Predicting black soldier fly larvae biomass and methionine accumulation using a kinetic model for batch cultivation and improving system performance using semi-batch cultivation.

Global demand for poultry and associated feed are projected to double over the next 30 years. Insect meal is a sustainable alternative to traditional feeds when produced on low-value high-volume agricultural byproducts. Black soldier fly (BSF) larvae (Hermetia illucens L.) are high in protein and contain methionine, an essential amino acid that is critical to poultry health. BSF larvae can be grown on many organic residues, however, larvae growth and quality vary based on feedstock and cultivation processes. Experiments were completed to monitor temporal changes in BSF larvae growth and composition using almond hulls as a growth substrate under batch and semi-batch processes and with varying substrate carbon to nitrogen ratio (C/N). A logistic kinetic growth model was developed to predict larval biomass and methionine accumulations during batch production. Estimated ranges of model parameters for larvae maximum specific growth rate and carrying capacity were 0.017-0.021 h-1 and 9.7-10.7 g larvae kg-1 hulls dry weight, respectively. Methionine content in larvae increased from 11.1 to 17.1 g kg-1 dry weight over a 30-day batch incubation period. Larvae-specific growth and yield increased by 168% and 268%, respectively, when cultivated in a semi-batch compared to a batch process. Increasing C/N ratio from 26 to 40 increased density of methionine content in larvae per unit feedstock by 25%. The findings demonstrate a logistic model can predict larvae biomass accumulation, harvest time can achieve specific methionine contents, and a semi-batch process is more favorable for larvae biomass accumulation compared to a batch process.

Algal photosynthetic aeration increases the capacity of bacteria to degrade organics in wastewater.

Wastewater treatment is an energy-intensive process and a net emitter of greenhouse gas emissions. A large fraction of these emissions is due to intensive aeration of aerobic bacteria to facilitate break-down of organic compounds. Algae can generate dissolved oxygen at levels in excess of saturation, and therefore hold the potential to partially displace or complement mechanical aeration in wastewater treatment processes. The objective of this study was to develop an internally consistent experimental and modeling approach to test the hypothesis that algal photosynthetic aeration can speed the removal of organic constituents by bacteria. This framework was developed using a simplified wastewater treatment process consisting of a model bacteria (Escherichia coli), a model algae (Auxenochlorella protothecoides), and a single carbon source that was consumable by bacteria only. This system was then tested both with and without the presence of algae. A MATLAB model that considered mass transfer and biological kinetics was used to estimate the production and consumption of O2 and CO2 by algae and bacteria. The results indicated that the presence of algae led to 18-66% faster removal of COD by bacteria, and that roughly one-third of biochemical oxygen demand was offset by algal photosynthetic aeration.

The effect of the microalgae-bacteria microbiome on wastewater treatment and biomass production.

The use of microalgae for wastewater treatment has been proposed as a cost-effective method to produce biofuels while remediating waste streams. This study examined the microalgae biomass production rate, wastewater treatment efficiency, and prokaryotic organism microbiome associated with microalgae Chlorella sorokiniana cultivated on anaerobic digestate effluent. Final microalgae biomass concentrations from nine photobioreactors were highly variable and had values that ranged between 0.14 g/L and 0.90 g/L. Nutrient removal efficiencies for TN (total nitrogen), N-NH4 (ammonium nitrogen), and COD (chemical oxygen demand) ranged from 34% to 67%, 65% to 97%, and-60% to 14%, respectively. Analysis of individual OTUs (operational taxonomic units) from the microbial community revealed that microalgae biomass concentrations were significantly correlated with the relative abundance of OTUs in the genus Pusillimonas. Predictive metagenomic analyses identified additional correlations associated with biomass production and nutrient removal. These results suggest that the microbial community present during microalgae cultivation on wastewater can impact the performance of the system for biomass production and wastewater treatment.

Characterization of digestate microbial community structure following thermophilic anaerobic digestion with varying levels of green and food wastes.

