Mobilization of a diatom mutator-like element (MULE) transposon inactivates the uridine monophosphate synthase (UMPS) locus in Phaeodactylum tricornutum.

Diatoms are photosynthetic unicellular microalgae that drive global ecological phenomena in the biosphere and are emerging as sustainable feedstock for an increasing number of industrial applications. Diatoms exhibit enormous taxonomic and genetic diversity, which often results in peculiar biochemical and biological traits. Transposable elements (TEs) represent a substantial portion of diatom genomes and have been hypothesized to exert a relevant role in enriching genetic diversity and making a core contribution to genome evolution. Here, through long-read whole-genome sequencing, we identified a mutator-like element (MULE) in the model diatom Phaeodactylum tricornutum, and we report the direct observation of its mobilization within the course of a single laboratory experiment. Under selective conditions, this TE inactivated the uridine monophosphate synthase (UMPS) gene of P. tricornutum, one of the few endogenous genetic loci currently targeted for selectable auxotrophy for functional genetics and genome-editing applications. We report the observation of a recently mobilized transposon in diatoms with unique features. These include the combined presence of a MULE transposase with zinc-finger SWIM-type domains and a diatom-specific E3 ubiquitin ligase of the zinc-finger UBR type, which are suggestive of a mobilization mechanism. Our findings provide new elements for the understanding of the role of TEs in diatom genome evolution and in the enrichment of intraspecific genetic variability.

Diverse RNA Viruses Associated with Diatom, Eustigmatophyte, Dinoflagellate, and Rhodophyte Microalgae Cultures.

Unicellular microalgae are of immense ecological importance with growing commercial potential in industries such as renewable energy, food, and pharmacology. Viral infections can have a profound impact on the growth and evolution of their hosts. However, very little is known of the diversity within, and the effect of, unicellular microalgal RNA viruses. In addition, identifying RNA viruses in these organisms that could have originated more than a billion years ago constitutes a robust data set to dissect molecular events and address fundamental questions in virus evolution. We assessed the diversity of RNA viruses in eight microalgal cultures, including representatives from the diatom, eustigmatophyte, dinoflagellate, red algae, and euglenid groups. Using metatranscriptomic sequencing combined with bioinformatic approaches optimized to detect highly divergent RNA viruses, we identified 10 RNA virus sequences, with nine constituting new viral species. Most of the newly identified RNA viruses belonged to the double-stranded Totiviridae, Endornaviridae, and Partitiviridae, greatly expanding the reported host range for these families. Two new species belonging to the single-stranded RNA viral clade Marnaviridae, commonly associated with microalgal hosts, were also identified. This study highlights that a substantial diversity of RNA viruses likely exists undetected within the unicellular microalgae. It also highlights the necessity for RNA viral characterization and for investigation of the effects of viral infections on microalgal physiology, biology, and growth, considering their environmental and industrial roles. IMPORTANCE Our knowledge of the diversity of RNA viruses infecting microbial algae-the microalgae-is minimal. However, describing the RNA viruses infecting these organisms is of primary importance at both the ecological and economic scales because of the fundamental roles these organisms play in aquatic environments and their growing value across a range of industrial fields. Using metatranscriptomic sequencing, we aimed to reveal the RNA viruses present in cultures of eight microalgae species belonging to the diatom, dinoflagellate, eustigmatophyte, rhodophyte, and euglena major clades of algae. Accordingly, we identified 10 new divergent RNA virus species belonging to RNA virus families as diverse as the double-stranded Totiviridae, Endornaviridae, and Partitiviridae and the single-stranded Marnaviridae. By expanding the known diversity of RNA viruses infecting unicellular eukaryotes, this study contributes to a better understanding of the early evolution of the virosphere and will inform the use of microalgae in industrial applications.

Effects of harvesting on morphological and biochemical characteristics of microalgal biomass harvested by polyacrylamide addition, pH-induced flocculation, and centrifugation.

The effects of microalgae harvesting methods on microalgal biomass quality were evaluated using three species namely the freshwater green alga Chlorella vulgaris, marine red alga Porphyridium purpureum and marine diatom Phaeodactylum tricornutum. Harvesting efficiencies of polyacrylamide addition, alkaline addition, and centrifugation ranged from 85 to 95, 59-92 and 100%, respectively, across these species. Morphology of the harvested cells (i.e. compromised cell walls) was significantly impacted by alkaline pH-induced flocculation for all three species. Over 50% of C. vulgaris cells were compromised with alkaline pH compared to < 10% with polyacrylamide and centrifugation. The metabolic profiles varied depending on harvesting methods. Species-specific decrease of certain metabolites was observed. These results suggest that the method of harvest can alter the metabolic profile of the biomass amongst the three harvesting methods, polyacrylamide addition showed higher harvesting efficiency with less compromised cells and higher retention of industry important biochemicals.

