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Santillan, E.

Publications and source records attributed to Santillan, E..

17 recordsLinked to original sources
bioRxiv2026

Dual-loop involving microbial single-cell protein production from soybean-processing wastewater and effluent-based refinement for circular bioeconomy applications

Nutrient-rich food-processing wastewaters represent valuable yet under-utilised side streams for sustainable protein production in the form of microbial biomass. Here we present an integrated dual-loop bioprocess that converts soybean-processing wastewater into microbial single-cell protein (SCP) while achieving substantial nutrient removal and product refinement. In the first loop, previously enriched microbial consortia were inoculated and cultivated in four parallel sequencing batch reactors (SBRs) for 44Ldays at a hydraulic retention time (HRT) of 3Ldays. This bioprocess configuration demonstrated features that support future scale-up while maintaining process stability, achieving a protein content of 33.3L{+/-}L3.2%, doubling the protein yield (15.32L{+/-}L3.49Lg dry weight per g soluble TKN) and quadrupling the production rate (0.29L{+/-}L0.06Lg dry weight LL{superscript 1} dL{superscript 1}) compared to operating reactors without inoculation (HRT: 7.2Ldays). Effluent treatment was stable, with 84% carbon and 78% nitrogen removal efficiencies, demonstrating efficient nutrient recovery. The SCP biomass was enriched in functional taxa, including Acidipropionibacterium, Lactococcus, Megasphaera, and Azospirillum, suggesting that reactor conditions and inoculum selection promoted a stable, protein-productive microbial community with potential probiotic benefits. In the second loop, bioreactor effluent was reused as aqueous matrix for heat treatment (60{degrees}C) of the SCP biomass, reducing the RNA content from 8.6% to 2.6%, with a 39% biomass loss accompanied by a 30% increase in total amino acid concentration. Hence, our valorisation approach integrates microbial biomass production, effluent reuse, and product refinement within a circular framework. The system provides a resource-efficient pathway for converting food-sector side streams into high-quality microbial community-based SCP, highlighting its potential scalability for sustainable nutrient and water management.

microbiology↗
bioRxiv2026

Microbial community-based single-cell protein supports partial fishmeal replacement in juvenile Asian seabass diets across feeding trials and production scales

The growing demand for sustainable aquafeeds has intensified interest in alternative protein ingredients capable of reducing reliance on fishmeal without compromising fish performance. Here, we evaluated microbial community-based single-cell protein (SCP) as a fishmeal substitute in juvenile Asian seabass (Lates calcarifer) diets in two independent feeding trials of juvenile fish conducted over 49 and 56 days, respectively and compared them to a previous study that lasted 24 days. SCP was produced from nutrient-rich soybean-processing side streams by microbial communities in fermenters and incorporated into experimental diets at inclusion levels ranging from 10% to 100% fishmeal replacement. In the 24-day trial, a diet containing 50% fishmeal replacement with lab-scale produced SCP achieved 100% survival and a feed conversion ratio (FCR), specific growth rate (SGR), and weight gain comparable to the fishmeal control diet. In the 49-day trial using pilot-scale produced SCP, a 50% fishmeal replacement also maintained an FCR and feed intake comparable to the control, whereas complete replacement reduced feed intake and growth performance. In a 56-day pilot-scale trial that used 500-L fish tanks, diets containing up to 50% fishmeal replacement maintained comparable survival, weight gain, and SGR, although moderately higher FCR values were observed at higher SCP inclusion levels. Proximate composition and essential amino acid profiles of fish fed control or SCP-containing diets were comparable. Genome-resolved metagenomic analyses revealed diverse microbial taxa associated with the SCP. Collectively, these findings support microbial community-based SCP as a scalable and reproducible alternative protein platform for aquaculture feeds across independent trials and production scales.

microbiology↗
bioRxiv2025

Microbial community-based protein enables sustainable fishmeal replacement and improved resilience in Pacific white shrimp (Litopenaeus vannamei)

