Carbon-rich Substrate Research Articles

Laser-Induced Graphene for Electrochemical Analysis of Food and Agricultural Contaminants

Carbon-nanomaterial sensors offer promising routes toward low-cost, high-throughput electrochemical sensors. Carbon nanomaterials, such as graphene and carbon nanotube (CNT), have been studied with various fabrication methods to produce sensors, including high-quality thin film graphene and CNT by chemical vapor deposition. However, the complexity and cost associated with their fabrication have hindered their implementation into single-use test strip sensors. Even high-yield mechanical and/or chemical exfoliation of graphene from graphite followed by solution phase printing of electrodes can be costly due to the complexity of ink formulation and the need for post-print annealing requirements. However, laser-induced graphene (LIG) avoids the need to exfoliate graphene from graphite or to create a solution-phase graphene ink by converting sp3 carbon found in carbon-rich substrates into conductive sp2-hybridized carbon found in graphene through a laser scribing technique. The resulting LIG also exhibits a high surface area and is porous with a high density of edge plane sites, which are beneficial for biofunctionalization and heterogeneous charge transport during electrochemical sensing. Furthermore, we have demonstrated that the surface area and wettability of the LIG can be tuned to greatly improve the sensitivity of electrochemical enzymatic biosensors (detection limits down to the picomolar range) and reduce the water layer buildup between the ion-selective membrane and the electrode, which improves the accuracy of the sensor readings. The fabricated LIG electrodes have been used in a wide variety of electrochemical sensing applications, including in food safety (foodborne pathogens and other contaminants), environmental monitoring (agrochemicals), and health monitoring sensors (salivary potassium and lactate, and urinary potassium and ammonium). Different electroanalytical methods are used, including amperometric, potentiometric, coulometric, and impedimetric, to quantify the analytes. We demonstrate how the ISE (ion-selective electrodes) can be functionalized with PVC-based ion-selective membranes for potassium, nitrate, nitrite, ammonium, sodium, and pH in various biological solutions, including soil and water for nutrient/fertilizer monitoring in farm fields. Additionally, we demonstrate how the hydrophilic LIG graphene can be used to create other highly sensitive electrochemical biosensors including enzymatic pesticide biosensors and Salmonella spp. immunosensors. All these electroanalytical sensors were validated in real-world samples and demonstrated to have limits of detection and linear sensing ranges that are relevant to their sensing applications. Results demonstrate the utility of LIG-based sensors for rapid and sensitive electrochemical measurement of contaminants as a readily modified platform for scalable production of electroanalytical devices applied toward point-of-service environmental and food monitoring.

Flexible Graphene-Based Sensors for Electrochemical Analysis of Human Saliva and Sweat

Flexible carbon nanomaterial sensors offer a low-cost, facile fabrication approach toward producing sensors at an industrial scale for electrochemical sensing. Graphene and carbon nanotube (CNT) based sensors have been studied, with various fabrication methods available, including producing high-quality thin film graphene and CNTs via chemical vapor deposition (CVD). However, CVD is a high-temperature vacuum process that is low-yielding and consequently costly, hindering its implementation into single-use test strip sensors. Other methods, such as inkjet or aerosol printing, are more cost-effective as they print graphene inks obtained from liquid-phase exfoliation of graphite, which can be performed using large batch processing techniques. Additionally, the laser-induced graphene (LIG) technique directly converts sp3 carbon found in carbon-rich substrates, such as polyimide, into conductive sp2-hybridized carbon found in graphene through a laser scribing technique. Saliva and sweat offer promising biological fluids for precision health monitoring and diagnostics due to the non-invasive nature of their sampling (no need for a blood draw at a laboratory or a fingerstick at home) and due to the abundance of medically relevant analytes. Saliva can also be used for monitoring infections such as COVID-19 infection. Herein, we introduce graphene-based flexible sensors fabricated using scalable manufacturing based on aerosol jet printing (AJP) using custom-formulated graphene inks and laser-induced graphene via direct writing. We report a label-free, high-sensitivity, and rapid-response-time graphene-based electrochemical immunosensor for quantitatively detecting the SARS-CoV-2 Spike Receptor-Binding Domain (RBD) as well as SARS-CoV-2 Spike S1 protein. We demonstrate LIG-based electrochemical sensors for real-time monitoring of glucose, lactate, and sodium in sweat, and potassium and lactate in saliva. Different electrochemical methods are used, including amperometric, potentiometric, coulometric, and impedimetric, to quantify the analytes. All these electrochemical sensors were validated in human saliva and sweat and demonstrated to have limits of detection and linear sensing ranges that are relevant to their sensing applications. Flexibility studies were also performed to evaluate the ability of the sensors to operate under stresses caused by bending, such as those that would be encountered by skin and implanted oral sensors. Results demonstrate the utility of AJP-graphene and LIG for rapid and sensitive measurement of biological analytes and pathogen infection as simple methods for scalable production of non-invasive salivary and perspiration (sweat) health sensors.

