Evaluation of planktonic cyanobacteria in Peruvian freshwater lentic water bodies: prevalence and regulatory framework to aid policy making.

Increasing reports of cyanobacteria or cyanotoxins around the world expose a major threat for the environment, animal, and human health. Current water treatment processes are ineffective at eliminating cyanotoxins; hence, risk management relies mostly on early detection and on the development of specific regulatory frameworks. In developed countries, well-documented monitoring activities offer a good assessment of the cyanobacterial and/or cyanotoxin status and are used to prevent intoxications. In developing countries such as Peru, despite their potential threat to the environment and public health, cyanobacteria and cyanotoxins are still poorly studied. We found that the regulatory measures regarding cyanobacteria and/or cyanotoxin are almost non-existent. We also present and discuss some examples of recent monitoring efforts underwent by isolated local authorities and scientific reports that, whereas limited, may provide some important insights to be considered nationally. A revision of the available information of planktonic cyanobacteria or cyanotoxins in Peruvian freshwater lentic water bodies revealed a total of 50 documented reports of 15 different genera across 19 water bodies, including the reported highly toxic Dolichospermum and Microcystis. A unique case of microcystin-LR has been documented. We propose some recommendations to be implemented to improve potential toxic cyanobacteria risk management that include incorporating a widespread monitoring of cyanobacterial communities in lakes and reservoirs used for human consumption via specific guidelines. Aligning Peruvian regulations on cyanobacteria and cyanotoxins to international standards may also support law enforcement and ensure compliance.

Evaluation of low-cost SARS-CoV-2 RNA purification methods for viral quantification by RT-qPCR and next-generation sequencing analysis: Implications for wider wastewater-based epidemiology adoption.

Based Epidemiology (WBE) consists of quantifying biomarkers in sewerage systems to derive real-time information on the health and/or lifestyle of the contributing population. WBE usefulness was vastly demonstrated in the context of the COVID-19 pandemic. Many methods for SARS-CoV-2 RNA determination in wastewater were devised, which vary in cost, infrastructure requirements and sensitivity. For most developing countries, implementing WBE for viral outbreaks, such as that of SARS-CoV-2, proved challenging due to budget, reagent availability and infrastructure constraints. In this study, we assessed low-cost methods for SARS-CoV-2 RNA quantification by RT-qPCR, and performed variant identification by NGS in wastewater samples. Results showed that the effect of adjusting pH to 4 and/or adding MgCl2 (25 mM) was negligible when using the adsorption-elution method, as well as basal physicochemical parameters in the sample. In addition, results supported the standardized use of linear rather than plasmid DNA for a more accurate viral RT-qPCR estimation. The modified TRIzol-based purification method in this study yielded comparable RT-qPCR estimation to a column-based approach, but provided better NGS results, suggesting that column-based purification for viral analysis should be revised. Overall, this work provides evaluation of a robust, sensitive and cost-effective method for SARS-CoV-2 RNA analysis that could be implemented for other viruses, for a wider WEB adoption.

Spatiotemporal Surveillance of SARS-CoV-2 in the Sewage of Three Major Urban Areas in Peru: Generating Valuable Data Where Clinical Testing Is Extremely Limited.

Peru has been severely affected by the COVID-19 pandemic. By January 2022, Peru had surpassed 200 000 COVID-19 deaths, constituting the highest death rate per capita worldwide. Peru has had several limitations during the pandemic: insufficient testing access, limited contact tracing, a strained medical infrastructure, and many economic hurdles. These limitations hindered the gathering of accurate information about infected individuals with spatial resolution in real time, a critical aspect of effectively controlling the pandemic. Wastewater monitoring for SARS-CoV-2 RNA offered a promising alternative for providing needed population-wide information to complement health care indicators. In this study, we demonstrate the feasibility and value of implementing a decentralized SARS-CoV-2 RNA wastewater monitoring system to assess the spatiotemporal distribution of COVID-19 in three major cities in Peru: Lima, Callao, and Arequipa. Our data on viral loads showed the same trends as health indicators such as incidence and mortality. Furthermore, we were able to identify hot spots of contagion within the surveyed urban areas to guide the efforts of health authorities. Viral decay in the sewage network of the cities studied was found to be negligible (<2%). Overall, our results support wastewater monitoring for SARS-CoV-2 as a valuable and cost-effective tool for monitoring the COVID-19 pandemic in the Peruvian context.