The properties of digestates generated through anaerobic digestion are influenced by interactions between the digester microbial communities, feedstock properties and digester operating conditions. This study investigated the effect of varying initial feedstock carbon to nitrogen (C/N) ratios on digestate microbiota and predicted abundance of genes encoding lignocellulolytic activity. The C/N ratio had a significant impact on the digestate microbiome. Feedstocks with intermediate C/N ratio (20-27) (where higher biomethane potential was observed) showed higher relative abundance of archaea compared to feedstocks with C/N ratios at 17 and 34. Within microbial networks, four microbial clusters and eight connector microorganisms changed significantly with the C/N ratio (P < 0.05). Feedstocks with C/N < 23 were richer in organisms from the family Thermotogaceae and genus Caldicoprobacter and enhanced potential for degradation of maltose, galactomannans, melobiose and lactose. This study provides new insights into how anaerobic digestion conditions relate to the structure and functional potential of digester microbial communities, which may be relevant to both digester performance and subsequent utilization of digestates for composting or amending soil.

Almond by-product composition impacts the rearing of black soldier fly larvae and quality of the spent substrate as a soil amendment.

BACKGROUND: Insect biomass is a sustainable alternative to traditional animal feeds, particularly when insects are produced on low-value high-volume agricultural by-products. Seven samples of almond by-product (hulls and shells) were obtained from processors in California and investigated for larvae production. Experiments were completed with and without larvae and spent substrate samples were assessed for their potential as soil amendments based on standard compost quality indicators. RESULTS: On average, specific larvae growth and average larval harvest weight were 158% and 109% higher, respectively, when larvae were reared on Monterey and pollinator hulls compared to nonpareil hulls and mixed shells. Larvae methionine and cystine contents were highest when larvae were reared on Monterey hulls and mixed shells, respectively. Available phytonutrients in spent substrate were affected by feedstock sample and larvae rearing. Spent nonpareil substrate without larvae had the highest NH4 -N levels and spent pollinator substrate incubated without larvae had the highest PO4 -P levels. Spent mixed shell substrate had the lowest availability of phytonutrients. CONCLUSION: The findings demonstrate that by-product composition has a significant impact on larvae growth and the properties of the spent substrate, and that spent substrate from larvae rearing requires further stabilization before application as a soil amendment. © 2020 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Cultivation of black soldier fly larvae on almond byproducts: impacts of aeration and moisture on larvae growth and composition.

BACKGROUND: The increasing production of almonds worldwide has resulted in the significant generation of byproduct streams that require end uses. One potential use for byproducts is for cultivation of additional food sources including insects. Studies were performed to determine if black soldier fly larvae (Hermetia illucens L.) could be cultivated on almond byproducts (hulls and shells) and to examine the effect of aeration and moisture on larvae growth and hull composition. RESULTS: Increasing aeration from 0.04 to 0.36 mL min-1 g dry weight-1 tripled the harvest weight of larvae and increased larvae yield by a factor of five. Larvae calcium content increased by 18% with an increase in aeration from 0.04 to 0.95 mL min-1 g dry weight-1 . Moisture content also affected harvest dry weight and yield; increasing moisture content from 480 g kg-1 (wet basis) to 680 g kg-1 increased harvest weight by 56% and yield by a factor of 2. Variables did not affect larvae methionine and cysteine content. Low moisture content and aeration rate yielded an environment that supported microbial consumption of hulls over larvae consumption and growth. CONCLUSIONS: The results demonstrate that almond hulls are a suitable feedstock for larvae production under controlled management of moisture content and aeration. © 2018 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Ionic liquid-tolerant microorganisms and microbial communities for lignocellulose conversion to bioproducts.