Valorisation of industrial hemp (Cannabis sativa L.) residues and cheese whey into volatile fatty acids for single cell protein production

The production of single cell protein (SCP) using lignocellulosic materials stands out as a promising route in the circular bioeconomy transition. However, multiple steps are necessary for lignocellulosics-to-SCP processes, involving chemical pretreatments and specific aerobic cultures. Whereas there are no studies that investigated the SCP production from lignocellulosics by using only biological processes and microbial biomass able to work both anaerobically and aerobically. In this view, the valorisation of industrial hemp (Cannabis sativa L.) biomass residues (HBRs), specifically hurds and a mix of leaves and inflorescences, combined with cheese whey (CW) was investigated through a semi-continuous acidogenic co-fermentation process (co-AF). The aim of this study was to maximise HBRs conversion into VFAs to be further used as carbon-rich substrates for SCP production. Different process conditions were tested by either removing CW or increasing the amount of HBRs in terms of VS (i.e., two and four times) to evaluate the performance of the co-AF process. Increasing HBRs resulted in a proportional increase in VFA production up to 3115 mg HAc L−1, with experimental production nearly 40% higher than theoretical predictions. The synergy between HBRs and CW was demonstrated, proving the latter as essential to improve the biodegradability of the former. The produced VFAs were subsequently tested as substrates for SCP synthesis in batch aerobic tests. A biomass concentration of 2.43 g TSS L−1 was achieved with a C/N ratio of 5.0 and a pH of 9.0 after two days of aerobic fermentation, reaching a protein content of 42% (g protein per g TSS). These results demonstrate the overall feasibility of the VFA-mediated HBR-to-SCP valorisation process.

A Review on the Anaerobic Co-Digestion of Livestock Manures in the Context of Sustainable Waste Management

As the worldwide demand for meat per person is continuously increasing, there is a corresponding rise in the number of livestock animals, leading to an increase in livestock manure. Selecting appropriate treatment technologies for livestock manures is still a complex task and considerable debates over this issue persist. To develop a more comprehensive understanding of the manure treatment framework, this review was undertaken to assess the most utilized manure management technologies and underscore their respective challenges. Anaerobic digestion has become a commercial reality for treating livestock manures. However, the mono-digestion of single substrates comes with certain drawbacks associated with manure characteristics. Anaerobic co-digestion, involving the utilization of multiple feedstocks, holds the potential to overcome these limitations. Extensive research and development have underscored numerous intrinsic benefits of co-digestion. These include improved digestibility resulting from the synergistic effects of co-substrates and enhanced process stability. This review underscores the limitations associated with the mono-digestion of livestock manures and critically evaluates the advantages of their co-digestion with carbon-rich substrates. Additionally, this review delves into key livestock manure management practices globally, emphasizing the significance of co-digesting livestock manures while addressing the progress and challenges in this field.

Flexible Phenanthracene Nanotubes for Explosive Detection.

Phenanthracene nanotubes with arylene-ethynylene-butadiynylene rims and phenanthracene walls are synthesized in a modular bottom-up approach. One of the rims carries hexadecyloxy side chains, mediating the affinity to highly oriented pyrolytic graphite. Molecular dynamics simulations show that the nanotubes are much more flexible than their structural formulas suggest: In 12, the phenanthracene units act as hinges that flip the two macrocycles relative to each other to one of two possible sites, as quantum mechanical models suggest and scanning tunneling microscopy investigations prove. Unexpectedly, both theory and experiment show for 13 that the three phenanthracene hinges are deflected from the upright position, accompanied by a deformation of both macrocycles from their idealized sturdy macroporous geometry. This flexibility together with their affinity to carbon-rich substrates allows for an efficient host-guest chemistry at the solid/gas interface opening the potential for applications in single-walled carbon nanotube-based sensing, and the applicability to build new sensors for the detection of 2,4,6-trinitrotoluene via nitroaromatic markers is shown.

