Impacts of metal stress on extracellular microbial products, and potential for selective metal recovery.

Harnessing microbial capabilities for metal recovery from secondary waste sources is an eco-friendly and sustainable approach for the management of metal-containing wastes. Soluble microbial products (SMP) and extracellular polymeric substances (EPS) are the two main groups of extracellular compounds produced by microorganisms in response to metal stress that are of great importance for remediation and recovery of metals. These include various high-, and low, molecular weight components, which serve various functional and structural roles. These compounds often contain functional groups with metal binding potential that can attenuate metal stress by sequestering metal ions, making them less bioavailable. Microorganisms can regulate the content and composition of EPS and SMP in response to metal stress in order to increase the compounds specificity and capacity for metal binding. Thus, EPS and SMP represent ideal candidates for developing technologies for selective metal recovery from complex wastes. To discover highly metal-sorptive compounds with specific metal binding affinity for metal recovery applications, it is necessary to investigate the metal binding affinity of these compounds, especially under metal stressed conditions. In this review we critically reviewed microbial EPS and SMP production as a response to metal stress with a particular emphasis on the metal binding properties of these compounds and their role in altering metal bioavailability. Furthermore, for the first time, we compiled the available data on potential application of these compounds for selective metal recovery from waste streams.

Dynamic modelling the effects of ionic strength and ion complexation on trace metal speciation during anaerobic digestion.

Dosing trace metals into anaerobic digestors is proven to improve biogas production rate and yield by stimulating microorganisms involved in the metabolic pathways. Trace metal effects are governed by metal speciation and bioavailability. Though chemical equilibrium speciation models are well-established and widely used to understand metal speciation, the development of kinetic models considering biological and physicochemical processes has recently gained attention. This work proposes a dynamic model for metal speciation during anaerobic digestion which is based on a system of ordinary differential equations aimed to describe the kinetics of biological, precipitation/dissolution, gas transfer processes and, a system of algebraic equations to define fast ion complexation processes. The model also considers ion activity corrections to define effects of ionic strength. Results from this study shows the inaccuracy in predicting trace metal effects on anaerobic digestion by typical metal speciation models and the significance of considering non-ideal aqueous phase chemistry (ionic strength and ion pairing/complexation) to define speciation and metal labile fractions. Model results show a decrease in metal precipitation and increase in metal dissolved fraction and methane production yield with increase in ionic strength. Capability of the model to dynamically predict trace metal effects on anaerobic digestion under different conditions, like changing dosing conditions and initial iron to sulphide ratio, was also tested and verified. Dosing iron increases methane production and decreases hydrogen sulphide production. However, when iron to sulphide ratio is greater than 1, methane production decreases due to increase in dissolved iron which reaches inhibitory concentration levels.

Effect of metals on mesophilic anaerobic digestion of strawberry extrudate in batch mode.

According to recent studies, the anaerobic digestion of strawberry extrudate is a promising option with potential in the berry industry biorefinery. However, the lack and/or unbalance of concentrations of metals in some agro-industrial residues could hamper methane production during the anaerobic digestion of these kinds of wastes. In this study, a fractional factorial design was applied to screen the supplementation requirements regarding six metals (Co, Ni, Fe, Cu, Mn, and Zn) for methane production from strawberry extrudate (SE). The logistic model was used to fit the experimental data of methane production-time. It allowed identifying two different stages in the anaerobic process and obtaining the kinetic parameters for each step. Maximum methane production obtained in the first (Bmax) kinetic stage, the methane production in the second stage (P), and the maximum methane production rates (Rmax) concluded a statistically significant effect for Ni and Zn. The second set of experiments was carried out with Ni and Zn through a central composite design to study the concentration effect in the anaerobic digestion process of the strawberry extrudate. The parameters P and Rmax demonstrated a positive interaction between Ni and Zn. Although, Bmax did not prove a statistically significant effect between Ni and Zn.