The growing demand for aquaculture feeds requires sustainable alternatives to fishmeal. Microbial community-based single-cell protein (SCP) produced from soybean-processing wastewater offers a circular approach that combines nutrient recovery with aquafeed production. We evaluated SCP as a fishmeal replacement for Pacific white shrimp (Litopenaeus vannamei) across four feeding trials lasting 42-84 days, involving shrimp from different hatcheries and SCP produced at laboratory and pilot scales. Diets containing 0-100% fishmeal replacement, corresponding to up to 220 g SCP kg-1 feed, were tested. Replacement of 50% was consistently supported across trials, while levels up to 90% maintained several key performance outcomes, including specific growth rate, feed conversion ratio, and survival, without significant differences from the fishmeal control. Complete replacement reduced growth in one trial, potentially reflecting the lower protein content and less favourable amino acid balance of SCP relative to fishmeal, particularly its lower lysine and methionine proportions. Shrimp tissue proximate and amino acid compositions showed no clear adverse responses to SCP inclusion in the trial in which they were assessed. Cross-trial analysis revealed a positive association between SCP replacement and survival after accounting for trial-specific baseline differences, with survival increasing by an estimated 1.53 percentage points for every 10-percentage-point increase in replacement (HC3 95% CI: 0.90-2.17; P < 0.001). Although the mechanisms underlying this association remain to be established, the findings support investigation of potential functional effects through targeted challenge and mechanistic studies. Overall, performance across four heterogeneous trials supports substantial fishmeal replacement by microbial community-based SCP and demonstrates its potential for practical application as a circular aquafeed ingredient.

microbiology↗
bioRxiv2025

Uncovering microbial life-history strategies under disturbance: a trait-based computational analysis of anaerobic systems

Trait-based approaches are helpful in simplifying ecosystem complexity to explore disturbance-diversity-function relationships. These frameworks classify organisms based on their functional characteristics--traits that influence growth, survival and reproduction--providing a mechanistic basis to understand how communities respond to changes in their environment. The application of these approaches has been successful in ecology, but to date has only been tested in a few microbial ecosystems, namely, soil microbial communities and aerobic bioreactors treating wastewater. Here, we employed Grimes competitor-stress-tolerant-ruderal framework in replicated mesophilic anaerobic bioreactors exposed to a disturbance (biomass removal) with varied frequencies at a constant number of disturbance events for 90 days. Bioreactors were inoculated with sludge from full-scale anaerobic digesters and fed with a mixture of primary and waste activated sludge. A genome-resolved metagenomics approach was utilised to assess the microbial communities. We found that communities across the disturbance range were clustered into three groups, suggesting the adoption of a three-way life-history strategy. This study demonstrates, for the first time, the applicability of trait-based life-history strategies in anaerobic microbial systems under disturbance, providing a new perspective for understanding and managing microbial ecosystems under disturbance conditions.

ecology↗
bioRxiv2025

Predicting microbial community responses to disturbance using genome-resolved trait-based life-history strategies

Understanding how microbial communities respond to disturbance remains a fundamental question in ecology, with broad implications for biodiversity, ecosystem function, and biotechnology. Trait-based approaches offer general rules to predict community responses by linking ecological strategies to measurable traits. While life-history strategy frameworks such as the competitor-ruderal-stress-tolerant (CSR) model are well established in plant and animal ecology, their application to microbial communities has been limited. Here, we experimentally tested how microbial communities shift across a gradient of disturbance frequency in replicated bioreactors treating synthetic wastewater. We applied six conditions by doubling the organic loading rate at different frequencies, from undisturbed to press disturbance, and monitored changes over 42 days using genome-resolved metagenomics, 16S rRNA gene sequencing, biomass quantification, and effluent chemistry. By integrating ordination, network analysis, and machine learning, we identified emergent community-level life-history strategies that aligned with increasing disturbance. These strategies were reflected in functional trade-offs, shifts in community composition, and genomic trait distributions. A simulation-based approach was used to generate a CSR classification of metagenome-assembled genomes, which was consistent with patterns observed in other microbial ecosystems. Our results demonstrate that life-history frameworks can capture predictable dynamics in microbial communities across disturbance regimes. This strategy provides a unifying tool for linking microbial structure, function, and traits across scales, helping to reconcile ecological theory with microbial resource management. More broadly, our findings support the integration of classical ecological theory with microbial genomics to uncover the trait-based principles that govern microbiome function and stability in both natural and engineered ecosystems.

