Optimisation of Protein Extraction Methods for Metaproteomics of Freshwater Microbial Communities

Recent developments in the molecular biology toolbox allow access to the genetic structure and diversity of microbial communities. More specifically, the advances in metaproteomics allow us to identify the functional expression of the genome and link this information to the biogeochemical processes of an ecosystem. One of the challenges, in this field, is the optimisation of protein extraction methods from environmental samples where protein concentrations can be low and the presence of interfering substances high. Experiments were conducted by extracting proteins from freshwater microbial communities utilising an array of physical and chemical methods. We used protein yield and 1D SDS-PAGE resolution as deciding factors. Liquid N2 grinding and freeze-thaw cycles resulted, after purification, in an enhanced resolution and protein yield. Furthermore, regarding sample purification, the use of detergent removal columns resulted in higher yields but literature indicates that acetone precipitation is more efficient in removing interfering substances

Competitive growth experiments with a high-lipid Chlamydomonas reinhardtii mutant strain and its wild-type to predict industrial and ecological risks.

Key microalgal species are currently being exploited as biomanufacturing platforms using mass cultivation systems. The opportunities to enhance productivity levels or produce non-native compounds are increasing as genetic manipulation and metabolic engineering tools are rapidly advancing. Regardless of the end product, there are both environmental and industrial risks associated to open pond cultivation of mutant microalgal strains. A mutant escape could be detrimental to local biodiversity and increase the risk of algal blooms. Similarly, if the cultivation pond is invaded by a wild-type (WT) microalgae or the mutant reverts to WT phenotypes, productivity could be impacted. To investigate these potential risks, a response surface methodology was applied to determine the competitive outcome of two Chlamydomonas reinhardtii strains, a WT (CC-124) and a high-lipid accumulating mutant (CC-4333), grown in mixotrophic conditions, with differing levels of nitrogen and initial WT to mutant ratios. Results of the growth experiments show that mutant cells have double the exponential growth rate of the WT in monoculture. However, due to a slower transition from lag phase to exponential phase, mutant cells are outcompeted by the WT in every co-culture treatment. This suggests that, under the conditions tested, outdoor cultivation of the C. reinhardtii cell wall-deficient mutant strains does not carry a significant environmental risk to its WT in an escape scenario. Furthermore, lipid results show the mutant strain accumulates over 200% more TAGs per cell, at 50 mg L(-1) NH4Cl, compared to the WT, therefore, the fragility of the mutant strain could impact on overall industrial productivity

Biotechnological conversion of methane to methanol: evaluation of progress and potential

Sources of methane are numerous, and vary greatly in their use and sustainable credentials. A Jekyll and Hyde character, it is a valuable energy source present as geological deposits of natural gas, however it is also potent greenhouse gas, released during many waste management processes. Gas-to-liquid technologies are being investigated as a means to exploit and monetise non-traditional and unutilised methane sources. The product identified as having the greatest potential is methanol due to it being a robust, commercially mature conversion process from methane and its beneficial fuel characteristics. Commercial methane to methanol conversion requires high temperatures and pressures, in an energy intensive and costly process. In contrast methanotrophic bacteria perform the desired transformation under ambient conditions, using methane monooxygenase (MMO) enzymes. Despite the great potential of these bacteria a number of biotechnical difficulties are hindering progress towards an industrially suitable process. We have identified five major challenges that exist as barriers to a viable conversion process that, to our knowledge, have not previously been examined as distinct process challenges. Although biotechnological applications of methanotrophic bacteria have been reviewed in part, no review has comprehensively covered progress and challenges for a methane to methanol process from an industrial perspective. All published examples to date of methanotroph catalysed conversion of methane to methanol are collated, and standardised to allow direct comparison. The focus will be on conversion of methane to methanol by whole-cell, wild type, methanotroph cultures, and the potential for their application in an industrially relevant process. A recent shift in the research community focus from a mainly biological angle to an overall engineering approach, offers potential to exploit methanotrophs in an industrially relevant biotechnological gas-to-liquid process. Current innovations and future opportunities are discussed

The use of natural infochemicals for sustainable and efficient harvesting of the microalgae Scenedesmus spp. for biotechnology: insights from a meta-analysis.