Chemical and physical pretreatment of biomass is a critical step in the conversion of lignocellulose to biofuels and bioproducts. Ionic liquid (IL) pretreatment has attracted significant attention due to the unique ability of certain ILs to solubilize some or all components of the plant cell wall. However, these ILs inhibit not only the enzyme activities but also the growth and productivity of microorganisms used in downstream hydrolysis and fermentation processes. While pretreated biomass can be washed to remove residual IL and reduce inhibition, extensive washing is costly and not feasible in large-scale processes. IL-tolerant microorganisms and microbial communities have been discovered from environmental samples and studies begun to elucidate mechanisms of IL tolerance. The discovery of IL tolerance in environmental microbial communities and individual microbes has lead to the proposal of molecular mechanisms of resistance. In this article, we review recent progress on discovering IL-tolerant microorganisms, identifying metabolic pathways and mechanisms of tolerance, and engineering microorganisms for IL tolerance. Research in these areas will yield new approaches to overcome inhibition in lignocellulosic biomass bioconversion processes and increase opportunities for the use of ILs in biomass pretreatment.

Enrichment of microbial communities tolerant to the ionic liquids tetrabutylphosphonium chloride and tributylethylphosphonium diethylphosphate.

The aims of this study were to identify thermophilic microbial communities that degrade green waste in the presence of the ionic liquids (IL) tetrabutylphosphonium chloride and tributylethylphosphonium diethylphosphate and examine preservation methods for IL-tolerant communities. High-solids incubations with stepwise increases in IL concentration were conducted to enrich for thermophilic IL-tolerant communities that decomposed green waste. 16S rRNA sequencing of enriched communities revealed microorganisms capable of tolerating high levels of IL. Cryogenic preservation of enriched communities reduced the IL tolerance of the community and decreased the relative abundance of IL-tolerant organisms. The use of cryoprotectants did not have an effect on microbial activity on green waste of the stored community. A successful approach was developed to enrich communities that decompose green waste in thermophilic high-solids environments in the presence of IL. Alternative community storage and revival methods are necessary for maintenance and recovery of IL-tolerant communities. The enriched communities provide a targeted source of enzymes for the bioconversion of IL-pretreated green waste for conversion to biofuels.

Co-culturing Chlorella minutissima with Escherichia coli can increase neutral lipid production and improve biodiesel quality.

Lipid productivity and fatty acid composition are important metrics for the production of high quality biodiesel from algae. Our previous results showed that co-culturing the green alga Chlorella minutissima with Escherichia coli under high-substrate mixotrophic conditions enhanced both culture growth and crude lipid content. To investigate further, we analyzed neutral lipid content and fatty acid content and composition of axenic cultures and co-cultures produced under autotrophic and mixotrophic conditions. We found that co-culturing C. minutissima with E. coli under high substrate conditions (10 g/L) increased neutral lipid content 1.9- to 3.1-fold and fatty acid content 1.5- to 2.6-fold compared to equivalent axenic C. minutissima cultures. These same co-cultures also exhibited a significant fatty acid shift away from trienoic and toward monoenoic fatty acids thereby improving the quality of the synthesized fatty acids for biodiesel production. Further investigation suggested that E. coli facilitates substrate uptake by the algae and that the resulting growth enhancement induces a nitrogen-limited condition. Enhanced carbon uptake coupled with nitrogen limitation is the likely cause of the observed neutral lipid accumulation and fatty acid profile changes.

Room-temperature storage of microalgae in water-in-oil emulsions: influence of solid particle type and concentration in the oil phase.

Water-in-oil emulsions containing silica nanoparticles (Aerosil R974) have the potential to stabilize microalgae for long-term storage. Studies were completed to determine if smectite clays could be used as an alternative to Aerosil R974. Emulsions were prepared with Aerosil R974, and hectorite and bentonite clays in the continuous phase and Chlorella sorokiniana was added to the aqueous phase to monitor the effects of solid particles on emulsion stability. Biological stability (cell viability) was determined using cell density measurements, and physical stability was measured from water droplet size distributions obtained by light scattering measurements and by examining phase separation over time. Measurements were also made to determine the effects of particles in the oil phase on emulsion viscosity. Particle concentrations greater than 0.25 wt% in the oil phase were required for maintaining physical stability. In emulsions containing 1 wt% solid particles and microalgae, biological stability of cells could be sustained for 340 days, regardless of particle type. At 1 wt% particles in the oil phase, apparent viscosity was 165% greater for samples containing hectorite and bentonite clays compared to samples containing Aerosil R974. The higher viscosity would need to be considered in large-scale production of emulsions for commercial application.