Advantages of using a carbon-rich substrate in a constructed wetland for agricultural water treatment: Carbon availability and biota development

Proper bed substrate selection is essential for the growth of microorganisms and plants, which are key elements in constructed wetlands (CWs) performance. This is even more important in CWs that treat irrigated agricultural drainage water characterised by a high nitrogen (N), but low carbon (C) and phosphorus (P) concentration. This nutrient imbalance compromises biota activity and limits water treatment processes. Three substrates were tested in a CW field-scale pilot plant for 2 years: gravel (100%) and two mixed substrates, gravel + 30% natural wetland soil and gravel + 10% biochar. While gravel is one of the most commonly used substrates in subsurface flow CWs, soil has hardly been employed, and field-scale biochar application studies are scarce. We analysed the effect of adding a C-rich substrate to gravel on both nutrient imbalance correction and the biotic performance of CWs. Adding natural soil or biochar increased P availability in beds. However, the dissolved organic C concentration in interstitial water was only enhanced by soil addition. Unlike gravel, microbial density and activity, and Phragmites australis plant growth, were higher in beds with soil, followed by those with biochar. A similar pattern was observed for plant tissue quality. Although biochar proved positive for biota, it had a short-term effect as a source of C and P. Therefore, employing a suitable gravel-soil mixture provides notable advantages for the irrigated agricultural water treatment challenge. Even after considering some limitations, our results represent an important step to design CWs that treat this wastewater type and for eutrophication control in sustainable agroecosystems.

Modelling atrazine sorption in carbon-rich substrates: a case study

Background The use of carbonaceous materials for environmental remediation is attractive due to low expense and high sorption capacity. However, their efficiency in contaminant removal is affected by substrate composition and sorption mechanism. Objective This study investigated atrazine sorption and desorption in three carbon-rich substrates. Data were evaluated considering isotherm models and sorbent chemical composition. Methods Sorption was performed by the batch equilibrium method with three substrates obtained from pig slurry composting with different proportions (w/w) of charcoal fines [...]

Effect of nutrients deficiency on biofilm formation and single cell protein production with a purple non-sulphur bacteria enriched culture

Purple non-sulphur bacteria (PNSB) are of interest for biorefinery applications to create biomolecules, but their production cost is expensive due to substrate and biomass separation costs. This research has utilized fuel synthesis wastewater (FSW) as a low-cost carbon-rich substrate to produce single-cell protein (SCP) and examines PNSB biofilm formation using this substrate to achieve a more efficient biomass-liquid separation. In this study, PNSB were grown in Ca, Mg, S, P, and N-deficient media using green shade as biofilm support material. Among these nutrient conditions, only N-deficient and control (nutrient-sufficient) conditions showed biofilm formation. Although total biomass growth of the control was 1.5 times that of the N-deficient condition and highest overall, the total biofilm-biomass in the N-deficient condition was 2.5 times greater than the control, comprising 49% of total biomass produced. Total protein content was similar between these four biomass samples, ranging from 35.0 ± 0.2% to 37.2 ± 0.0%. The highest protein content of 44.7 ± 1.3% occurred in the Mg-deficient condition (suspended biomass only) but suffered from a low growth rate. Overall, nutrient sufficient conditions are optimal for overall protein productivity and dominated by suspended growth, but where fixed growth systems are desired for cost-effective harvesting, N-deficient conditions provide an effective means to maximize biofilm production without sacrificing protein content.

Bioremediation and bioelectricity from Himalayan rock soil in sediment-microbial fuel cell using carbon rich substrates

In sediment microbial fuel cells soil microbes are responsible for bioremediation by degradation of pollutants present in it. The microbial metabolism produces bioelectricity in the process. This work reports the use of Himalayan rock soil for such evaluations. Three substrates including organic matter, glucose and sucrose were utilized to evaluate its feasibility. Open circuit voltage was achieved was highest with glucose as substrate at 500 ± 15 mV. The electrode distances were changed to evaluate its effect on the bioelectricity production at 2, 3, 4, and 6 cm respectively. The study also showed the survival of electrogenic microbes in acidic environment of the soil. Carbon oxidation rates were reported for each of the s-MFC reactors with the three substrates. 0.105 mmol C/m−2 d−1 for the organic matter, 0.689 mmol C/m−2 d−1 for glucose, and 0.507 mmol C/m−2 d−1 for sucrose. This shows the ability of the soil microbes to oxidize the soil complexes by degrading it and release energy. Additionally, FTIR analysis confirmed the potential for degrading soil carbonates, nitrates, and other organic substances for each of the substrate-based s-MFC. This suggests the use of s-MFCs for remediation of pollutants that can be difficult to break by the conventional method and the production of useful energy from the waste.