Unassembled cell wall proteins form aggregates in the extracellular space of Chlamydomonas reinhardtii strain UVM4.

The green microalga Chlamydomonas reinhardtii is emerging as a promising cell biofactory for secreted recombinant protein (RP) production. In recent years, the generation of the broadly used cell wall-deficient mutant strain UVM4 has allowed for a drastic increase in secreted RP yields. However, purification of secreted RPs from the extracellular space of C. reinhardtii strain UVM4 is challenging. Previous studies suggest that secreted RPs are trapped in a matrix of cell wall protein aggregates populating the secretome of strain UVM4, making it difficult to isolate and purify the RPs. To better understand the nature and behaviour of these extracellular protein aggregates, we analysed and compared the extracellular proteome of the strain UVM4 to its cell-walled ancestor, C. reinhardtii strain 137c. When grown under the same conditions, strain UVM4 produced a unique extracellular proteomic profile, including a higher abundance of secreted cell wall glycoproteins. Further characterization of high molecular weight extracellular protein aggregates in strain UVM4 revealed that they are largely comprised of pherophorins, a specific class of cell wall glycoproteins. Our results offer important new insights into the extracellular space of strain UVM4, including strain-specific secreted cell wall proteins and the composition of the aggregates possibly related to impaired RP purification. The discovery of pherophorins as a major component of extracellular protein aggregates will inform future strategies to remove or prevent aggregate formation, enhance purification of secreted RPs, and improve yields of recombinant biopharmaceuticals in this emerging cell biofactory. KEY POINTS: • Extracellular protein aggregates hinder purification of recombinant proteins in C. reinhardtii • Unassembled cell wall pherophorins are major components of extracellular protein aggregates • Known aggregate composition informs future strategies for recombinant protein purification.

Methyl Jasmonate and Methyl-ß-Cyclodextrin Individually Boost Triterpenoid Biosynthesis in Chlamydomonas Reinhardtii UVM4.

The commercialisation of valuable plant triterpenoids faces major challenges, including low abundance in natural hosts and costly downstream purification procedures. Endeavours to produce these compounds at industrial scale using microbial systems are gaining attention. Here, we report on a strategy to enrich the biomass of the biotechnologically-relevant Chlamydomonas reinhardtii strain UVM4 with valuable triterpenes, such as squalene and (S)-2,3-epoxysqualene. C. reinhardtii UVM4 was subjected to the elicitor compounds methyl jasmonate (MeJA) and methyl-ß-cyclodextrine (MßCD) to increase triterpene yields. MeJA treatment triggered oxidative stress, arrested growth, and altered the photosynthetic activity of the cells, while increasing squalene, (S)-2,3-epoxysqualene, and cycloartenol contents. Applying MßCD to cultures of C. reinhardtii lead to the sequestration of the two main sterols (ergosterol and 7-dehydroporiferasterol) into the growth medium and the intracellular accumulation of the intermediate cycloartenol, without compromising cell growth. When MßCD was applied in combination with MeJA, it counteracted the negative effects of MeJA on cell growth and physiology, but no synergistic effect on triterpene yield was observed. Together, our findings provide strategies for the triterpene enrichment of microalgal biomass and medium.

A comprehensive analysis of an effective flocculation method for high quality microalgal biomass harvesting.

Flocculation is a low-cost harvesting technique for microalgae biomass production, but flocculation efficiency is species dependent. In this study, we investigated the efficacy of two synthetic (polyacrylamide) and one natural (chitosan) flocculants against three algal species: the cyanobacterium Synechocystis sp., the freshwater Chlorella vulgaris, and the marine Phaeodactylum tricornutum at laboratory and pilot scales to evaluate harvesting efficiency, biomass integrity and media recycling. Growth phase affected the harvesting efficiency of the eukaryotic microalgae. The flocculation was optimal at stationary phase with high flocculation efficiency achieved using polyacrylamides at 24-36 mg/g dry weight. The effect of the flocculants on the harvested biomass was investigated. The flocculated Synechocystis sp. showed a higher proportion of compromised cells compared to C. vulgaris and P. tricornutum likely due to differences in cell walls composition. Compromised cells could lead to the release of valuable products into the surrounding growth media during flocculation. The residual culture media was recycled once with no impact on cell growth for all treatments and algal species. The flocculation technique was demonstrated at pilot-scale using 350 L microalgal suspension, showing an efficiency of 82-90% at a polyacrylamide dosage of 6.5-10 mg/L. This efficiency and the biomass quality are comparable to the laboratory-scale results. Overall, results indicate that polyacrylamide flocculants work on a wide range of species without the need for pre-treatment. The information generated in this study can contribute to making the microalgae industry more competitive.