Interrelating EPS, soluble microbial products and metal solubility in a methanogenic consortium stressed by nickel and cobalt.

The relationships between extracellular polymeric substances (EPS), soluble microbial product production, metal solubility, and methanogenic activity were investigated. The individual, and joint, toxic effects of nickel and cobalt on methanogenic consortia fed with glucose as model substrate were studied using biomethane potential assays. Cobalt was found to be less toxic to methanogens than nickel at each concentration tested, and the combined effects of Ni and Co on methane production in the bimetal experiment was higher than the sum of the effects of each metal alone. The protein content of EPS, and extracellular soluble protein fractions, decreased with increasing concentrations of total metals. Meanwhile, no significant change in response to metal stress was apparent for carbohydrate content of EPS or extracellular soluble carbohydrate. Decreasing protein content of EPS was accompanied by reduced methanogenic activity and an increase in the soluble metal fraction. The strong associations observed between these variables could be due to the critical role of EPS in protecting microbial cells against nickel and cobalt stress, possibly by capturing metal cations through their functional groups, thus reducing metal availability to the microbial cells in the methanogenic consortia underpinning the anaerobic digestion process.

Evaluation of the influence of trace metals on methane production from domestic sewage, using the Plackett-Burman experimental design.

Methanogenesis involves several enzymes with trace metal components that catalyze major metabolic pathways and, therefore, requires a sufficient supply of micronutrients such as iron, nickel or cobalt. The statistically-based Plackett-Burman experimental design was adopted in this study to identify which trace metal have a statistically significant effect on the maximum methane production from domestic sewage. The addition of Barium (Ba), Cobalt (Co), Copper (Cu), Iron (Fe), Manganese (Mn), Nickel (Ni) and Selenium (Se) was tested in batch reactors using domestic sewage as the substrate and sewage sludge as the inoculum. The results showed that the addition of Fe was statistically significant, positively affecting the maximum CH4 production (p-value 0.05). The results are expressed in L of CH4 per g of Chemical Oxygen Demand added, increasing it from 0.10 to 0.13 LCH4 gCODadd-1. In L of CH4 per g of Volatile Solids, increased it from 0.031 to 0.040 LCH4 gVS-1. The addition of Se was statistically significant, but with a negative effect on the maximum methane production (p-value 0.002), which decreased from 0.13 to 0.10 LCH4.gCODadd-1. Moreover, six different Fe concentrations (0, 40, 80, 120, 160 and 200 mgL-1) were tested, showing that the addition of 120 mgL-1 had the greatest effect for statistically improving the maximum methane production, with 33% improvement (0.12 ± 0.003 to 0.16 ± 0.012 LCH4 gCODadd-1) compared to no addition of Fe and the specific CH4 production to 0.040 ± 0.001 LCH4 gVS-1.

Biogas Potential of the Side Streams Obtained in a Novel Phenolic Extraction System from Olive Mill Solid Waste.

The olive oil production is an important industrial sector in many Mediterranean areas, but it is currently struggled by the necessity of a proper valorisation of the olive mill solid waste or alperujo. The alperujo is the main by-product generated during the two-phase olive oil extraction, accounting for up to 80% of the initial olive mass. The alperujo is a source of valuable compounds, such as the pomace olive oil or highly interesting phenolic compounds. In the present research, a novel biorefinery approach has been used for phenolic compounds recovery. However, the extraction of these valuables compounds generates different exhausted phases with high organic matter content that are required to be managed. This study consists of the evaluation of the anaerobic biodegradability of the different fractions obtained in a novel biorefinery approach for the integral valorisation of alperujo. The results show that the different phases obtained during the biorefinery of the alperujo can be effectively subjected to anaerobic digestion and no inhibition processes were detected. The highest methane yield coefficients were obtained for the phases obtained after a two-months storages, i.e., suspended solids and liquid phase free of suspended solids, which generated 366 ± 7 mL CH4/g VS and 358 ± 6 mL CH4/g VS, respectively. The phenol extraction process reduced the methane yield coefficient around 25% due to the retention of biodegradable compounds during the extraction process. Regardless of this drop, the anaerobic digestion is a suitable technology for the stabilization of the different generated residual phases, whereas the high market price of the extracted phenols can largely compensate the slight decrease in the methane generation.