ecology↗
bioRxiv2025

Enhanced Resistance and Resilience of Anaerobic Digestion Microbiome after Single and Dual Short-Term Disturbances

Anaerobic digesters are operated at high solids retention times (SRTs) of 20 d or longer to mitigate accidental substrate overloading. However, the long-term effects of such disturbances on digester performance and microbiome remain unclear. This study investigates whether short-term pulse disturbances (temporary SRT reductions) can enhance microbial resilience and accelerate recovery from sustained overloading conditions. We explored the feasibility of anaerobic digestion at shorter SRTs by introducing one or two pulse SRT disturbances (SRT reduction from 15 to 5 d) in four mesophilic anaerobic digesters treating wastewater sludge, followed by sustained operation (press disturbance) 5 d SRT. Dual pulse disturbances showed faster process recovery compared to digesters receiving a single pulse disturbance (60 d vs 104 d) with reduced volatile fatty acids levels during the subsequent press disturbance periods. Microbial community redundancy and resilience, with shifts between the slow-growing, resource-efficient taxa (K-strategists) and fast-growing, opportunistic taxa (r-strategists), were crucial in ensuring minimal disruptions during disturbance and recovery periods. Overall, pulse disturbances may serve as a practical tool for enhancing microbiome resilience in anaerobic digestion, enabling stable performance under fluctuating substrate loading conditions. SYNOPSISControlled pulse disturbances enhance anaerobic digestion resilience by priming microbial communities, enabling stable performance at short solids retention times and high organic loading conditions.

microbiology↗
bioRxiv2024

MicroEcoTools: an R package for comprehensive theoretical microbial ecology analysis

BackgroundEcological theory aids in understanding how disturbances affect ecosystems. However, experimental data are often complex, with multiple post-disturbance theories potentially applying simultaneously to the same ecosystem. This emphasizes the need for tools to experimentally test these theoretical predictions. ResultsWe introduce MicroEcoTools, an R package designed to test ecological framework predictions using microbial community data. It assesses microbial diversity and evaluates the relative impacts of stochastic and deterministic assembly mechanisms through a taxa-based null model approach for replicated designs. Specifically, the package allows application of Grimes trait-based life-history categories--competitor, stress-tolerant, and ruderal (CSR)-- to taxa, functional traits, and ecosystem functions within microbial communities. MicroEcoTools also includes relevant statistical tests, numeric simulations, and publicly available datasets for demonstration. ConclusionsIn conclusion, MicroEcoTools facilitates the application of ecological frameworks, including community assembly mechanisms, diversity analysis, and life-history strategies, to microbial ecosystems under disturbance. This R package, along with its source code, can be freely accessed on GitHub at https://www.github.com/Soheil-A-Neshat/MicroEcoTools.

ecology↗
bioRxiv2023

Microbial community-based bacterial protein from soybean-processing wastewater as a value-added alternative fish feed ingredient for Asian sea bass (Lates calcarifer)

As the global demand for food increases, aquaculture plays a key role as the fastest growing animal protein sector. However, existing aquafeeds contain protein ingredients that are not sustainable under current production systems. We evaluated the use of microbial community-based single cell protein (SCP), produced from soybean processing wastewater, as a partial fishmeal protein substitute in juvenile Asian seabass (Lates calcarifer). A 24-day feeding trial was conducted with a control fishmeal diet and a 50% fishmeal replacement with microbial community-based SCP as an experimental group, in triplicate tanks containing 20 fish each. Both diets met the protein, essential amino acids (except for lysine), and fat requirements for juvenile Asian sea bass. The microbial composition of the SCP was dominated by the genera Acidipropionibacterium and Propioniciclava, which have potential as probiotics and producers of valuable metabolites. The growth performance in terms of percent weight gain, feed conversion ratio (FCR), specific growth rate (SGR), and survival were not significantly different between groups after 24 days. The experimental group had less variability in terms of weight gain and FCR than the control group. Overall, microbial community-based protein produced from soybean processing wastewater has potential as a value-added feed ingredient for sustainable aquaculture feeds.

microbiology↗
bioRxiv2022

Microbial community-based production of single cell protein from soybean-processing wastewater of variable chemical composition