Open raceway ponds are regarded as the most economically viable option for large-scale cultivation of microalgae for low to mid-value bio-products, such as biodiesel. However, improvements are required including reducing the costs associated with harvesting biomass. There is now a growing interest in exploiting natural ecological processes within biotechnology. We review how chemical cues produced by algal grazers induce colony formation in algal cells, which subsequently leads to their sedimentation. A statistical meta-analysis of more than 80 studies reveals that Daphnia grazers can induce high levels of colony formation and sedimentation in Scenedesmus obliquus and that these natural, infochemical induced sedimentation rates are comparable to using commercial chemical equivalents. These data suggest that natural ecological interactions can be co-opted in biotechnology as part of a promising, low energy and clean harvesting method for use in large raceway systems

A metaproteomic analysis of the response of a freshwater microbial community under nutrient enrichment.

Eutrophication can lead to an uncontrollable increase in algal biomass, which has repercussions for the entire microbial and pelagic community. Studies have shown how nutrient enrichment affects microbial species succession, however details regarding the impact on community functionality are rare. Here, we applied a metaproteomic approach to investigate the functional changes to algal and bacterial communities, over time, in oligotrophic and eutrophic conditions, in freshwater microcosms. Samples were taken early during algal and cyanobacterial dominance and later under bacterial dominance. 1048 proteins, from the two treatments and two timepoints, were identified and quantified by their exponentially modified protein abundance index. In oligotrophic conditions, Bacteroidetes express extracellular hydrolases and Ton-B dependent receptors to degrade and transport high molecular weight compounds captured while attached to the phycosphere. Alpha- and Beta-proteobacteria were found to capture different substrates from algal exudate (carbohydrates and amino acids, respectively) suggesting resource partitioning to avoid direct competition. In eutrophic conditions, environmental adaptation proteins from cyanobacteria suggested better resilience compared to algae in a low carbon nutrient enriched environment. This study provides insight into differences in functional microbial processes between oligo- and eutrophic conditions at different timepoints and highlights how primary producers control bacterial resources in freshwater environments

A systems biology approach to investigate the response of Synechocystis sp. PCC6803 to a high salt environment.

BACKGROUND: Salt overloading during agricultural processes is causing a decrease in crop productivity due to saline sensitivity. Salt tolerant cyanobacteria share many cellular characteristics with higher plants and therefore make ideal model systems for studying salinity stress. Here, the response of fully adapted Synechocystis sp. PCC6803 cells to the addition of 6% w/v NaCl was investigated using proteomics combined with targeted analysis of transcripts. RESULTS: Isobaric mass tagging of peptides led to accurate relative quantitation and identification of 378 proteins, and approximately 40% of these were differentially expressed after incubation in BG-11 media supplemented with 6% salt for 9 days. Protein abundance changes were related to essential cellular functional alterations. Differentially expressed proteins involved in metabolic responses were also analysed using the probabilitistic tool Mixed Model on Graphs (MMG), where the role of energy conversion through glycolysis and reducing power through pentose phosphate pathway were highlighted. Temporal RT-qPCR experiments were also run to investigate protein expression changes at the transcript level, for 14 non-metabolic proteins. In 9 out of 14 cases the mRNA changes were in accordance with the proteins. CONCLUSION: Synechocystis sp. PCC6803 has the ability to regulate essential metabolic processes to enable survival in high salt environments. This adaptation strategy is assisted by further regulation of proteins involved in non-metabolic cellular processes, supported by transcriptional and post-transcriptional control. This study demonstrates the effectiveness of using a systems biology approach in answering environmental, and in particular, salt adaptation questions in Synechocystis sp. PCC6803

