Real-Time Sensor Data Profile-Based Deep Learning Method Applied to Open Raceway Pond Microalgal Productivity Prediction.

Microalgal biotechnology holds the potential for renewable biofuels, bioproducts, and carbon capture applications due to unparalleled photosynthetic efficiency and diversity. Outdoor open raceway pond (ORP) cultivation enables utilization of sunlight and atmospheric carbon dioxide to drive microalgal biomass synthesis for production of bioproducts including biofuels; however, environmental conditions are highly dynamic and fluctuate both diurnally and seasonally, making ORP productivity prediction challenging without time-intensive physical measurements and location-specific calibrations. Here, for the first time, we present an image-based deep learning method for the prediction of ORP productivity. Our method is based on parameter profile plot images of sensor parameters, including pH, dissolved oxygen, temperature, photosynthetically active radiation, and total dissolved solids. These parameters can be remotely monitored without physical interaction with ORPs. We apply the model to data we generated during the Unified Field Studies of the Algae Testbed Public-Private-Partnership (ATP3 UFS), the largest publicly available ORP data set to date, which includes millions of sensor records and 598 productivities from 32 ORPs operated in 5 states in the United States. We demonstrate that this approach significantly outperforms an average value based traditional machine learning method (R2 = 0.77 ≫ R2 = 0.39) without considering bioprocess parameters (e.g., biomass density, hydraulic retention time, and nutrient concentrations). We then evaluate the sensitivity of image and monitoring data resolutions and input parameter variations. Our results demonstrate ORP productivity can be effectively predicted from remote monitoring data, providing an inexpensive tool for microalgal production and operational forecasting.

An integrated digital microfluidic bioreactor for fully automatic screening of microalgal growth and stress-induced lipid accumulation.

Algae are the promising feedstock of biofuel. The screening of competent species and proper fertilizer supply is of the most important tasks. To accelerate this rather slow and laborious step, we developed an integrated high-throughput digital microfluidic (DMF) system that uses a discrete droplet to serve as a microbioreactor, encapsulating microalgal cells. On the basis of fundamental understanding of various droplet hydrodynamics induced by the existence of different sorts of ions and biological species, incorporation of capacitance-based position estimator, electrode-saving-based compensation, and deterministic splitting approach, was performed to optimize the DMF bioreactor. Thus, it enables all processes (e.g., nutrient gradient generation, algae culturing, and analyzing of growth and lipid accumulation) occurring automatically on-chip especially in a high-fidelity way. The ability of the system to compare different microalgal strains on-chip was investigated. Also, the Chlorella sp. were stressed by various conditions and then growth and oil accumulation were analyzed and compared, which demonstrated its potential as a powerful tool to investigate microalgal lipid accumulation at significantly lower laborites and reduced time.

Purification and photobiochemical profile of photosystem 1 from a high-salt tolerant, oleaginous Chlorella (Trebouxiophycaea, Chlorophyta).

The eukaryotic green alga Chlamydomonas reinhardtii has been studied extensively within the biofuel industry as a model organism, as researchers look towards algae to provide chemical feedstocks (i.e., lipids) for the production of liquid transportation fuels. C. reinhardtii, however, is unsuitable for high-level production of such precursors due to its relatively poor lipid accumulation and fresh-water demand. In this study we offer insight into the primary light harvesting and electron transfer reactions that occur during phototropic growth in a high-salt tolerant strain of Chlorella (a novel strain introduced here as NE1401), a single-celled eukaryotic algae also in the phylum Chlorophyta. Under nutrient starvation many eukaryotic algae increase dramatically the amount of lipids stored in lipid bodies within their cell interiors. Microscopy and lipid analyses indicate that Chlorella sp. NE1401 may become a superior candidate for algal biofuels production. We have purified highly active Photosystem 1 (PS1) complexes to study in vitro, so that we may understand further the photobiochemisty of this promising biofuel producer and how its characteristics compare and contrast with that of the better understood C. reinhardtii. Our findings suggest that the PS1 complex from Chlorella sp. NE1401 demonstrates similar characteristics to that of C. reinhardtii with respect to light-harvesting and electron transfer reactions. We also illustrate that the relative extent of the light state transition performed by Chlorella sp. NE1401 is smaller compared to C. reinhardtii, although they are triggered by the same dynamic light stresses.