Assessing cellulose microfibrillar structure changes due to cellulase action.

There is a need to understand how cellulose structural properties impact productive cellulase-cellulose interactions toward solving the mechanisms of the heterogeneous reaction. We coupled biochemical studies of cellulose hydrolysis by a purified Trichoderma reesei Cel7A (TrCel7A) cellobiohydrolase with atomic force microscopy (AFM) to study the impact of the cellulolytic activity on the fibrillar structure of cellulose. Bacterial cellulose (BC) fibrils were hydrolyzed by TrCel7A then immobilized by hydrophobic interactions on glass for AFM imaging. Commonly used methods to culture and isolate cellulose fibrils resulted in significant oxidation of the reducing-ends but minimal oxidation along the fibrils. We observed extensive fibrillation of BC fibrils to ∼3 nm microfibrils during the course of hydrolysis by TrCel7A, leaving thinned un-fibrillated recalcitrant fibrils at >80% hydrolysis extents. Additionally, this remaining fraction appeared to be segmented along the fibril length.

Starch self-processing in transgenic sweet potato roots expressing a hyperthermophilic α-amylase.

Sweet potato is a major crop in the southeastern United States, which requires few inputs and grows well on marginal land. It accumulates large quantities of starch in the storage roots and has been shown to give comparable or superior ethanol yields to corn per cultivated acre in the southeast. Starch conversion to fermentable sugars (i.e., for ethanol production) is carried out at high temperatures and requires the action of thermostable and thermoactive amylolytic enzymes. These enzymes are added to the starch mixture impacting overall process economics. To address this shortcoming, the gene encoding a hyperthermophilic α-amylase from Thermotoga maritima was cloned and expressed in transgenic sweet potato, generated by Agrobacterium tumefaciens-mediated transformation, to create a plant with the ability to self-process starch. No significant enzyme activity could be detected below 40°C, but starch in the transgenic sweet potato storage roots was readily hydrolyzed at 80°C. The transgene did not affect normal storage root formation. The results presented here demonstrate that engineering plants with hyperthermophilic glycoside hydrolases can facilitate cost effective starch conversion to fermentable sugars. Furthermore, the use of sweet potato as an alternative near-term energy crop should be considered.

Plant cell calcium-rich environment enhances thermostability of recombinantly produced alpha-amylase from the hyperthermophilic bacterium Thermotoga maritime.

In the industrial processing of starch for sugar syrup and ethanol production, a liquefaction step is involved where starch is initially solubilized at high temperature and partially hydrolyzed with a thermostable and thermoactive alpha-amylase. Most amylases require calcium as a cofactor for their activity and stability, therefore calcium, along with the thermostable enzyme, are typically added to the starch mixture during enzymatic liquefaction, thereby increasing process costs. An attractive alternative would be to produce the enzyme directly in the tissue to be treated. In a proof of concept study, tobacco cell cultures were used as model system to test in planta production of a hyperthermophilic alpha-amylase from Thermotoga maritima. While comparable biochemical properties to recombinant production in Escherichia coli were observed, thermostability of the plant-produced alpha-amylase benefited significantly from high intrinsic calcium levels in the tobacco cells. The plant-made enzyme retained 85% of its initial activity after 3 h incubation at 100 degrees C, whereas the E. coli-produced enzyme was completely inactivated after 30 min under the same conditions. The addition of Ca(2+) or plant cell extracts from tobacco and sweetpotato to the E. coli-produced enzyme resulted in a similar stabilization, demonstrating the importance of a calcium-rich environment for thermostability, as well as the advantage of producing this enzyme directly in plant cells where calcium is readily available.