The use of food-processing wastewaters to produce microbial biomass-derived single cell protein (SCP) is a sustainable way to meet the global food demand. Yet, despite the potential benefits of lower costs and greater resource recovery compared to pure cultures, bioconversion processes relying on microbial community-based approaches to SCP production have received scarce attention. Here, we evaluated SCP production from soybean-processing wastewaters under controlled reactor conditions using the existent microbial communities in these wastewaters. Six sequencing batch reactors of 4.5-L working volume were operated at 30 {square} for 34 d in cycles consisting of 3-h anaerobic and 9-h aerobic phases. Four reactors received no microbial inoculum and the remaining two were amended with a 1.5 L of mixed culture from a prior microbial community-based SCP production. Microbial characterization was done via 16S rRNA gene metabarcoding. Influent wastewater batches had variable chemical characteristics but a similar microbial composition. Reactors produced more SCP when fed with wastewaters of higher soluble total Kjeldahl nitrogen (sTKN) content and a lower carbon-to-nitrogen ratio (sCOD:sTKN). The biomass protein yield ranged from 0.24 to 3.13 g protein/g sTKN, with a maximum protein content of 50%. An average of 92% of sCOD and 73% of sTN removal was achieved. Distinct microbial communities were enriched in all six bioreactors after 34 d, where the prevailing genera included Azospirillum, Rhodobacter, Lactococcus, Novosphingobium, and Acidipropionibacterium. In contrast, the microbial community of influent wastewaters was dominated by Lactococcus and Weissella. We showed that constituents in soybean wastewater can be converted to SCP through microbial community-based growth processes and demonstrated the effect of variable influent wastewater composition on SCP production.

microbiology↗
bioRxiv2021

Microbiome assembly predictably shapes diversity across a range of disturbance frequencies

Diversity is frequently linked to the functional stability of ecological communities. However, its association with assembly mechanisms remains largely unknown, particularly under fluctuating disturbances. Here, we subjected complex bacterial communities in bioreactor microcosms to different frequencies of organic loading shocks, tracking temporal dynamics in their assembly, structure and function. Null modelling revealed a stronger role of stochasticity at intermediate disturbance frequencies, preceding the formation of a peak in -diversity. Communities at extreme ends of the disturbance range had the lowest -diversity and highest within-treatment similarity in terms of {beta}-diversity, with stronger deterministic assembly. Stochasticity prevailed during the initial successional stages, coinciding with better specialized function (nitrogen removal). In contrast, general functions (carbon removal and microbial aggregate settleability) benefited from stronger deterministic processes. We showed that changes in assembly processes predictably precede changes in diversity under a gradient of disturbance frequencies, advancing our understanding of the mechanisms behind disturbance-diversity-function relationships.

ecology↗
bioRxiv2021

Metagenomics and Metatranscriptomics Reveal Pathway of 3-Chloroaniline Degradation in Wastewater Reactors

Biological wastewater treatment systems are often affected by major shifts in influent quality, including the input of various toxic chemicals. Yet the mechanisms underlying adaptation of activated sludge process performance when challenged with a sustained toxin input are rarely studied in a controlled and replicated experimental setting. Three replicate bench-scale bioreactors were subjected to a chemical disturbance in the form of 3-chloroaniline (3-CA) over 132 days, after an acclimation period of 58 days, while three control reactors received no 3-CA input. Nitrification was initially affected by 3-CA but the microbial communities in all three treatment reactors adapted to biologically degrade 3-CA within three weeks of the experiment, resulting in partial nitrification recovery. Combining process and microbial community data from amplicon sequencing with potential functions gleaned from assembled metagenomics and metatranscriptomics data, two putative degradation pathways for 3-CA were identified. The first pathway proceeds via a phenol monooxygenase followed by ortho-cleavage of the aromatic ring, and the second one involves a benzoate dioxygenase and subsequent meta-cleavage of the aromatic ring. The genera Gemmatimonas, OLB8, and Taibaiella correlated significantly with 3-CA degradation. Metagenome-assembled genome data also showed the genus OLB8 to be differentially enriched in treatment reactors, making it a strong candidate as 3-CA degrader. Using replicated reactors, this study has demonstrated the impact of a sustained stress on the activated sludge community and processes carried out by its members, followed by process recovery. By a combination of techniques, we showed that microbial communities can develop degradative capacity following a sustained xenobiotic input, and that targeted culture-independent approaches can suggest plausible mechanisms for 3-CA degradation and identify the taxa potentially contributing to it.