Microalgal assemblages in a poikilohaline pond.

Microalgal strains for algal biofuels production in outdoor ponds will need to have high net growth rates under diverse environmental conditions. A small, variable salinity pond in the San Elijo Lagoon estuary in southern California was chosen to serve as a model pond due to its routinely high chlorophyll content. Profiles of microalgal assemblages from water samples collected from April 2011 to January 2012 were obtained by constructing 18S rDNA environmental clone libraries. Pond assemblages were found to be dominated by green algae Picochlorum sp. and Picocystis sp. throughout the year. Pigment analysis suggested that the two species contributed most of the chlorophyll a of the pond, which ranged from 21.9 to 664.3 µg · L(-1) with the Picocystis contribution increasing at higher salinities. However, changes of temperature, salinity or irradiance may have enabled a bloom of the diatom Chaetoceros sp. in June 2011. Isolates of these microalgae were obtained and their growth rates characterized as a function of temperature and salinity. Chaetoceros sp. had the highest growth rate over the temperature test range while it showed the most sensitivity to high salinity. All three strains showed the presence of lipid bodies during nitrogen starvation, suggesting they have potential as future biofuels strains.

Storage and Algal Association of Bacteria That Protect Microchloropsis salina from Grazing by Brachionus plicatilis.

Loss of algal production from the crashes of algal mass cultivation systems represents a significant barrier to the economic production of microalgal-based biofuels. Current strategies for crash prevention can be too costly to apply broadly as prophylaxis. Bacteria are ubiquitous in microalgal mass production cultures, however few studies investigate their role and possible significance in this particular environment. Previously, we demonstrated the success of selected protective bacterial communities to save Microchloropsis salina cultures from grazing by the rotifer Brachionus plicatilis. In the current study, these protective bacterial communities were further characterized by fractionation into rotifer-associated, algal-associated, and free-floating bacterial fractions. Small subunit ribosomal RNA amplicon sequencing was used to identify the bacterial genera present in each of the fractions. Here, we show that Marinobacter, Ruegeria, and Boseongicola in algae and rotifer fractions from rotifer-infected cultures likely play key roles in protecting algae from rotifers. Several other identified taxa likely play lesser roles in protective capability. The identification of bacterial community members demonstrating protective qualities will allow for the rational design of microbial communities grown in stable co-cultures with algal production strains in mass cultivation systems. Such a system would reduce the frequency of culture crashes and represent an essentially zero-cost form of algal crop protection.

Production of chemicals and utilities in-house improves the environmental sustainability of phytoplankton-based biorefinery.

Life cycle assessment was used to evaluate the environmental impacts of phytoplanktonic biofuels as possible sustainable alternatives to fossil fuels. Three scenarios were examined for converting planktonic biomass into higher-value commodities and energy streams using the alga Scenedesmus sp. and the cyanobacterium Arthrospira sp. as the species of interest. The first scenario (Sc-1) involved the production of biodiesel and glycerol from the planktonic biomass. In the second scenario (Sc-2), biodiesel and glycerol were generated from the planktonic biomass, and biogas was produced from the residual biomass. The process also involved using a catalyst derived from snail shells for biodiesel production. The third scenario (Sc-3) was similar to Sc-2 but converted CO2 from the biogas upgrading to methanol, which was then used in synthesizing biodiesel. The results indicated that Sc-2 and Sc-3 had a reduced potential (up to 60 % less) for damaging human health compared to Sc-1. Sc-2 and Sc-3 had up to 61 % less environmental impact than Sc-1. Sc-2 and Sc-3 reduced the total cumulative exergy demand by up to 44 % compared to Sc-1. In conclusion, producing chemicals and utilities within the biorefinery could significantly improve environmental sustainability, reduce waste, and diversify revenue streams.