Trace elements effect on hydrolytic stage towards biogas production of model lignocellulosic substrates.

The effect and the response of several trace elements (TE) addition to the anaerobic degradation of key compounds of lignocellulosic biomass were evaluated. Lignin, cellulose and xylose were selected as principal compounds of lignocellulosic biomass. Lignin degradation was only improved by the addition of 1000 mg Fe/L, which allowed an improvement on the methane yield coefficient of 28% compared to control. SEM images from an abiotic assay showed that this effect is more likely related with a chemical effect induced by the Fe solution, instead of an enzymatic response. Pre-treatments focused on breaking the recalcitrant structure of the lignin could be more promising than TE addition for rich lignin-content substrates. Unlike to the response observed with lignin, cellulose showed a clear effect of the TE addition on methane production rate, indicating a higher preponderance of the enzymatic activity compared to the lignin biomethanization. Experiments with xylose resulted in a strong accumulation of volatile fatty acids. TE addition should be adapted to the substrate composition given the different response of each lignocellulosic compound to the different TE addition.

Full-scale agricultural biogas plant metal content and process parameters in relation to bacterial and archaeal microbial communities over 2.5 year span.

A start-up of 4 MW agricultural biogas plant in Vucja vas, Slovenia, was monitored from 2011 to 2014. The start-up was carried out in 3 weeks with the intake of biomass from three operating full-scale 1-2 MW donor agricultural biogas plants. The samples were taken from donor digesters and from two serial digesters during the start-up over the course of 2.5 years. Bacterial and Archaeal microbial communities progressively diverged from the composition of donor digesters during the start-up phase. The rate of change of Bacterial community decreased exponentially over the first 2.5 years as dynamics within the first 70 days was comparable to that of the next 1.5 years, whereas approximately constant rate was observed for Archaea. Despite rearrangements, the microbial communities remained functionally stable and produced biogas throughout the whole 2.5 years of observation. All systems parameters measured were ordered according to their Kernel density (Gaussian function) ranging from the most dispersed (substrate categories used as cosubstrates, quantities of each cosubstrate, substate dry and volatile matter, process parameters) towards progressively least dispersed (trace metal and ion profiles, aromatic-polyphenolic compounds, biogas plant functional output (energy)). No deficiency was detected in trace metal content as the distribution of metals and elements fluctuated within the suggested limits for biogas over 2.5 year observation. In contrast to the recorded process variables, Bacterial and Archaeal microbial communities exhibited directed changes oriented in time. Variation partitioning showed that a large fraction of variability in the Bacterial and Archaeal microbial communities (55% and 61%, respectively) remained unexplained despite numerous measured variables (n = 44) and stable biogas production. Our results show that the observed reorganization of microbial communities was not directly associated with impact on the full-scale biogas reactor performance. Novel parameters need to be determined to elucidate the variables directly associated with the reorganization of microbial communities and those relevant for sustained function such as the more in-depth interaction between TSOC, trace metal profiles, aromatic-polyphenolic compounds and ionic strength (e.g. electrical conductivity).

Sequential adaptation of Nannochloropsis gaditana to table olive processing water.