Harvesting Porphyridium purpureum using polyacrylamide polymers and alkaline bases and their impact on biomass quality.

This study aims to examine the flocculation efficiency of Porphyridium purpureum (i.e. a red marine microalga with high content of pigments and fatty acids) grown in seawater medium using polyacrylamide polymers and alkaline flocculation. Polymers Flopam™ and FO3801 achieved the highest flocculation efficiency of over 99% at the optimal dose of 21 mg per g of dry biomass through charge neutralisation and bridging mechanism. The addition of sodium hydroxide, potassium hydroxide, and sodium carbonate also achieved flocculation efficiency of 98 and 91%, respectively, but high doses were required (i.e. > 500 mg per g of dry biomass). Calcium hydroxide was not as effective and could only achieve 75% flocculation efficiency. Precipitation of magnesium hydroxide was identified as the major cause of hydroxide-induced flocculation. On the other hand, sodium carbonate addition induced flocculation via both magnesium and calcium carbonate co-precipitation. The large mass of precipitates caused a sweeping effect and enmeshed the microalgal cells to trigger sedimentation. Cell membrane integrity analysis of flocculated P. purpureum indicated that polyacrylamide polymers led to significant compromised cells (i.e. 96%), compared to the alkaline bases (70-96% compromised cells). These results appear to be the first to demonstrate the high efficiency of polyacrylamide polymer and alkaline flocculation of P. purpureum but at the expense of the biomass quality.

Biomethane production from anaerobic co-digestion at wastewater treatment plants: A critical review on development and innovations in biogas upgrading techniques.

Anaerobic co-digestion (AcoD) can utilise spare digestion capacity at existing wastewater treatment plants (WWTP) to generate surplus biogas beyond the plant's internal energy requirement. Data from industry reports and the peer-reviewed literature show that through AcoD, numerous examples of WWTPs have become net energy producers, necessitating other high-value applications for surplus biogas. A globally emerging trend is to upgrade biogas to biomethane, which can then be used as town gas or transport fuel. Water, organic solvent and chemical scrubbing, pressure swing adsorption, membrane separation, and cryogenic technology are commercially available CO2 removal technologies for biogas upgrade. Although water scrubbing is currently the most widely applied technology due to low capital and operation cost, significant market growth in membrane separation has been seen over the 2015-2019 period. Further progress in materials engineering and sciences is expected and will further enhance the membrane separation competitiveness for biogas upgrading. Several emerging biotechnologies to i) improve biogas quality from AcoD; ii) accelerate the absorption rate, and iii) captures CO2 in microalgal culture have also been examined and discussed in this review. Through a combination of AcoD and biogas upgrade, more WWTPs are expected to become net energy producers.

Emerging Technologies in Algal Biotechnology: Toward the Establishment of a Sustainable, Algae-Based Bioeconomy.

Mankind has recognized the value of land plants as renewable sources of food, medicine, and materials for millennia. Throughout human history, agricultural methods were continuously modified and improved to meet the changing needs of civilization. Today, our rapidly growing population requires further innovation to address the practical limitations and serious environmental concerns associated with current industrial and agricultural practices. Microalgae are a diverse group of unicellular photosynthetic organisms that are emerging as next-generation resources with the potential to address urgent industrial and agricultural demands. The extensive biological diversity of algae can be leveraged to produce a wealth of valuable bioproducts, either naturally or via genetic manipulation. Microalgae additionally possess a set of intrinsic advantages, such as low production costs, no requirement for arable land, and the capacity to grow rapidly in both large-scale outdoor systems and scalable, fully contained photobioreactors. Here, we review technical advancements, novel fields of application, and products in the field of algal biotechnology to illustrate how algae could present high-tech, low-cost, and environmentally friendly solutions to many current and future needs of our society. We discuss how emerging technologies such as synthetic biology, high-throughput phenomics, and the application of internet of things (IoT) automation to algal manufacturing technology can advance the understanding of algal biology and, ultimately, drive the establishment of an algal-based bioeconomy.