Adapting the algal microbiome for growth on domestic landfill leachate

We aimed to improve algal growth rate on leachate by optimising the algal microbiome. An algal-bacterial consortium was enriched from landfill leachate and subjected to 24 months of adaptive laboratory evolution, increasing the growth rate of the dominant algal strain, Chlorella vulgaris, almost three-fold to 0.2 d^−1. A dramatic reduction in nitrate production suggested a shift in biological utilisation of ammoniacal-N, supported by molecular 16S rRNA taxonomic analyses, where Nitrosomonas numbers were not detected in the adapted consortium. A PICRUSt approach predicted metagenomic functional content and revealed a high number of sequences belonging to bioremediation pathways, including degradation of aromatic compounds, benzoate and naphthalene, as well as pathways known to be involved in algal-bacterial symbiosis. This study enhances our understanding of beneficial mechanisms in algal-bacterial associations in complex effluents, and ultimately enables the bottom-up design of optimised algal microbiomes for exploitation within industry

Outdoor pilot-scale cultivation and techno economic assessment of a novel omega-3 eicosapentaenoic acid over-producing Nannochloropsis oculata strain

Nannochloropsis oculata is a microalga that produces a significant amount of eicosapentaenoic acid (EPA). Cultivating strains in photobioreactors commercially can be challenging. We cultivated a mutant N. oculata strain, capable of accumulating >40 % EPA more than the wild-type strain at laboratory scale, in an outdoor 300 L pilot-scale photobioreactor, in the U.K. The mutant N. oculata recorded the highest amount of EPA quantity at 129.87 mg/g dry cell weight (DCW) compared to the wild-type strain, which accumulated 75.43 mg/g DCW. A techno-economic assessment (TEA) evaluated the feasibility of the study. Using specific combined cultivation scenarios, the process resulted in a positive net present value (NPV) and return on investment (ROI) at £52,156,484.46 and 607.22 % after 10 years, respectively. The TEA of the improved EPA quantity by M1 N. oculata showed the high processing cost could be overcome by high yields and an optimised operational strategy

Isolation and characterization of microsatellite loci from two inbreeding bark beetle species (Coccotrypes)

We developed 14 microsatellite markers in Coccotrypes carpophagus and 14 in C. dactyliperda. These loci will be used for studying genetic structure and the level of inbreeding in populations in the Canary Islands and Madeira. As a result of long-term inbreeding, genetic variability is relatively low in these bark beetle species. We found one to five alleles per locus in 29 C. carpophagus and 41 C. dactyliperda from various localities. Eleven of the markers developed for C. carpophagus amplified in C. dactyliperda and seven of the markers developed for C. dactyliperda amplified in C. carpophagus

Circular economy fertilization: Testing micro and macro algal species as soil improvers and nutrient sources for crop production in greenhouse and field conditions

Nutrient losses from agricultural land to freshwater and marine environments contribute to eutrophication and often to the growth of algal blooms. However, the potential benefits of recycling this algal biomass back to agricultural land for soil quality and crop nutrition in a “circular-economy” has received little attention. We tested the effects of algal additions to arable soil in greenhouse-grown garden peas, and field plots of spring wheat, on plant growth and nutrition and physical and chemical properties of the soil. Representatives of five algal species, which contrasted in elemental composition, were applied at 0.2, 2 and 4 g kg−1in the greenhouse and at 24 g m2in the field. These included the cyanobacteria Arthrospira platensis (Spirulina), the unicellular green algae Chlorella sp., the red seaweed Palmaria palmata, and the brown seaweeds Laminaria digitata and Ascophyllum nodosum. In the greenhouse at the highest application rates (4 g kg−1), Chlorella sp., and Spirulina increased soil total nitrogen and available phosphorus, and Spirulina also increased soil nitrate concentrations. P. palmata and L. digitata significantly increased soil inorganic (NH4+and NO3−) concentrations under all three application rates. Chlorella sp. significantly increased soil total P, N and C, available P, NH4+-N, and pea yield. Soil water-stable aggregates were unchanged by the algal additions in both the greenhouse and field study. In the field, 4 species (Chlorella sp. Spirulina, P. palmata and L. digitata) increased soil inorganic nitrogen concentrations, confirming their potential to recycle mineralizable nitrogen to agricultural soils, but no significant effects were found on wheat yields under the application rates tested