The main objective of this study was to evaluate the suitability of Nannochloropsis gaditana to grow by sequential adaptation to TOPW (Table olive processing water) at increased substrate concentrations (10-80%). Sequential adaptation allows growing Nannochloropsis gaditana up to 80% TOPW, although the maximum microalgae biomass productions were achieved for percentages of 20-40%, i.e. 0.308 ± 0.005 g VSS (Volatile Suspended Solids)/L. In all growth experiments, proteins were the majority compound in the grown microalgae biomass (0.44 ± 0.05 g/g VSS), whereas phenols were retained up to a mean concentration of 12.1 ± 1.9 mg total phenols/g VSS. The highest microalgae biomass production rate at rate of 80% TOPW took place in the first two days when most nutrients were also removed. Average removal efficiencies at this percentage of TOPW were 69.1%, 50.9%, 54.3% and 71.8% for total organic carbon, total soluble nitrogen, phosphate and total phenols, respectively. Sequential adaptation can ensure the obtaining of a sustainable microalgae culture as a treatment method for TOPW.

Batch anaerobic co-digestion of waste activated sludge and microalgae (Chlorella sorokiniana) at mesophilic temperature.

The microalgae Chlorella sorokiniana are used as co-substrate for waste activated sludge (WAS) anaerobic digestion. The specific objective of this research was to evaluate the feasibility of improving methane production from anaerobic digestion of WAS in co-digestion with this microalga, based on an optimized mixture percentage. Thus, the anaerobic co-digestion of both substrates aims to overcome the drawbacks of the anaerobic digestion of single WAS, simultaneously improving its management. Different co-digestion mixtures (0% WAS-100% microalgae; 25% WAS-75% microalgae; 50% WAS-50% microalgae; 75% WAS-25% microalgae; 100% WAS-0% microalgae) were studied. The highest methane yield (442 mL CH4/g VS) was obtained for the mixture with 75% WAS and 25% microalgae. This value was 22% and 39% higher than that obtained in the anaerobic digestion of the sole substrates WAS and microalgae, respectively, as well as 16% and 25% higher than those obtained for the co-digestion mixtures with 25% WAS and 75% microalgae and 50% WAS and 50% microalgae, respectively. The kinetic constant of the process increased 42%, 42% and 12%, respectively, for the mixtures with 25%, 50% and 75% of WAS compared to the substrate without WAS. Anaerobic digestion of WAS, together with C. sorokiniana, has been clearly improved by ensuring its viability, suitability and efficiency.

Screening of biomethane production potential from dominant microalgae.

The use of microalgae for biomethane production has been considerably increasing during the recent years. In this study, four dominant species belonging to the genera Scenedesmus, Chlorella, Dunaliella and Nostoc were selected. The influence of different genera with several morphological, structural and physicochemical characteristics on methane production was assessed in biochemical methane potential (BMP) tests. The ultimate methane yield values were 332 ± 24, 211 ± 2, 63 ± 17 and 28 ± 10 mL CH4/g VSadded for Scenedesmus obliquus, Chlorella sorokiniana, Dunaliella salina and Nostoc sp., respectively. The highest methane production was achieved by microalga species that had no complex cell wall or wall basically composed by proteins and simple sugars such as in S. obliquus, whereas lower methane yields were found for D. salina and Nostoc sp., due to the salinity effects and cell wall composition in terms of complex polysaccharide and glycolipid layers, respectively. Kinetic constant values obtained in the BMP tests ranged between 1.00 ± 0.08 and 0.097 ± 0.005 days(-1) for D. salina and S. obliquus, respectively.

Model based analysis of trace metal dosing strategies to improve methane yield in anaerobic digestion systems.

Favourable effects of trace metals (TMs) on regulating anaerobic digestion (AD) performance are extensively utilised to improve methane yield. This study discusses a model-based approach to find out the best TM dosing strategies. The model has been applied to compare continuous, preloading, pulse dosing and in-situ loading. Simulations were also carried out to comprehend appropriate dosing form, dosing time and quantity of metals to be dosed. Model results show that the best way to dose TMs is repeated pulse dosing at low concentration levels in the optimum range with high frequency. Best dosing strategy for the system in this study was found to be 5 µM pulse loading at 5 days intervals as it gave maximum methane production and low effluent metal loss. Preferable dosing form depends on reactor configuration and this has been verified after model calibration with experimental data. Easily dissociable metal chlorides are ideal for continuous reactors.