microbiology↗
bioRxiv2020

Press xenobiotic disturbance favors deterministic assembly with a shift in function and structure of bacterial communities in sludge bioreactors

Disturbance is thought to affect community assembly mechanisms, which in turn shape community structure and the overall function of the ecosystem. Here, we tested the effect of a continuous (press) xenobiotic disturbance on the function, structure, and assembly of bacterial communities within a wastewater treatment system. Two sets of four-liter sequencing batch reactors were operated in triplicate with and without the addition of 3-chloroaniline for a period of 132 days, following 58 days of acclimation after inoculation with sludge from a full-scale treatment plant. Temporal dynamics of bacterial community structure were derived from 16S rRNA gene amplicon sequencing. Community function, structure and assembly differed between press disturbed and undisturbed reactors. Temporal partitioning of assembly mechanisms via phylogenetic and non-phylogenetic null modelling analysis revealed that deterministic assembly prevailed for disturbed bioreactors, while the role of stochastic assembly was stronger for undisturbed reactors. Our findings are relevant because research spanning various disturbance types, environments and spatiotemporal scales is needed for a comprehensive understanding of the effects of press disturbances on assembly mechanisms, structure, and function of microbial communities. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/341966v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@115e7d3org.highwire.dtl.DTLVardef@126da82org.highwire.dtl.DTLVardef@1d3c4caorg.highwire.dtl.DTLVardef@1cb2ea_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗
bioRxiv2019

Press disturbance unveils different community structure, function and assembly of bacterial taxa and functional genes in mesocosm-scale bioreactors

Sustained perturbations, or press disturbances, are of interest in microbial ecology as they can drive systems to alternative stable states. Here, we tested the effect of a sustained doubling of organic carbon loading on structure, assembly and function of bacterial communities. Two sets of replicate 5-liter sequencing batch reactors were operated at two different organic carbon loading levels (323 and 629 mg COD L-1) for a period of 74 days, following 53 days of acclimation after inoculation with sludge from a full-scale treatment plant. Temporal dynamics of community taxonomic and functional gene structure were derived from metagenomics and 16S rRNA gene metabarcoding data. Assembly mechanisms were assessed through a mathematical null model on the effective bacterial turnover expressed as a proportion of total bacterial diversity. Disturbed reactors exhibited different community function, structure and assembly compared to the undisturbed reactors. Bacterial taxa and functional genes showed dissimilar alpha-diversity and community assembly patterns. Deterministic assembly mechanisms were generally stronger in disturbed reactors, associated with common taxa. Stochastic assembly was more important for functional genes and was driven by rare genes. We urge caution when assessing microbial community assembly mechanisms, as results can vary depending on the approach.

microbiology↗
bioRxiv2019

Sustained organic loading perturbation inhibits nitrite oxidation in bioreactors, unveiling variable resistance and resilience of nitrification and nitrifiers

Sustained disturbances are relevant for environmental biotechnology as they can lead to alternative stable states in a system that may not be reversible. Here, we tested the effect of a sustained organic loading alteration (food-to-biomass ratio, F:M, and carbon-to-nitrogen ratio, C:N) on activated sludge bioreactors, focusing on the stability of nitrification and nitrifiers. Two sets of replicate 5-liter sequencing batch reactors were operated at different, low and high, F:M (0.19-0.36 mgCOD/mgTSS/d) and C:N (3.5-6.3 mgCOD/mgTKN) conditions for a period of 74 days, following 53 days of sludge acclimation. Recovery and resilience were tested during the last 14 days by operating all reactors at low F:M and C:N (henceforth termed F:M-C:N). Stable nitrite accumulation (77%) was achieved through high F:M-C:N loading with a concurrent reduction in the abundance of Nitrospira. Subsequently, only two of the three reactors experiencing a switch back from high to low F:M-C:N recovered the nitrite oxidation function, with an increase in Nitrobacter as the predominant NOB, without a recovery of Nitrospira. The AOB community was more diverse, resistant and resilient than the NOB community. We showed that functional recovery and resilience can vary across replicate reactors, and that nitrification recovery need not coincide with a return to the initial nitrifying community structure.