Study on Selection of Bacillus Sp. Strain Has Active Carbon Metabolism for Peat Treatment

The objective of selecting strains of Bacillus sp. has high activity in carbon metabolism for peat treatment, firstly focusing on the ability to degrade lignin, cellulose, phosphorus and protein. From the gene pool, by surveying the microbial strains capable of degrading carbon-rich substrates including lignin, cellulose, insoluble phosphate, proteolytic, we have identified two Bacillus sp. strains contain Bacillus polyfermenticus and Bacillus subtilis strains have strong ability to metabolize carbon-rich substrates and decompose phosphorus in peat. The Bacillus polyfermenticus strain had cellulose, lignin, organophosphorus and starch-degrading ring diameters of 41, 40, 23 and 38 mm, respectively. The Bacillus subtilis strain had cellulose, lignin, organophosphorus and starch-degrading ring diameters of 40, 41, 24 and 29 mm, respectively. In addition, the content of humic acids formed after 48 hours of fermentation increased 2.5 times compared to the original without the addition of Bacillus polyfermenticus strain and increased 2.8 times compared to the original without the addition of Bacillus subtilis strain. Therefore, two strains of Bacillus sp. selected with high efficiency in producing humic acid products. At the same time, the selected microbial strains have a high biosafety level (level of 1, the safest level in the biosafety scale).

Closing the loop in bioproduction: Spent microbial biomass as a resource within circular bioeconomy

Successful transition to a circular bioeconomy relies on the availability and efficient use of organic feedstocks such as agricultural and food waste. Advances in industrial biotechnology provide novel tools to valorize these feedstocks differently. Less attention, however, has been directed towards assessment of the organic side-residues arising from industrial biotechnology, such as spent microbial biomass (SMB). This study aims to reflect the current state of SMB within bioeconomy and create awareness of this growing industrial resource. Data from a range of published fermentation processes is used to estimate the amount of SMB formed per product (weight per weight, wt/wt) across different types of bioproducts, namely organic acids, alcohols, polymers, amino acids, antibiotics, protein and vitamins. Varying amounts of SMB are generated depending on the bioproducts and bioprocess, where bulk bioproducts, e.g. alcohols, generate less SMB than bioproduction of high-value low-volume specialty products, e.g. vitamins. It is estimated that more than 50 million tons of nutrient-rich SMB was generated in 2013, with SMB from bulk and specialty bioproduction accounting for roughly equal amounts. Furthermore, the composition of six industrially relevant organisms is summarized and compared, highlighting the general features of SMB as a carbon-rich substrate mainly consisting of protein. The results indicate that SMB is a growing resource with a reliable supply and predictable composition. The predictable nature of SMB could make it a favorable substrate for further innovation in industrial applications and nutrient circulation within the bioeconomy, for example, by using it as a co-substrate for valorization of other biomasses.

Deadwood mass and microclimate affect labile soil carbon and nitrogen under thinning of a naturally regenerated oak forest

Context Labile soil carbon and nitrogen are important indicators for evaluating carbon sequestration and nutrient availability under forest management such as thinning. Aims This study assessed the effects of thinning on labile soil carbon and nitrogen indices in a naturally regenerated oak forest. Methods Un-thinned control, 15% thinning, and 30% thinning were set up, and labile soil carbon and nitrogen (microbial biomass, permanganate oxidisable carbon, dissolved carbon and nitrogen, and inorganic nitrogen) and C:N balance indices (C:N ratio and threshold element ratio) were measured after 6 years. Key results Thinning generally increased labile soil carbon and nitrogen concentrations (P < 0.01); however, their fraction within the total soil carbon and nitrogen decreased under thinning. Redundancy analysis revealed that the remaining deadwood mass was the most influential factor affecting such differences in labile soil carbon and nitrogen indices across the three treatments (P < 0.01), although soil moisture had an effect as well (P < 0.05). Furthermore, the dissolved C:N ratio was higher than threshold element ratio only in 15% and 30% thinnings, indicating that thinning could shift the limiting factor for microbial growth from carbon to nitrogen by adding deadwoods, which provided carbon-rich substrates. Conclusions Overall, results demonstrate that deadwood retention and soil microclimatic changes could be the significant factors affecting soil carbon and nitrogen storage and the associated microbial affinity in thinned forests. Implications Therefore, sustainable forestry strategies for thinning residue management should be addressed to reinforce soil carbon and nitrogen conditions and the forest ecosystem functions.