Perspectives for Glyco-Engineering of Recombinant Biopharmaceuticals from Microalgae.

Microalgae exhibit great potential for recombinant therapeutic protein production, due to lower production costs, immunity to human pathogens, and advanced genetic toolkits. However, a fundamental aspect to consider for recombinant biopharmaceutical production is the presence of correct post-translational modifications. Multiple recent studies focusing on glycosylation in microalgae have revealed unique species-specific patterns absent in humans. Glycosylation is particularly important for protein function and is directly responsible for recombinant biopharmaceutical immunogenicity. Therefore, it is necessary to fully characterise this key feature in microalgae before these organisms can be established as industrially relevant microbial biofactories. Here, we review the work done to date on production of recombinant biopharmaceuticals in microalgae, experimental and computational evidence for N- and O-glycosylation in diverse microalgal groups, established approaches for glyco-engineering, and perspectives for their application in microalgal systems. The insights from this review may be applied to future glyco-engineering attempts to humanize recombinant therapeutic proteins and to potentially obtain cheaper, fully functional biopharmaceuticals from microalgae.

Genome sequencing as a new window into the microbial community of membrane bioreactors - A critical review.

Recent developed sequencing techniques have resulted in a new and unprecedented way to study biological wastewater treatment, in which most organisms are uncultivable. This review provides (i) an insight on state-of-the-art sequencing techniques and their limitations; (ii) a critical assessment of the microbial community in biological reactor and biofouling layer in a membrane bioreactor (MBR). The data from high-throughput sequencing has been used to infer microbial growth conditions and metabolisms of microorganisms present in MBRs at the time of sampling. These data shed new insight to two fundamental questions about a microbial community in the MBR process namely the microbial composition (who are they?) and the functions of each specific microbial assemblage (what are their function?). The results to date also highlight the complexity of the microbial community growing on MBRs. Environmental conditions are dynamic and diverse, and can influence the diversity and structural dynamics of any given microbial community for wastewater treatment. The benefits of understanding the structure of microbial communities on three major aspects of the MBR process (i.e. nutrient removal, biofouling control, and micropollutant removal) were symmetrically delineated. This review also indicates that the deployment of microbial community analysis for a practical engineering context, in terms of process design and system optimization, can be further realized.

Application of a novel molecular technique to characterise the effect of settling on microbial community composition of activated sludge.

Activated sludge (AS) and return activated sludge (RAS) microbial communities from three full-scale municipal wastewater treatment plants (denoted plant A, B and C) were compared to assess the impact of sludge settling (i.e. gravity thickening in the clarifier) and profile microorganisms responsible for nutrient removal and reactor foaming. The results show that all three plants were dominated with microbes in the phyla of Proteobacteria, Bacteroidetes, Verrucomicrobia, Actinobacteria, Chloroflexi, Firmicutes, Nitrospirae, Spirochaetae, Acidobacteria and Saccharibacteria. AS and RAS shared above 80% similarity in the microbial community composition, indicating that sludge thickening does not significantly alter the microbial composition. Autotrophic and heterotrophic nitrifiers were present in the AS. However, the abundance of autotrophic nitrifiers was significantly lower than that of the heterotrophic nitrifiers. Thus, ammonium removal at these plants was achieved mostly by heterotrophic nitrification. Microbes that can cause foaming were at 3.2% abundance, and this result is well corroborated with occasional aerobic biological reactor foaming. By contrast, these microbes were not abundant (<2.1%) at plant A and C, where aerobic biological reactor foaming has not been reported.

Impacts of mixing on foaming, methane production, stratification and microbial community in full-scale anaerobic co-digestion process.

This study investigated the impact of mixing on key factors including foaming, substrate stratification, methane production and microbial community in three full scale anaerobic digesters. Digester foaming was observed at one plant that co-digested sewage sludge and food waste, and was operated without mixing. The lack of mixing led to uneven distribution of total chemical oxygen demand (tCOD) and volatile solid (VS) as well as methane production within the digester. 16S rRNA gene-based community analysis clearly differentiated the microbial community from the top and bottom. By contrast, foaming and substrate stratification were not observed at the other two plants with internal circulation mixing. The abundance of methanogens (Methanomicrobia) at the top was about four times higher than at the bottom, correlating to much higher methane production from the top verified by ex-situ biomethane assay, causing foaming. This result is consistent with foaming potential assessment of digestate samples from the digester.

