Background Chlamydomonas reinhardtii is gaining recognition as a promising expression system for the production of recombinant proteins. However, its performance as a cellular biofactory remains suboptimal, especially with respect to consistent expression of heterologous genes. Gene silencing mechanisms, position effect, and low nuclear transgene expression are major drawbacks for recombinant protein production in this model system. To unveil the molecular changes following transgene insertion, retention, and expression in this species, we genetically engineered C. reinhardtii wild type strain 137c (strain cc-125 mt+) to express the fluorescent protein mVenus and subsequently analysed its intracellular proteome. Results The obtained transgenic cell lines showed differences in abundance in more than 400 proteins, with multiple pathways altered post-transformation. Proteins involved in chromatin remodelling, translation initiation and elongation, and protein quality control and transport were found in lower abundance. On the other hand, ribosomal proteins showed higher abundance, a signal of ribosomal stress response. Conclusions These results provide new insights into the modifications of C. reinhardtii proteome after transformation, highlighting possible pathways involved in gene silencing. Moreover, this study identifies multiple protein targets for future genetic engineering approaches to improve the prospective use of C. reinhardtii as cell biofactory for industrial applications.

SummaryDiatoms are photosynthetic unicellular microalgae that drive global ecological phenomena in the biosphere and are emerging sustainable feedstock for an increasing number of industrial applications. Diatoms exhibit enormous taxonomic and genetic diversity, which often result in peculiar biochemical and biological traits. Transposable elements (TE) represent a substantial portion of diatom genomes and have been hypothesized to exert a relevant role in enriching genetic diversity and centrally contribute to genome evolution. Here, through long-read whole genome sequencing, we identified a novel Mutator-Like Element (MULE) in the model diatomPhaeodactylum tricornutum,and we report the direct observation of its mobilization within the course of one single laboratory experiment. Under selective conditions, this novel TE inactivated theuridine monophosphate synthase(UMPS) geneof P. tricornutum,one of the two only endogenous genetic loci currently targeted for selectable auxotrophy in functional genetics and genome editing applications.We report the first, real-time observation of the mobilization of a transposon in diatoms that possesses novel peculiar features. These include the combined presence of a MULE transposase domain with Zinc finger, SWIM-type domains, and of a diatom-specific E3 ubiquitin ligase of the zinc finger UBR type, which indicate a novel 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. Ultimately, this raises relevant concerns on the targeting of loci such asUMPSas selectable markers for functional genetics and biotechnological applications in diatoms.Significance StatementWe identified a novel DNA transposon in the diatomPhaeodactylum tricornutum. This new Mutator-Like Element encodes a transposase and a diatom-specific E3 ubiquitin ligase, which suggest a novel mobilization mechanism. We documented independent insertions in real-time, which spontaneously inactivated theuridine monophosphate synthase(UMPS) locus, a common selectable marker. We provide new insights on the role of transposons in diatom genome dynamics and evolution and on the unsuitability ofUMPSas selection locus in diatoms.

SUMMARY 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.

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.

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.

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

Cyanobacteria offer a good alternative to fungi for laccase production at industrial scales. Random mutagenesis approaches with ethyl methanesulfonate were used in combination with enzymatic assays screenings to select a mutant of Synechocystis sp. PCC 6803, A2, with enhanced extracellular laccase-like activity. Anthraquinone dye decolourisation assays revealed 7 % enhanced decolourisation in A2 relative to the wild type after 24 h. The comparison of microbiome structure and diversity of the wild type and A2 strains confirmed that the improved traits were due to the mutation(s) and not the associated bacteria. The newly isolated Synechocystis mutant is the first example of successful random mutagenesis of cyanobacteria for laccase production. Attempts to characterise the biochemical reaction and putative laccase in A2 strain were unsuccessful but will be the subject of further research. This study suggests that random mutagenesis as a powerful approach for generating cyanobacterial strains with enhanced laccase-like activity for prospective commercial applications.

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-β-cyclodextrine (MβCD) 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 MβCD 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 MβCD 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.

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.

A hybrid process combining a single compartment aerobic membrane bioreactor (MBR) and a membrane microalgal reactor (MMR) was evaluated for nutrient removal and microalgal biomass production.

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.

Microalgae are increasingly being considered for recombinant protein production because of low cultivation costs, absence of endotoxins and insusceptibility to human infectious agents. Despite these advantages, the yield of recombinant protein produced in microalgae is still low compared to more established expression systems and optimization at the genetic and cultivation levels is required for this new system to be economically viable. This study investigates the effect of biphasic temperature regimes on the yield of recombinant human interferon alpha 2a (IFN-alpha 2a), a therapeutic protein known for its anti-cancer and anti-viral properties, produced by the model green alga Chlamydomonas reinhardtii (Cr.IFN-alpha 2a). Biphasic growth is commonly employed to increase recombinant protein production in mammalian cell lines used for commercial production of therapeutic proteins, with a lowering of the temperature resulting in higher yields. In this study, lowering the temperature from 25 degrees C to 15 degrees C in mid-exponential growth phase increased the accumulation of Cr.IFN-alpha 2a by 3.3-fold while it slowed down the growth of the three C. reinhardtii transgenic lines tested. In contrast, a rise of temperature from 25 degrees C to 35 degrees C accelerated cell growth, while negatively impacting the production of Cr.IFN-alpha 2a. After a two-step chromatography purification, the Cr.IFN-alpha 2a produced was estimated to be 53% pure with a yield of 90 mu g/L of culture. The amino acid sequence of Cr.IFN-alpha 2a was confirmed by mass spectrometry. However, the anti-viral activity of Cr.IFN-alpha 2a was found to be 10 times lower than the human IFN-alpha 2a standard produced using E. coli when challenged in a cytopathic effect (CPE) assay, likely due to the formation of aggregates. While the molecular mechanisms driving the accumulation of Cr.IFN-alpha 2a at lower temperature remains unclear, our results support that reducing the temperature at the peak of expression is a valid strategy to increase the yield of recombinant Cr.IFN-alpha 2a in C. reinhardtii.

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.

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.

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.

A simple, efficient, and fast settling flocculation technique to harvest microalgal biomass was demonstrated using a proprietary cationic polyacrylamide flocculant for a freshwater (Chlorella vulgaris) and a marine (Phaeodactylum tricornutum) microalgal culture at their mid-stationary growth phase. The optimal flocculant doses were 18.9 and 13.7 mg/g of dry algal biomass for C. vulgaris and P. tricornutum, respectively (equivalent to 7 g per m3 of algal culture for both species). The obtained optimal dose was well corroborated with changes in cell surface charge, and culture solution optical density and turbidity. At the optimal dose, charge neutralization of 64 and 86% was observed for C. vulgaris and P. tricornutum algal cells, respectively. Algae recovery was independent of the culture solution pH in the range of pH 6 to 9. Algal biomass recovery was achieved of 100 and 90% for C vulgaris and P. tricornutum respectively, and over 98% medium recovery was achievable by simple decanting.

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.

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.

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.