Solids Residence Time Impacts Carbon Dynamics and Bioenergy Feedstock Potential in Phototrophic Wastewater Treatment Systems.

The use of wastewater-grown microalgae has the potential to reduce the cost of algae-derived biofuels while simultaneously advancing nutrient recovery at water resource recovery facilities (WRRFs). However, a significant barrier has been the low yield and high protein content of phototrophic biomass. Here, we examine the use of solids residence time (SRT) as a selective pressure in driving biochemical composition, yield, biofuel production, and WRRF nutrient management cost. We cultivated mixed phototrophic communities in controlled, laboratory-scale photobioreactors on the local WRRF secondary effluent to link SRT with biochemical composition and techno-economic analysis to yield insights into biomass composition and downstream processing effects on minimum fuel selling price. SRT significantly impacted biochemical composition, with total and dynamic carbohydrates the highest at low SRT (total carbohydrates being 0.60 and 0.32 mg-carbohydrate·mg-protein-1 at SRT 5 and 15 days, respectively). However, there were distinct differences between extant, steady-state performance and intrinsic potential, and longer SRT communities were able to accumulate significant fractions (51% on an ash-free dry weight basis, AFDW %) of carbohydrate reserves under nutrient starvation. Overall, hydrothermal liquefaction (HTL) was found to be more suitable than lipid extraction for hydrotreating (LEH) and combined algal processing (CAP) for conversion of biomass to fuels, but LEH and CAP became more competitive when intrinsic carbon storage potential was realized. The results suggest that the use of algae for nutrient recovery could reduce the nutrient management cost at WRRFs through revenue from algal biofuels, with HTL resulting in a net revenue.

CO2 utilization: Turning greenhouse gas into fuels and valuable products.

This study critically reviews the recent developments and future opportunities pertinent to the conversion of CO2 as a potent greenhouse gas (GHG) to fuels and valuable products. CO2 emissions have reached an alarming level of around 410 ppm and have become the primary driver of global warming and climate change leading to devastating events such as droughts, hurricanes, torrential rains, floods, tornados and wildfires across the world. These events are responsible for thousands of deaths and have adversely affected the economic development of many countries, loss of billions of dollars, across the globe. One of the promising choices to tackle this issue is carbon sequestration by pre- and post-combustion processes and oxyfuel combustion. The captured CO2 can be converted into fuels and valuable products, including methanol, dimethyl ether (DME), and methane (CH4). The efficient use of the sequestered CO2 for the desalinization might be critical in overcoming water scarcity and energy issues in developing countries. Using the sequestered CO2 to produce algae in combination with wastewater, and producing biofuels is among the promising strategies. Many methods, like direct combustion, fermentation, transesterification, pyrolysis, anaerobic digestion (AD), and gasification, can be used for the conversion of algae into biofuel. Direct air capturing (DAC) is another productive technique for absorbing CO2 from the atmosphere and converting it into various useful energy resources like CH4. These methods can effectively tackle the issues of climate change, water security, and energy crises. However, future research is required to make these conversion methods cost-effective and commercially applicable.

Ten years of algal biofuel and bioproducts: gains and pains.

MAIN CONCLUSION: It has been proposed that future efforts should focus on basic studies, biotechnology studies and synthetic biology studies related to algal biofuels and various high-value bioproducts for the economically viable production of algal biof uels. In recognition of diminishing fossil fuel reserves and the worsening environment, microalgal biofuel has been proposed as a renewable energy source with great potential. Algal biofuel thus became one of the hottest topics in renewable energy research in the new century, especially over the past decade. Between 2007 and 2017, research related to microalgal biofuels experienced a dramatic, three-stage development, rising, growing exponentially, and then declining rapidly due to overheating of the subject. However, biofuel-driven algal biotechnology and bioproducts research has been thriving since 2010. To clarify the gains (and pains) of the past decade and detail prospects for the future, this review summarizes the extensive scientific progress and substantial technical advances in algal biofuel over the past decade, covering basic biology, applied research, as well as the production of value-added natural products. Even after 10 years of hard work and billions of dollars in investments, its unacceptably high cost remains the ultimate bottleneck for the industrialization of algal biofuel. To maximize the total research benefits, both economically and socially, it has been proposed that future efforts should focus on basic studies to characterize oilgae, on biotechnology studies into various high-value bioproducts. Moreover, the development of synthetic biology provides new possibilities for the economically viable production of biofuels via the directional manufacture of microalgal bioproducts in algal cell factories.