Microplate assay for quantitation of neutral lipids in extracts from microalgae.

Lipid quantitation is widespread in the algae literature, but popular methods such as gravimetry, gas chromatography and mass spectrometry (GC-MS), and Nile red cell staining suffer drawbacks, including poor quantitation of neutral lipids, expensive equipment, and variable results among algae species, respectively. A high-throughput microplate assay was developed that uses Nile red dye to quantify neutral lipids that have been extracted from algae cells. Because the algal extracts contained pigments that quenched Nile red fluorescence, a mild bleach solution was used to destroy pigments, resulting in a nearly linear response for lipid quantities in the range of 0.75 to 40 µg. Corn oil was used as a standard for quantitation, although other vegetable oils displayed a similar response. The assay was tested on lipids extracted from three species of Chlorella and resulted in close agreement with triacylglycerol (TAG) levels determined by thin layer chromatography. The assay was found to more accurately measure algal lipids conducive to biodiesel production and nutrition applications than the widely used gravimetric assay. Assay response was also consistent among different species, in contrast to Nile red cell staining procedures.

The impact of elevated CO2 concentration on the quality of algal starch as a potential biofuel feedstock.

Cultured microalgae are viewed as important producers of lipids and polysaccharides, both of which are precursor molecules for the production of biofuels. This study addressed the impact of elevated carbon dioxide (CO2) on Chlorella sorokiniana production of starch and on several properties of the starch produced. The production of C. sorokiniana biomass, lipid and starch were enhanced when cultures were supplied with 2% CO2. Starch granules from algae grown in ambient air and 2% CO2 were analyzed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The granules from algae grown in 2% CO2 were disk-shaped and contained mainly stromal starch; granules from cultures grown in ambient air were cup-shaped with primarily pyrenoid starch. The granules from cells grown in 2% CO2 had a higher proportion of the accumulated starch as the highly branched, amylopectin glucan than did granules from cells grown in air. The rate of hydrolysis of starch from 2% CO2-grown cells was 1.25 times greater than that from air-grown cells and 2-11 times higher than the rates of hydrolysis of starches from cereal grains. These data indicate that culturing C. sorokiniana in elevated CO2 not only increases biomass yield but also improves the structure and composition of starch granules for use in biofuel generation. These modifications in culture conditions increase the hydrolysis efficiency of the starch hydrolysis, thus providing potentially important gains for biofuel production.

Managing the cultivation and processing of microalgae to prolong storage in water-in-oil emulsions.

Producing biofuel from microalgae on a large scale will require high biomass productivity using systems such as high-rate raceway ponds. The vast scale of proposed raceway ponds, spanning 247 to 988 acres per farm, suggests practices currently used in commercial monoculture agricultural systems will need to be adopted for cultivation of algae. In commercial crop production, monoculture is facilitated by a well-established seed production, distribution, and delivery system. Currently, no such system exists for microalgae. The aims of this study were to investigate the application of water-in-oil (W/O) emulsions for the storage of microalgae and the management steps required to prolong cell viability. Water-in-oil emulsions were prepared with Chlorella sorokiniana, C. minutissima, C. vulgaris var. vulgaris, and C. vulgaris to investigate the impacts of cell cultivation medium and cell acclimation prior to emulsification on cell viability during storage. For emulsions prepared with C. sorokiniana, cells that received an acclimation treatment 24 h between cell separation from the cultivation medium and emulsification survived over 100 days longer than cells that did not receive an acclimation treatment. Emulsions prepared with C. sorokiniana grown in medium containing 29.7 mM KNO3, 1.66 mM MgSO4 · 7H2O, and 0.85 mM FeSO4 · 2H2O had higher levels of viable cells after 100 days of storage compared to cells grown in medium containing 9.90 mM KNO3 and 0.20 mM MgSO4 · 7H2O with no FeSO4 · 2H2O. The results indicate that processing of cells can be managed to increase the stability of microalgae in W/O emulsions.