Improved Reference Genome for Cyclotella cryptica CCMP332, a Model for Cell Wall Morphogenesis, Salinity Adaptation, and Lipid Production in Diatoms (Bacillariophyta).

The diatom, Cyclotella cryptica, is a well-established model species for physiological studies and biotechnology applications of diatoms. To further facilitate its use as a model diatom, we report an improved reference genome assembly and annotation for C. cryptica strain CCMP332. We used a combination of long- and short-read sequencing to assemble a high-quality and contaminant-free genome. The genome is 171 Mb in size and consists of 662 scaffolds with a scaffold N50 of 494 kb. This represents a 176-fold decrease in scaffold number and 41-fold increase in scaffold N50 compared to the previous assembly. The genome contains 21,250 predicted genes, 75% of which were assigned putative functions. Repetitive DNA comprises 59% of the genome, and an improved classification of repetitive elements indicated that a historically steady accumulation of transposable elements has contributed to the relatively large size of the C. cryptica genome. The high-quality C. cryptica genome will serve as a valuable reference for ecological, genetic, and biotechnology studies of diatoms.

Central composite design parameterization of microalgae/cyanobacteria co-culture pretreatment for enhanced lipid extraction using an external clamp-on ultrasonic transducer.

Lipids extracted from algal biomass could provide an abundant, rapidly growing, high yield feedstock for bio-diesel and other green fuels to supplement current fossil-based sources. Ultrasound pretreatment is a mechanical cell disruption method that has been shown to enhance lipid recovery from algae due to cavitation effects that disrupt algae cell walls. In this study, a locally grown mixture of Chlorella vulgaris/Cyanobacteria leptolyngbya was sonicated in an ultrasonic reactor with a clamp-on transducer prior to solvent lipid extraction. This configuration allows for a non-contact delivery method of ultrasonic energy with improved operational advantages (no fouling of transducer, continuous operation, and fully scalable design). A central composite design (CCD) was implemented to statistically analyze and evaluate the effect of ultrasonic power (350-750 W) and treatment time (5-30 min) on lipid yield. Lipid recovery was found to increase with both ultrasonic power and treatment time. Total lipid yields (on dry biomass basis) extracted via the Bligh and Dyer method from Chlorella vulgaris/cyanobacteria co-culture ranged from 8.3% for untreated algae to 16.9% for algae sonicated with 750 W power for 30 min, which corresponds to more than a doubling of lipid recovery due to ultrasound pretreatment. Increased power and treatment times were found to increase the degree of cell disruption as observed in the SEM and TEM images after ultrasonic pretreatment. Additionally, hexane (1:1 v/v) was evaluated as an alternative to the standard Bligh & Dyer (2:2:1.8 v/v/v chloroform/methanol/cell suspension) lipid extraction solvent system. On average, the Bligh and Dyer method extracted on average over twice the amount of lipids compared to hexane extraction. The lipid profile of the algae extracts indicates high concentrations of lauric acid (12:0), palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1), and linoleic acid (18:2). This particular configuration of an ultrasonic system proved to be a viable method for the pretreatment of algae for enhanced lipid yields. Future research should focus on identifying alternative extraction solvents and expanding the range of treatment conditions to optimize the ultrasonic power and treatment times for maximum lipid recovery.

Enhancement of microalgal growth and biocomponent-based transformations for improved biofuel recovery: A review.

Microalgal biomass has received much attention as feedstock for biofuel production due to its capacity to accumulate a substantial amount of biocomponents (including lipid, carbohydrate, and protein), high growth rate, and environmental benefit. However, commercial realization of microalgal biofuel is a challenge due to its low biomass production and insufficient technology for complete utilization of biomass. Recently, advanced strategies have been explored to overcome the challenges of conventional approaches and to achieve maximum possible outcomes in terms of growth. These strategies include a combination of stress factors; co-culturing with other microorganisms; and addition of salts, flue gases, and phytohormones. This review summarizes the recent progress in the application of single and combined abiotic stress conditions to stimulate microalgal growth and its biocomponents. An innovative schematic model is presented of the biomass-energy conversion pathway that proposes the transformation of all potential biocomponents of microalgae into biofuels.

