this strain as well and compared it in detail to strain PCC 7712. Comparative genome analysis revealed 257 and 80 unique protein-coding sequences for strains PCC 7601 and PCC 7712, respectively. Clustering genomes based on average nucleotide identity (ANI) and 16S rRNA homology showed 99.98% similarity and only minor distance, respectively, between the two strains in contrast to 21 other cyanobacterial genomes. Despite these high similarities, both strains differ in the ability to fix atmospheric nitrogen and show specific sequence variations, which are discussed in the paper.

Evaluation of self-sustaining cyanobacterial biofilms for technical applications.

Cyanobacteria are potent microorganisms for sustainable photo-biotechnological production processes, as they are depending mainly on water, light, and carbon dioxide. Persisting challenges preventing their application include low biomass, as well as insufficient process stability and productivity. Here, we evaluate different cyanobacteria to be applied in a novel capillary biofilm reactor. Cultivated as biofilms, the organisms self-immobilize to the reactor walls, reach high biomass and enable long and robust production processes. As 'best performer' Tolypothrix sp. PCC 7712 emerged from this study. It reached the highest biomass in the reactors with 62.6 ± 6.34 gBDWL-1, produced 0.14 µmole H2 mgChl a -1h-1 under N2-fixing conditions, showed optimal surface coverage of the available growth surface, and only minor detachment in contrast to other tested species, highlighting its potential for photobiotechnology in the near future.

Fungal bioaugmentation of anaerobic digesters fed with lignocellulosic biomass: What to expect from anaerobic fungus Orpinomyces sp.

Energy-efficient biogas reactors are often designed and operated mimicking natural microbial ecosystems such as the digestive tracts of ruminants. Anaerobic fungi play a crucial role in the degradation of lignocellulose-rich fiber thanks to their high cellulolytic activity. Fungal bioaugmentation is therefore at the heart of our understanding of enhancing anaerobic digestion (AD). The efficiency of bioaugmentation with anaerobic fungus Orpinomyces sp. was evaluated in lignocellulose-based AD configurations. Fungal bioaugmentation increased the methane yield by 15-33% during anaerobic co-digestion of cow manure and selected cereal crops/straws. Harvesting stage of the crops was a decisive parameter to influence methane production together with fungal bioaugmentation. A more efficient fermentation process in the bioaugmented digesters was distinguished by relatively-higher abundance of Synergistetes, which was mainly represented by the genus Anaerobaculum. On the contrary, the composition of the methanogenic archaea did not change, and the majority of methanogens was assigned to Methanosarcina.

Bioaugmentation with Clostridium thermocellum to enhance the anaerobic biodegradation of lignocellulosic agricultural residues.

This study aimed to improve biomethane production from lignocellulosic biomass by assessing the impact of bioaugmentation with Clostridium thermocellum on the performance of anaerobic digesters at different inoculation ratios. The outputs of the digestion experiments revealed that bioaugmentation strategies with C. thermocellum increased the methane yield up to 39%. The sequencing analysis indicated that the indigenous microbial community was modified by the bioaugmentation. During the process of bioaugmentation, in the digester that was inoculated at the ratio of 20% (v:v), an increase in the abundance of Ruminococcaceae family led to a decrease in the Bacteroidaceae and Synergistaceae families. Furthermore, the metabolic products of the bioaugmented strains greatly influenced the diversity of the archaeal community and an increase in the abundance of Methanomicrobiales was observed.

Application of next-generation sequencing methods for microbial monitoring of anaerobic digestion of lignocellulosic biomass.

The anaerobic digestion of lignocellulosic wastes is considered an efficient method for managing the world's energy shortages and resolving contemporary environmental problems. However, the recalcitrance of lignocellulosic biomass represents a barrier to maximizing biogas production. The purpose of this review is to examine the extent to which sequencing methods can be employed to monitor such biofuel conversion processes. From a microbial perspective, we present a detailed insight into anaerobic digesters that utilize lignocellulosic biomass and discuss some benefits and disadvantages associated with the microbial sequencing techniques that are typically applied. We further evaluate the extent to which a hybrid approach incorporating a variation of existing methods can be utilized to develop a more in-depth understanding of microbial communities. It is hoped that this deeper knowledge will enhance the reliability and extent of research findings with the end objective of improving the stability of anaerobic digesters that manage lignocellulosic biomass.