Addition of a Carbon-Rich Substrate to a Constructed Wetland Favours Carbon and Phosphorus Availability and Biota Development: Relevance for Treating Agricultural Water

Addition of a Carbon-Rich Substrate to a Constructed Wetland Favours Carbon and Phosphorus Availability and Biota Development: Relevance for Treating Agricultural Water

Green hydrogen and platform chemicals production from acidogenic conversion of brewery spent grains co-fermented with cheese whey wastewater: adding value to acidogenic CO2

The biotechnological production of fuel and chemicals from renewable, organic carbon-rich substrates offers a sustainable way to meet the increasing demand for energy.

Carbon-rich substrates altered microbial communities with indication of carbon metabolism functional shifting in a degraded salt marsh of the Yellow River Delta, China

Coastal salt marshes have been significantly suffering degradation worldwide, turning these wetlands from carbon sink to carbon source. The additions of carbon-rich substrates in degraded coastal wetlands have been documented to alleviate this situation; however, the microbial responses as well as their carbon metabolisms to the input are not well explored. Here, we investigated the effects of different addition levels (0.1%, 1%, and 3%) of reed straw and biochar addition on microbial communities (bacteria and fungi) and predicted functions of carbon metabolism in degraded coastal salt marshes. The effects of the carbon-rich substrates on microbial communities are more pronounced under 3% of reed straw and biochar than other levels, straw increased opportunistic bacteria (e.g., Bacillus and Cellvibrionaceae) and biochar increased low carbon turnover bacteria (e.g., Acidobacteria and Actinobacteria). The addition of higher doses of reed straw and biochar promoted the carbon metabolism of soil bacteria as predicted by the PICRUSt 2. Interestingly, the addition of reed straw increased the abundance involved in fungal carbon metabolism, but this was not the case for the biochar input. The addition of reed straw and biochar promoted the growth of some bacteria (e.g., Rhizobiales and Bacillus) that are beneficial to plant growth, and decreased some fungal pathogen (e.g., Magnaporthaceae and Phaeosphaeria). Our findings revealed that the carbon-rich substrates addition can reshape the microbial structure and shift their carbon metabolisms, specially, biochar has a higher potential to improve the ecosystem services, such as blue carbon sequestration, of these degraded coastal salt marshes.

Relative evaluation of acid, alkali, and hydrothermal pretreatment influence on biochemical methane potential of date biomass

Date biomass is a carbon-rich renewable resource that can be considered a potential carbon-rich substrate for energy generation over anaerobic digestion (AD). However, due to its complex nature, appropriate pretreatment is necessary to achieve a higher methane yield. Hence, the current study was envisioned to evaluate the influence of three different pretreatment strategies, namely acid, alkali, and hydrothermal pretreatment on biochemical methane potential (BMP) of seven diverse sorts of Algerian date biomass, namely Pedicels, Fibrilium, Petiole, Fruit bunch, Spath, Palm, and its mixture. Among all the pretreatment conditions, alkaline pretreatment highly influenced the lignin composition of date biomass and showed higher BMP. Among all sorts of biomass, higher BMP was detected through Palm as 295.9 mL CH4/g-TS, whereas the lowest BMP values were recorded with Petiole as 226.74 mL CH4/g-TS. Among all the experimental variations, ammonium pretreated Palm biomass documented the highest substrate conversion efficiency (63.80%), which correlates well with the observed higher BMP values. Nevertheless, there was a very marginal improvement in BMP detected in the case of other pretreatment strategies compared to alkaline pretreatment. This might be due to the efficacy of the applied pretreatment method on delignification of date biomass.

Enhancement of euryhaline Asterarcys quadricellulare biomass production for improving biogas generation through anaerobic co-digestion with carbon rich substrate.

The online version contains supplementary material available at 10.1007/s13205-021-02792-x.