Down-Selection and Outdoor Evaluation of Novel, Halotolerant Algal Strains for Winter Cultivation.

Algae offer promising feedstocks for the production of renewable fuel and chemical intermediates. However, poor outdoor winter cultivation capacity currently limits deployment potential. In this study, 300 distinct algal strains were screened in saline medium to determine their cultivation suitability during winter conditions in Mesa, Arizona. Three strains, from the genera Micractinium, Chlorella, and Scenedesmus, were chosen following laboratory evaluations and grown outdoors in 1000 L raceway ponds during the winter. Strains were down-selected based on doubling time, lipid and carbohydrate amount, final biomass accumulation capacity, cell size and phylogenetic diversity. Algal biomass productivity and compositional analysis for lipids and carbohydrates show successful outdoor deployment and cultivation under winter conditions for these strains. Outdoor harvest-yield biomass productivities ranged from 2.9 to 4.0 g/m2/day over an 18 days winter cultivation trial, with maximum productivities ranging from 4.0 to 6.5 g/m2/day, the highest productivities reported to date for algal winter strains grown in saline media in open raceway ponds. Peak fatty acid levels ranged from 9 to 26% percent of biomass, and peak carbohydrate levels ranged from 13 to 34% depending on the strain. Changes in the lipid and carbohydrate profile throughout outdoor growth are reported. This study demonstrates that algal strain screening under simulated outdoor environmental conditions in the laboratory enables identification of strains with robust biomass productivity and biofuel precursor composition. The strains isolated here represent promising winter deployment candidates for seasonal algal biomass production when using crop rotation strategies.

Algal polycultures enhance coproduct recycling from hydrothermal liquefaction.

The aim of this study was to determine if polycultures of algae could enhance tolerance to aqueous-phase coproduct (ACP) from hydrothermal liquefaction (HTL) of algal biomass to produce biocrude. The growth of algal monocultures and polycultures was characterized across a range ACP concentrations and sources. All of the monocultures were either killed or inhibited by 2% ACP, but polycultures of the same species were viable at up to 10%. The addition of ACP increased the growth rate (up to 25%) and biomass production (53%) of polycultures, several of which were more productive in ACP than any monoculture was in the presence or absence of ACP. These results suggest that a cultivation process that applies biodiversity to nutrient recycling could produce more algae with less fertilizer consumption.

Life cycle assessment and nutrient analysis of various processing pathways in algal biofuel production.

This study focuses on analyzing nutrient distributions and environmental impacts of nutrient recycling, reusing, and discharging in algal biofuels production. The three biomass conversion pathways compared in this study were: hydrothermal liquefaction technology (HTL), hydrothermal hydrolysis pretreatment +HTL (HTP), and wet lipid extraction (WLE). Carbon, nitrogen, and phosphorous (C, N, P) flows were described in each pathway. A primary cost analysis was conducted to evaluate the economic performance. The LCA results show that the HTP reduced life cycle NOx emissions by 10% from HTL, but increased fossil fuel use, greenhouse gas emissions, and eutrophication potential by 14%, 5%, and 28% respectively. The cost of per gallon biodiesel produced in HTP was less than in HTL. To further reduce emissions, efforts should be focused on improving nutrient uptake rates in algae cultivation, increasing biomass carbon detention in hydrothermal hydrolysis, and/or enhancing biomass conversion rates in the biooil upgrading processes.

Parasites in algae mass culture.

Parasites are now known to be ubiquitous across biological systems and can play an important role in modulating algal populations. However, there is a lack of extensive information on their role in artificial ecosystems such as algal production ponds and photobioreactors. Parasites have been implicated in the demise of algal blooms. Because individual mass culture systems often tend to be unialgal and a select few algal species are in wide scale application, there is an increased potential for parasites to have a devastating effect on commercial scale monoculture. As commercial algal production continues to expand with a widening variety of applications, including biofuel, food and pharmaceuticals, the parasites associated with algae will become of greater interest and potential economic impact. A number of important algal parasites have been identified in algal mass culture systems in the last few years and this number is sure to grow as the number of commercial algae ventures increases. Here, we review the research that has identified and characterized parasites infecting mass cultivated algae, the techniques being proposed and or developed to control them, and the potential impact of parasites on the future of the algal biomass industry.