Metabolic engineering of lipid catabolism increases microalgal lipid accumulation without compromising growth.

Biologically derived fuels are viable alternatives to traditional fossil fuels, and microalgae are a particularly promising source, but improvements are required throughout the production process to increase productivity and reduce cost. Metabolic engineering to increase yields of biofuel-relevant lipids in these organisms without compromising growth is an important aspect of advancing economic feasibility. We report that the targeted knockdown of a multifunctional lipase/phospholipase/acyltransferase increased lipid yields without affecting growth in the diatom Thalassiosira pseudonana. Antisense-expressing knockdown strains 1A6 and 1B1 exhibited wild-type-like growth and increased lipid content under both continuous light and alternating light/dark conditions. Strains 1A6 and 1B1, respectively, contained 2.4- and 3.3-fold higher lipid content than wild-type during exponential growth, and 4.1- and 3.2-fold higher lipid content than wild-type after 40 h of silicon starvation. Analyses of fatty acids, lipid classes, and membrane stability in the transgenic strains suggest a role for this enzyme in membrane lipid turnover and lipid homeostasis. These results demonstrate that targeted metabolic manipulations can be used to increase lipid accumulation in eukaryotic microalgae without compromising growth.

Combining ABE fermentation and anaerobic digestion to treat with lipid extracted algae for enhanced bioenergy production.

As a downstream process output, biobutanol can be produced via acetone, butanol, and ethanol (ABE) fermentation from lipid-extracted algae (LEA), but the leftover residue has not been treated for additional value. In current study, LEA were acid hydrolyzed to extract glucose into the hydrolysate, which was then used for ABE fermentation to produce butanol. In the meantime, anaerobic digestion was performed on the hydrolysis residue to produce methane and release nutrients for algae recultivation. To optimize butanol and methane production, several carbon or nitrogen supplements were applied. The results showed that the hydrolysate produced a high butanol concentration of 8.5 g/L with bean cake supplemented, and the residue co-digested with wastepaper had a higher methane production compared to the direct anaerobic digestion of LEA. The causes of the enhanced performances were discussed. The digestates were reused for algae recultivation and were proved to be effective for algae and oil reproduction. The combined process of ABE fermentation and anaerobic digestion was thus proved a promising technique to treat LEA for economic benefit.

Identification of Transcription Factors and the Regulatory Genes Involved in Triacylglycerol Accumulation in the Unicellular Red Alga Cyanidioschyzon merolae.

Microalgal triacylglycerols (TAGs) are a good feedstock for liquid biofuel production. Improving the expression and/or function of transcription factors (TFs) involved in TAG accumulation may increase TAG content; however, information on microalgae is still lacking. In this study, 14 TFs in the unicellular red alga Cyanidioschyzon merolae were identified as candidate TFs regulating TAG accumulation using available transcriptome and phosphoproteome data under conditions driving TAG accumulation. To investigate the roles of these TFs, we constructed TF-overexpression strains and analyzed lipid droplet (LD) formation and TAG contents in the cells grown under standard conditions. Based on the results, we identified four TFs involved in LD and TAG accumulation. RNA-Seq analyses were performed to identify genes regulated by the four TFs using each overexpression strain. Among the TAG biosynthesis-related genes, only the gene encoding the endoplasmic reticulum-localized lysophosphatidic acid acyltransferase 1 (LPAT1) was notably increased among the overexpression strains. In the LPAT1 overexpression strain, TAG accumulation was significantly increased compared with the control strain under normal growth conditions. These results indicate that the four TFs positively regulate TAG accumulation by changing their target gene expression in C. merolae.

Natural plant extracts as an economical and ecofriendly alternative for harvesting microalgae.