Physiological response of onion (Allium cepa L.) seedlings to shungite application under two soil water regimes

The application of carbon-rich substrates to agricultural soils is discussed as a strategy to improve soil properties and fertility, which can affect plant physiological traits and enhance agricultural crop yield. The aim of this study was to evaluate if shungite, carbon-rich sedimentary-volcanic rock, may improve plant ecophysiological traits under sufficient water supply as well as soil water deficit. A pot culture experiment was conducted with onion (Allium cepa L.) seedlings, using four shungite concentrations (0, 5, 10 and 20 g kg−1) in an Umbric Podzols and two water regimes: well-watered and drying-wetting cycles. Soil water deficit decreased root nutrient content, depressed seedlings growth, net CO2 assimilation rate (An), stomatal conductance (gs) and the respiration rates in both darkness (Rd) and the light (Rl), but increased water use efficiency (WUE) at leaf level. Shungite application decreased the leaf necrosis under both water regimes and increased total leaf length of DW seedlings. Compared with the well-watered conditions, under drying-wetting cycle shungite stimulated the increase of the An rate and WUE at low measurement temperature. No significant effect of shungite was found for Rd, Rl, Rl/Rd, Rd/Ag (Ag = An + Rl) and Rl/Ag regardless soil water regimes. Shungite application was not so successful to eliminate the negative effects of soil water deficit on growth and physiological processes of A. cepa. The observed positive effects of shungite on the physiological traits of onion seedlings were more likely associated with the increase in the content of nutrients than with the improvement in soil water properties.

Insight into the roles of ferric chloride on short-chain fatty acids production in anaerobic fermentation of waste activated sludge: Performance and mechanism

• The effect of ferric chloride on short chain fatty acids production was evaluated. • Solubilization, hydrolysis, and acidogenesis were promoted by ferric chloride. • Ferric chloride promoted acetogenesis and high molecular organic acids conversion. • Ferric chloride changed the acid-fermentation to propionic acid-type fermentation. • Excessive ferric chloride inhibited acid-fermentation by strong aggregation. Ferric chloride (FC) is widely used in sewage treatment and sludge conditioning, which could be inevitably accumulated in waste-activated sludge (WAS). However, its effect on short-chain fatty acids (SCFAs) production from WAS anaerobic fermentation has yet to be thoroughly investigated. This study aims to reveal how different dosages of FC affect the production of SCFAs and elucidate the corresponding mechanism. Experimental results showed that 16 mg Fe/g total suspended solids (TSS) of FC was favorable for SCFAs production, with the maximum production was 3.3 times of the control. Mechanistic study revealed that FC induced dissimilatory iron reduction (DIR) and enhanced hydrolase activities, contributing to the disintegration of WAS flocs and the hydrolysis of complex organics. Moreover, FC stimulated the productivity of HAc by promoting the release of carbon-rich substrates and accepting the intermediate electrons generated in acetogenesis process. Further study indicated FC inhibited the methanogenesis and changed the acid-fermentation type by affecting pH and ORP, which was conducive to SCFAs accumulation. Microbial community analysis confirmed that FC enriched the bacterial microorganisms related to hydrolysis and acidogenesis, and increased the abundance of Fe(III)-reducing genera. However, excessive FC (>16 mg Fe/g TSS) inhibited SCFAs production, due to the strong aggregation increased mass transfer resistance and the “over-acidification” damaged the microbial community.

Improvement of methane production at alkaline and neutral pH from anaerobic co-digestion of microalgal biomass and cheese whey

Over the past decades, biofuels from microalgae have gained increasing attention. In the present work, methane production from mono-digestion of thermochemical microalgal hydrolysate and co-digestion with cheese whey was evaluated in an anaerobic sequencing batch reactor. The reactor was fed with 5 g total chemical oxygen demand (TCOD)/L and operated either at alkaline or neutral pH. The average methane content in the biogas was higher at alkaline pH (83 %) than at neutral pH (61 %), whereas the methane yield from co-digestion was similar at alkaline (233 N mL/g TCOD) and neutral pH (226 N mL/g TCOD), being 1.8-fold higher than the mono-digestion of the microalgal hydrolysate. After increasing stepwise the organic load up to 12 g TCOD/L, the methane production rate reached peaks around 1000 N mL/L-d, being between 200−800 mL/L-d in those stages with lower organic loads. The methane production from the particulate fraction of the microalgal hydrolysate and its co-digestion with cheese whey demonstrated that the particulate fraction was indeed used to produce methane, but co-digestion did not improve methane yield. The results showed that microalgal hydrolysate could be efficiently co-digested with a carbon-rich substrate enhancing methane production.