Microorganism viability influences internal phase droplet size changes during storage in water-in-oil emulsions.

Water-in-oil emulsions provide an alternative for long-term stabilization of microorganisms. Maintaining physical stability of the emulsion and cell viability is critical for large-scale application. Water-in-oil (W/O) emulsions were prepared with the biolarvacide Lagenidium giganteum and the green alga Chlorella vulgaris. Physical stability was measured via light scattering measurements of the internal phase droplets and cell viability was measured by plating and enumerating colony forming units. Emulsions were demonstrated to stabilize L. giganteum and C. vulgaris for more than 4 months without refrigeration. Introducing nutrients into the internal phase of W/O emulsions without cells had no significant effect on changes in aqueous phase droplet size dynamics. Internal phase droplet size changes that occurred over time were greater in the presence of cells. Increases in droplet size were correlated with cell death indicating measurement of internal phase droplet size changes may be an approach for monitoring declines in cell viability during storage.

Improving the efficiency of enzyme utilization for sugar beet pulp hydrolysis.

Sugar beet pulp (SBP) is a carbohydrate-rich residue of table sugar processing. It shows promise as a feedstock for fermentable sugar and biofuel production via enzymatic hydrolysis and microbial fermentation. This research focused on the enzymatic hydrolysis of SBP and examined the effects of solid loading (2-10 %, dry basis), enzyme preparation, and enzyme recycle on the production of fermentable sugars. The enzyme partitioning to the solid and liquid phases during SBP enzymatic hydrolysis and loss during recycling were investigated using SDS-PAGE and Zymogram analysis. Without considering product inhibition, the cellulase added initially to the SBP hydrolysis lost only 6 % filter paper activity and negligible carboxymethyl cellulose activity upon multiple cycles of SBP hydrolysis. It was found that enzyme dosage can be reduced by 50 % while maintaining similar, and in some cases higher fermentable sugar yield. The removal of hydrolysis products will further improve enzymatic hydrolysis of SBP for biofuel production.

The effect of circular soil biosolarization treatment on the physiology, metabolomics, and microbiome of tomato plants under certain abiotic stresses.

Soil biosolarization (SBS) is an alternative technique for soil pest control to standard techniques such as soil fumigation and soil solarization (SS). By using both solar heating and fermentation of organic amendments, faster and more effective control of soilborne pathogens can be achieved. A circular economy may be created by using the residues of a given crop as organic amendments to biosolarize fields that produce that crop, which is termed circular soil biosolarization (CSBS). In this study, CSBS was employed by biosolarizing soil with amended tomato pomace (TP) residues and examining its impact on tomato cropping under conditions of abiotic stresses, specifically high salinity and nitrogen deficiency. The results showed that in the absence of abiotic stress, CSBS can benefit plant physiological performance, growth and yield relative to SS. Moreover, CSBS significantly mitigated the impacts of abiotic stress conditions. The results also showed that CSBS impacted the soil microbiome and plant metabolome. Mycoplana and Kaistobacter genera were found to be positively correlated with benefits to tomato plants health under abiotic stress conditions. Conversely, the relative abundance of the orders RB41, MND1, and the family Ellin6075 and were negatively correlated with tomato plants health. Moreover, several metabolites were significantly affected in plants grown in SS- and CSBS-treated soils under abiotic stress conditions. The metabolite xylonic acid isomer was found to be significantly negatively correlated with tomato plants health performance across all treatments. These findings improve understanding of the interactions between CSBS, soil ecology, and crop physiology under abiotic stress conditions.

Glycoside hydrolase activities of thermophilic bacterial consortia adapted to switchgrass.