The study investigated the ability of plant based natural coagulants from Azadirachta indica; Ficus indica; Moringa oleifera; Citrus sinensis; Punica granatum and Musa acuminata to harvest the microalgal biomass. Influence of eluent type (water and NaCl) and concentration (1-5 N) on coagulant extraction; coagulant dosage (1-5 g) and volume (20-100 ml); pH (6-12) and algal concentration (0.1-1 g l-1) on harvesting were analyzed. The results obtained were compared with alum and chitosan. FTIR and biochemical analysis confirmed the presence of bioactive compounds to aid coagulation. Biomass removal efficiency of 75.50% was obtained with M. oleifera extracts (8 mg ml-1) at pH 7.5-7.8, within 100 min. The harvesting efficiency increased to 95.76% when 4 mg ml-1M. oleifera extracts was combined with 0.75 mg ml-1 chitosan. The life cycle and cost analysis acknowledged the eco-friendly coagulants as strong alternative for conventional coagulants used in microalgal harvesting, thereby improvising the overall bioprocess.

Comprehensive techno-economic analysis of wastewater-based algal biofuel production: A case study.

Combining algae cultivation and wastewater treatment for biofuel production is considered the feasible way for resource utilization. An updated comprehensive techno-economic analysis method that integrates resources availability into techno-economic analysis was employed to evaluate the wastewater-based algal biofuel production with the consideration of wastewater treatment improvement, greenhouse gases emissions, biofuel production costs, and coproduct utilization. An innovative approach consisting of microalgae cultivation on centrate wastewater, microalgae harvest through flocculation, solar drying of biomass, pyrolysis of biomass to bio-oil, and utilization of co-products, was analyzed and shown to yield profound positive results in comparison with others. The estimated break even selling price of biofuel ($2.23/gallon) is very close to the acceptable level. The approach would have better overall benefits and the internal rate of return would increase up to 18.7% if three critical components, namely cultivation, harvest, and downstream conversion could achieve breakthroughs.

Hybrid life-cycle assessment of algal biofuel production.

The objective of this work is to establish whether algal bio-crude production is environmentally, economically and socially sustainable. To this end, an economic multi-regional input-output model of Australia was complemented with engineering process data on algal bio-crude production. This model was used to undertake hybrid life-cycle assessment for measuring the direct, as well as indirect impacts of producing bio-crude. Overall, the supply chain of bio-crude is more sustainable than that of conventional crude oil. The results indicate that producing 1 million tonnes of bio-crude will generate almost 13,000 new jobs and 4 billion dollars' worth of economic stimulus. Furthermore, bio-crude production will offer carbon sequestration opportunities as the production process is net carbon-negative.

A Lipid-Accumulating Alga Maintains Growth in Outdoor, Alkaliphilic Raceway Pond with Mixed Microbial Communities.

Algal biofuels and valuable co-products are being produced in both open and closed cultivation systems. Growing algae in open pond systems may be a more economical alternative, but this approach allows environmental microorganisms to colonize the pond and potentially infect or outcompete the algal "crop." In this study, we monitored the microbial community of an outdoor, open raceway pond inoculated with a high lipid-producing alkaliphilic alga, Chlorella vulgaris BA050. The strain C. vulgaris BA050 was previously isolated from Soap Lake, Washington, a system characterized by a high pH (∼9.8). An outdoor raceway pond (200 L) was inoculated with C. vulgaris and monitored for 10 days and then the culture was transferred to a 2,000 L raceway pond and cultivated for an additional 6 days. Community DNA samples were collected over the 16-day period in conjunction with water chemistry analyses and cell counts. Universal primers for the SSU rRNA gene sequences for Eukarya, Bacteria, and Archaea were used for barcoded pyrosequence determination. The environmental parameters that most closely correlated with C. vulgaris abundance were pH and phosphate. Community analyses indicated that the pond system remained dominated by the Chlorella population (93% of eukaryotic sequences), but was also colonized by other microorganisms. Bacterial sequence diversity increased over time while archaeal sequence diversity declined over the same time period. Using SparCC co-occurrence network analysis, a positive correlation was observed between C. vulgaris and Pseudomonas sp. throughout the experiment, which may suggest a symbiotic relationship between the two organisms. The putative relationship coupled with high pH may have contributed to the success of C. vulgaris. The characterization of the microbial community dynamics of an alkaliphilic open pond system provides significant insight into open pond systems that could be used to control photoautotrophic biomass productivity in an open, non-sterile environment.