Effect of nitrogen concentration on lipid productivity and fatty acid composition of Monoraphidium sp.

Green algae, Monoraphidium sp. T4X, was isolated locally, in New Delhi, India and identified as a potential source of biofuel. The study focuses on the effect of nutritional amendments and their uptake rates with respect to growth and change in fatty acid composition of the species. The lipid productivity and fatty acid profile were investigated and compared under six different nitrogen concentrations. Of the tested concentrations, cultures with nitrate concentration 0.36 g/l exhibited higher lipid productivity (0.18 g/l/day) with optimum content of all fatty acid compositions (SFA=37.22, MUFA=39.19, PUFA=23.60) with appropriate biodiesel properties. The right phase for harvesting microalgae was also investigated on the basis of the growth curve.

Improved Free Fatty Acid Production in Cyanobacteria with Synechococcus sp. PCC 7002 as Host.

Microbial free fatty acids (FFAs) have been proposed as a potential feedstock for renewable energy. The ability to directly convert carbon dioxide into FFAs makes cyanobacteria ideal hosts for renewable FFA production. Previous metabolic engineering efforts using the cyanobacterial hosts Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 have demonstrated this direct conversion of carbon dioxide into FFAs; however, FFA yields in these hosts are limited by the negative impact of FFA production on the host cell physiology. This work investigates the use of Synechococcus sp. PCC 7002 as a cyanobacterial host for FFA production. In comparison to S. elongatus PCC 7942, Synechococcus sp. PCC 7002 strains produced and excreted FFAs at similar concentrations but without the detrimental effects on host physiology. The enhanced tolerance to FFA production with Synechococcus sp. PCC 7002 was found to be temperature-dependent, with physiological effects such as reduced photosynthetic yield and decreased photosynthetic pigments observed at higher temperatures. Additional genetic manipulations were targeted for increased FFA production, including thioesterases and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Overexpression of non-native RuBisCO subunits (rbcLS) from a psbAI promoter resulted in more than a threefold increase in FFA production, with excreted FFA concentrations reaching >130 mg/L. This work illustrates the importance of host strain selection for cyanobacterial biofuel production and demonstrates that the FFA tolerance of Synechococcus sp. PCC 7002 can allow for high yields of excreted FFA.

Constraints to commercialization of algal fuels.

Production of algal crude oil has been achieved in various pilot scale facilities, but whether algal fuels can be produced in sufficient quantity to meaningfully displace petroleum fuels, has been largely overlooked. Limitations to commercialization of algal fuels need to be understood and addressed for any future commercialization. This review identifies the major constraints to commercialization of transport fuels from microalgae. Algae derived fuels are expensive compared to petroleum derived fuels, but this could change. Unfortunately, improved economics of production are not sufficient for an environmentally sustainable production, or its large scale feasibility. A low-cost point supply of concentrated carbon dioxide colocated with the other essential resources is necessary for producing algal fuels. An insufficiency of concentrated carbon dioxide is actually a major impediment to any substantial production of algal fuels. Sustainability of production requires the development of an ability to almost fully recycle the phosphorous and nitrogen nutrients that are necessary for algae culture. Development of a nitrogen biofixation ability to support production of algal fuels ought to be an important long term objective. At sufficiently large scale, a limited supply of freshwater will pose a significant limitation to production even if marine algae are used. Processes for recovering energy from the algal biomass left after the extraction of oil, are required for achieving a net positive energy balance in the algal fuel oil. The near term outlook for widespread use of algal fuels appears bleak, but fuels for niche applications such as in aviation may be likely in the medium term. Genetic and metabolic engineering of microalgae to boost production of fuel oil and ease its recovery, are essential for commercialization of algal fuels. Algae will need to be genetically modified for improved photosynthetic efficiency in the long term.