Industrial-scale biofuel production requires robust enzymatic cocktails to produce fermentable sugars from lignocellulosic biomass. Thermophilic bacterial consortia are a potential source of cellulases and hemicellulases adapted to harsher reaction conditions than commercial fungal enzymes. Compost-derived microbial consortia were adapted to switchgrass at 60°C to develop thermophilic biomass-degrading consortia for detailed studies. Microbial community analysis using small-subunit rRNA gene amplicon pyrosequencing and short-read metagenomic sequencing demonstrated that thermophilic adaptation to switchgrass resulted in low-diversity bacterial consortia with a high abundance of bacteria related to thermophilic paenibacilli, Rhodothermus marinus, and Thermus thermophilus. At lower abundance, thermophilic Chloroflexi and an uncultivated lineage of the Gemmatimonadetes phylum were observed. Supernatants isolated from these consortia had high levels of xylanase and endoglucanase activities. Compared to commercial enzyme preparations, the endoglucanase enzymes had a higher thermotolerance and were more stable in the presence of 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]), an ionic liquid used for biomass pretreatment. The supernatants were used to saccharify [C2mim][OAc]-pretreated switchgrass at elevated temperatures (up to 80°C), demonstrating that these consortia are an excellent source of enzymes for the development of enzymatic cocktails tailored to more extreme reaction conditions.

High-throughput analysis of hexosamine using a colorimetric method.

A 96-well plate method was developed for analysis of total hexosamine content in biological samples. Four hexosamine monomer derivatives-glucosamine hydrochloride, glucosamine sulfate, galactosamine hydrochloride, and mannosamine hydrochloride-were examined for the linearity of their spectra in the concentration range specified in the assay. The hexosamine concentration analysis range was linear from 0.1 to 1 mM. The quantification of hexosamines from chitin and chitosan upon acid hydrolysis was also tested. Accurate quantification of glucosamine content in chitin and chitosan with different molecular sizes and degrees of acetylation was demonstrated using the new method.

Bioenergy feedstock-specific enrichment of microbial populations during high-solids thermophilic deconstruction.

Thermophilic microbial communities that are active in a high-solids environment offer great potential for the discovery of industrially relevant enzymes that efficiently deconstruct bioenergy feedstocks. In this study, finished green waste compost was used as an inoculum source to enrich microbial communities and associated enzymes that hydrolyze cellulose and hemicellulose during thermophilic high-solids fermentation of the bioenergy feedstocks switchgrass and corn stover. Methods involving the disruption of enzyme and plant cell wall polysaccharide interactions were developed to recover xylanase and endoglucanase activity from deconstructed solids. Xylanase and endoglucanase activity increased by more than a factor of 5, upon four successive enrichments on switchgrass. Overall, the changes for switchgrass were more pronounced than for corn stover; solids reduction between the first and second enrichments increased by a factor of four for switchgrass while solids reduction remained relatively constant for corn stover. Amplicon pyrosequencing analysis of small-subunit ribosomal RNA genes recovered from enriched samples indicated rapid changes in the microbial communities between the first and second enrichment with the simplified communities achieved by the third enrichment. The results demonstrate a successful approach for enrichment of unique microbial communities and enzymes active in a thermophilic high-solids environment.

Factorial experimental designs for enhancement of concurrent poly(hydroxyalkanoate) production and brewery wastewater treatment.

The influence of four main process parameters--solids retention time (SRT), hydraulic retention time (HRT), anoxic-oxic cycling, and carbon-to-nitrogen ratio (C/N ratio)--on poly(hydroxyalkanoate) (PHA) production, while treating brewery wastewater, was studied. Two sets of two-level, three-factor experimental designs were implemented to (1) determine the effects and interactions among process parameters, (2) assess their significance to PHA production, and (3) approximate optimal operational conditions. The HRT and SRT were found to be the crucial operational parameters affecting PHA production. The highest PHA content of 55% (on a cell-weight basis) was produced at a 4-day HRT and 4-day SRT, whereas a maximum PHA concentration of 907 mg/L was obtained at a 2-day HRT and 12-day SRT. The effect of anoxic conditions on PHA production was insignificant. The C/N ratio played a more important role in the PHA concentration in the system than in the PHA content in the biomass.