Molecular physiology reveals ammonium uptake and related gene expression in the seagrass Zostera muelleri.

Seagrasses are important marine foundation species, which are presently threatened by coastal development and global change worldwide. The molecular mechanisms that drive seagrass responses to anthropogenic stresses, including elevated levels of nutrients such as ammonium, remains poorly understood. Despite the evidence that seagrasses can assimilate ammonium by using glutamine synthetase (GS)/glutamate synthase (glutamine-oxoglutarate amidotransferase or GOGAT) cycle, the regulation of this fundamental metabolic pathway has never been studied at the gene expression level in seagrasses so far. Here, we combine (i) reverse transcription quantitative real-time PCR (RT-qPCR) to measure expression of key genes involved in the GS/GOGAT cycle, and (ii) stable isotope labelling and mass spectrometry to investigate 15N-ammonium assimilation in the widespread Australian species Zostera muelleri subsp. capricorni (Z. muelleri). We demonstrate that exposure to a pulse of ammonium in seawater can induce changes in GS gene expression of Z. muelleri, and further correlate these changes in gene expression with 15N-ammonium uptake rate in above- and below-ground tissue.

Influence of inoculum and anode surface properties on the selection of Geobacter-dominated biofilms.

This study evaluated the impact of inoculum source and anode surface modification (carboxylate -COO(-) and sulfonamide -SO2NH2 groups) on the microbial composition of anode-respiring biofilms. These two factors have not previously been considered in detail. Three different inoculum sources were investigated, a dry aerobic soil, brackish estuarine mud and freshwater sediment. The biofilms were selected using a poised anode (-0.36 V vs Ag/AgCl) and acetate as the electron donor in a three-electrode configuration microbial fuel cell (MFC). Population profiling and cloning showed that all biofilms selected were dominated by Geobacter sp., although their electrochemical properties varied depending on the source inoculum and electrode surface modification. These findings suggest that Geobacter sp. are widespread in soils, even those that do not provide a continuously anaerobic environment, and are better at growing in the MFC conditions than other bacteria.

Maintenance of Geobacter-dominated biofilms in microbial fuel cells treating synthetic wastewater.

Geobacter-dominated biofilms can be selected under stringent conditions that limit the growth of competing bacteria. However, in many practical applications, such stringent conditions cannot be maintained and the efficacy and stability of these artificial biofilms may be challenged. In this work, biofilms were selected on low-potential anodes (-0.36 V vs Ag/AgCl, i.e. -0.08 V vs SHE) in minimal acetate or ethanol media. Selection conditions were then relaxed by transferring the biofilms to synthetic wastewater supplemented with soil as a source of competing bacteria. We tracked community succession and functional changes in these biofilms. The Geobacter-dominated biofilms showed stability in their community composition and electrochemical properties, with Geobacter sp. being still electrically active after six weeks in synthetic wastewater with power densities of 100±19 mW·m(-2) (against 74±14 mW·m(-2) at week 0) for all treatments. After six weeks, the ethanol-selected biofilms, despite their high taxon richness and their efficiency at removing the chemical oxygen demand (0.8 g·L(-1) removed against the initial 1.3 g·L(-1) injected), were the least stable in terms of community structure. These findings have important implications for environmental microbial fuel cells based on Geobacter-dominated biofilms and suggest that they could be stable in challenging environments.

Influence of anode potentials on selection of Geobacter strains in microbial electrolysis cells.

Through their ability to directly transfer electrons to electrodes, Geobacter sp. are key organisms for microbial fuel cell technology. This study presents a simple method to reproducibly select Geobacter-dominated anode biofilms from a mixed inoculum of bacteria using graphite electrodes initially poised at -0.25, -0.36 and -0.42 V vs. Ag/AgCl. The biofilms all produced maximum power density of approximately 270 m Wm(-2) (projected anode surface area). Analysis of 16S rRNA genes and intergenic spacer (ITS) sequences found that the biofilm communities were all dominated by bacteria closely related to Geobacter psychrophilus. Anodes initially poised at -0.25 V reproducibly selected biofilms that were dominated by a strain of G. psychrophilus that was genetically distinct from the strain that dominated the -0.36 and -0.42 V biofilms. This work demonstrates for the first time that closely related strains of Geobacter can have very different competitive advantages at different anode potentials.