Simultaneous nutrient removal, optimised CO2 mitigation and biofuel feedstock production by Chlorogonium sp. grown in secondary treated non-sterile saline sewage effluent.

The phycoremediation process has great potential for effectively addressing environmental pollution. To explore the capabilities of simultaneous algal nutrient removal, CO2 mitigation and biofuel feedstock production from spent water resources, a Chlorogonium sp. isolated from a tilapia pond in Hong Kong was grown in non-sterile saline sewage effluent for a bioremediation study. With high removal efficiencies of NH3-N (88.35±14.39%), NO3(-)-N (85.39±14.96%), TN (93.34±6.47%) and PO4(3-)-P (91.80±17.44%), Chlorogonium sp. achieved a CO2 consumption rate of 58.96 mg L(-1) d(-1), which was optimised by the response surface methodology. Under optimised conditions, the lipid content of the algal biomass reached 24.26±2.67%. Overall, the isolated Chlorogonium sp. showed promising potential in the simultaneous purification of saline sewage effluent in terms of tertiary treatment and CO2 sequestration while delivering feedstock for potential biofuel production in a waste-recycling manner.

Algal recycling enhances algal productivity and settleability in Pediastrum boryanum pure cultures.

Recycling a portion of gravity harvested algae (i.e. algae and associated bacteria biomass) has been shown to improve both algal biomass productivity and harvest efficiency by maintaining the dominance of a rapidly-settleable colonial alga, Pediastrum boryanum in both pilot-scale wastewater treatment High Rate Algal Ponds (HRAP) and outdoor mesocosms. While algal recycling did not change the relative proportions of algae and bacteria in the HRAP culture, the contribution of the wastewater bacteria to the improved algal biomass productivity and settleability with the recycling was not certain and still required investigation. P. boryanum was therefore isolated from the HRAP and grown in pure culture on synthetic wastewater growth media under laboratory conditions. The influence of recycling on the productivity and settleability of the pure P. boryanum culture was then determined without wastewater bacteria present. Six 1 L P. boryanum cultures were grown over 30 days in a laboratory growth chamber simulating New Zealand summer conditions either with (Pr) or without (Pc) recycling of 10% of gravity harvested algae. The cultures with recycling (Pr) had higher algal productivity than the controls (Pc) when the cultures were operated at both 4 and 3 d hydraulic retention times by 11% and 38% respectively. Furthermore, algal recycling also improved 1 h settleability from ∼60% to ∼85% by increasing the average P. boryanum colony size due to the extended mean cell residence time and promoted formation of large algal bio-flocs (>500 µm diameter). These results demonstrate that the presence of wastewater bacteria was not necessary to improve algal productivity and settleability with algal recycling.

The effects of alternative pretreatment strategies on anaerobic digestion and methane production from different algal strains.

The effect of various pretreatment strategies on methane yields following anaerobic digestion (AD) of five different microalgal strains was investigated. Pavlova_cf sp., Tetraselmis sp. and Thalassiosira weissflogii exhibited substantial methane yields of 0.4-0.5L/g volatile solids (VS) without pretreatment, providing up to 75-80% of theoretical values. In contrast, methane yields from Chlorella sp. and Nannochloropsis sp. were around 0.35L/g VS, or 55-60% of the theoretical values, respectively. Alkali treatment was not effective and thermal pretreatment only enhanced Nannochloropsis methane yields. Thermochemical pretreatment had the strongest impact on biomass solubilization with methane yields increasing by 30% and 40% for Chlorella and Nannochloropsis, respectively. The lipid content had a strong beneficial impact on the theoretical and observed methane yields as compared to protein and carbohydrate content. Other features such as cell-wall composition are also likely to be important factors dictating algal biodegradability and methane yields addressed in part by thermochemical pretreatment.

Algal cell disruption using microbubbles to localize ultrasonic energy.