microbiology↗
bioRxiv2019

Trait-based life-history strategies explain succession scenario for complex bacterial communities under varying disturbance

Trait-based approaches are increasingly gaining importance in community ecology, as a way of finding general rules for the mechanisms driving changes in community structure and function under the influence of perturbations. Frameworks for life-history strategies have been successfully applied to describe changes in plant and animal communities upon disturbance. To evaluate their applicability to complex bacterial communities, we operated replicated wastewater treatment bioreactors for 35 days and subjected them to eight different disturbance frequencies of a toxic pollutant (3-chloroaniline), starting with a mixed inoculum from a full-scale treatment plant. Relevant ecosystem functions were tracked and microbial communities assessed through metagenomics and 16S rRNA gene sequencing. Combining a series of ordination, statistical and network analysis methods, we associated different life-history strategies with microbial communities across the disturbance range. These strategies were evaluated using tradeoffs in community function and genotypic potential, and changes in bacterial genus composition. We further compared our findings with other ecological studies and adopted a semi-quantitative CSR (competitors, ruderals, stress-tolerants) classification. The framework reduces complex datasets of microbial traits, functions, and taxa into ecologically meaningful components to help understand the system response to disturbance, and hence represents a promising tool for managing microbial communities. Originality-Significance StatementThis study establishes, for the first time, CSR life-history strategies in the context of bacterial communities. This framework is explained using community aggregated traits in an environment other than soil, also a first, using a combination of ordination methods, network analysis, and genotypic information from shotgun metagenomics and 16S rRNA gene amplicon sequencing.

microbiology↗
bioRxiv2018

Frequency of disturbance alters diversity, function, and underlying assembly mechanisms of complex bacterial communities

Disturbance is known to affect ecosystem structure, but predicting its outcomes remains elusive. Similarly, community diversity is believed to relate to ecosystem functions, yet the underlying mechanisms are poorly understood. Here, we tested the effect of disturbance on the structure, diversity, and ecosystem function of complex microbial communities within an engineered system. We carried out a microcosm experiment where activated sludge bioreactors were subjected to a range of disturbances in the form of a toxic pollutant, tracking changes in ecosystem function. Microbial communities were assessed by combining distance-based methods, general linear multivariate models, -diversity indices, and null model analyses on metagenomics and 16S rRNA gene amplicon data. A stronger temporal decrease in -diversity at the extreme, undisturbed and press-disturbed, ends of the disturbance range led to a hump-backed pattern, with the highest diversity found at intermediate levels of disturbance. Undisturbed and press-disturbed levels displayed the highest community and functional similarity across replicates, suggesting deterministic processes were dominating. The opposite was observed amongst intermediately disturbed levels, indicating stronger stochastic assembly mechanisms. Tradeoffs were observed in community function between organic carbon removal and both nitrification and biomass productivity, as well as between diversity and these functions. Hence, not every ecosystem function was favoured by higher community diversity. Our results show that the assessment of changes in diversity, along with the underlying stochastic-niche assembly processes, is essential to understanding the impact of disturbance in complex microbial communities.\n\nImportanceMicrobes drive the Earths biogeochemical cycles, yet how they respond to perturbations like anthropogenic pollutants is poorly understood. As human impact continues to increase worldwide, foreseeing how disturbances will affect microbial communities and the ecosystem services they provide is key for ecosystem management and conservation efforts. Employing laboratory-scale wastewater treatment bioreactors, this study shows that changes in community diversity accompany variations in the underlying deterministic-stochastic assembly mechanisms. Disturbances could promote stochastic community structuring, which despite harboring higher diversity could lead to variable overall function, possibly explaining why after similar perturbations the process outcome differs. A conceptual framework, termed the intermediate stochasticity hypothesis is proposed to theoretically predict bacterial community shifts in diversity and ecosystem function, given a range of possible disturbance types, in a well-replicated time-series experiment. Our findings are relevant for managing complex microbial systems, which could display similar responses to disturbance, like oceans, soils or the human gut.

microbiology↗