Microbubbles were added to an algal solution with the goal of improving cell disruption efficiency and the net energy balance for algal biofuel production. Experimental results showed that disruption increases with increasing peak rarefaction ultrasound pressure over the range studied: 1.90 to 3.07 MPa. Additionally, ultrasound cell disruption increased by up to 58% by adding microbubbles, with peak disruption occurring in the range of 10(8)microbubbles/ml. The localization of energy in space and time provided by the bubbles improve efficiency: energy requirements for such a process were estimated to be one-fourth of the available heat of combustion of algal biomass and one-fifth of currently used cell disruption methods. This increase in energy efficiency could make microbubble enhanced ultrasound viable for bioenergy applications and is expected to integrate well with current cell harvesting methods based upon dissolved air flotation.

Action spectra of oxygen production and chlorophyll a fluorescence in the green microalga Nannochloropsis oculata.

The first complete action spectrum of oxygen evolution and chlorophyll a fluorescence was measured for the biofuel candidate alga Nannochloropsis oculata. A novel analytical procedure was used to generate a representative and reproducible action spectrum for microalgal cultures. The action spectrum was measured at 14 discrete wavelengths across the visible spectrum, at an equivalent photon flux density of 60 µmol photon sm(-2) s(-1). Blue light (∼ 414 nm) was absorbed more efficiently and directed to photosystem II more effectively than red light (∼ 679 nm) at light intensities below the photosaturation limit. Conversion of absorbed photons into photosynthetic oxygen evolution was maximised at 625 nm; however, this maximum is unstable since neighbouring wavelengths (646 nm) resulted in the lowest photosystem II operating efficiency. Identifying the wavelength-dependence of photosynthesis has clear implications to optimising growth efficiency and hence important economic implications to the algal biofuels and bioproducts industries.

Investigating the life-cycle and growth rate of Pediastrum boryanum and the implications for wastewater treatment high rate algal ponds.

The colonial alga Pediastrum boryanum has beneficial characteristics for wastewater treatment High Rate Algal Ponds (HRAP) including high biomass productivity and settleability. Our previous work has shown that these characteristics are enhanced when a portion of gravity harvested algae is recycled back to the pond. To help understand the mechanisms behind the improved performance of P. boryanum dominated HRAP with algal recycling, this study investigated the life-cycle of P. boryanum. Experiments determined the exact timing and growth rate of P. boryanum life-cycle stages ('juvenile', 'growth' and 'reproductive') under four combinations of light and temperature (250 or 120 µMol/m(2)/s; 20 or 10 °C). Single juvenile 16-celled colonies were grown in microcosms on an inverted microscope and photographed every 15 min until reproduction ceased. Two asexual life-cycles and a rarely occurring sexual life-cycle were observed. The time required to achieve asexual reproductive maturity increased from 52 h (high light and temperature) to 307 h (low light and temperature), indicating that the minimum hydraulic retention time or mean cell residence time (MCRT) must be higher than these values to sustain a P. boryanum HRAP culture under ambient conditions. The net growth rate of a P. boryanum colony varied between life-cycle stages (growth > juvenile > reproductive). This suggests that the higher biomass productivity measured in HRAP with algal recycling could be due to both the increased MCRT and an increase in the net growth rate of the HRAP culture by 'seeding' with faster growing colonies.

Comparison of Synechocystis sp. PCC6803 and Nannochloropsis salina for lipid production using artificial seawater and nutrients from anaerobic digestion effluent.

The potential use of Synechocystis sp. PCC6803 (Synechocystis sp.) for lipid production using artificial seawater (ASW) medium supplemented with anaerobic digestion effluent (ADE) was investigated and compared to marine microalgae, Nannochloropsis salina (N. salina). Synechocystis sp. showed growth rates 83% and 20% higher than N. salina at 3% and 6% ADE loading ratios, respectively, achieving the highest biomass productivity of 212 mg L(-1) d(-1) in semi-continuous cultivation. The rapid growth of Synechocystis sp. was offset by its low lipid content, resulting in lipid productivities 7-28% lower than N. salina. The lipid productivity of Synechocystis sp. may be further improved by decreasing the harvesting interval during semi-continuous cultivation. Fatty acid analysis showed that lipids extracted from Synechocystis sp. contained higher palmitic acid (60.3±2.0%) and linoleic acid (20.0±1.6%), and had a higher cetane number and oxidative stability than those from N. salina.