Valorization of strawberry extrudate waste: Recovery of phenolic compounds by direct-hydrothermal treatment and subsequent methane production by mesophilic semi-continuous anaerobic digestion.

Strawberry extrudate (SE) is an underused by-product from strawberry industry. Recovery of the phenolic compounds present in SE would represent a very interesting valorisation option. Two main challenges need to be solved, firstly, the solubilisation and recovery of the phenolic compounds contained in SE, and, after that, the stabilisation of the resulted de-phenolized SE. The present research evaluates the potential of a biorefinery process combining a hydrothermal pre-treatment, followed by a phenolic extraction process and, finally, the anaerobic digestion of the remaining SE for producing energy that will contribute to compensate the energy requirements of the whole system. Following the hydrothermal pre-treatment at 170 °C for 60 min, an extraction of 0.6 ± 0.1 g of gallic acid per kilogram of SE was achieved using an adsorbent resin, representing a recovery rate of 64 %. Long-term semi-continuous anaerobic digestion of de-phenolized SE was evaluated at different organic loading rates to evaluate the stability of the process. The anaerobic digestion of pre-treated SE achieved a stable methane production value of 243 ± 34 mL CH4·g volatile solids-1·d-1 at an organic loading rate (ORL) of 1.25 g volatile solids·L-1·d-1. During the operation at this ORL, the control parameters including pH, alkalinity, soluble chemical organic demand (sCOD), and volatile fatty acid (VFA) remained stable and consistently constant. Specifically, the VFA in the reactor during this stable period achieved a value of 102 ± 128 mg O2/L. Also, an economic balance showed that the minimal price of the generated phenolic extract for having benefited from the proposed biorefinery system was 0.812 €·(g of gallic acid equivalents)-1, a price within the range of phenolic compounds used in the food industry.

Enhanced wastewater bioremediation by a sulfur-based copolymer as scaffold for microalgae immobilization (AlgaPol).

In recent years, there has been an increasing concern related to the contamination of aqueous ecosystems by heavy metals, highlighting the need to improve the current techniques for remediation. This work intends to address the problem of removing heavy metals from waterbodies by combining two complementary methodologies: adsorption to a copolymer synthesized by inverse vulcanization of sulfur and vegetable oils and phytoremediation by the microalga Chlorella sorokiniana to enhance the metal adsorption. After studying the tolerance and growth of Chlorella sorokiniana in the presence of the copolymer, the adsorption of highly concentrated Cd2+ (50 mg L-1) by the copolymer and microalgae on their own and the combined immobilized system (AlgaPol) was compared. Additionally, adsorption studies have been performed on mixtures of the heavy metals Cd2+ and Cu2+ at a concentration of 8 mg L-1 each. AlgaPol biofilm is able to remove these metals from the growth medium by more than 90%. The excellent metal adsorption capacity of this biofilm can be kinetically described by a pseudo-second-order model.

Modeling the antimicrobial effects of olive mill waste extract, rich in hydroxytyrosol, on the growth of lactic acid bacteria using response surface methodology.

The objective of this study is to assess the inhibitory effects of an aqueous extract from olive oil mill waste (alperujo) on the growth of a lactic acid bacteria (LAB) cocktail consisting of various strains of Lactiplantibacillus pentosus and Lactiplantibacillus plantarum species. For this purpose, response surface methodology was employed using two independent variables (pH levels 3.5-5.55; hydroxytyrosol concentration ranging from 0.93-2990 ppm). The response variable was the average inhibition per treatment on the LAB cocktail (expressed as a percentage). The developed model identified significant terms, including the linear effect of hydroxytyrosol and pH, their interaction, and the quadratic effect of pH. Maximum inhibition of the LAB cocktail was observed at progressively higher concentrations of hydroxytyrosol and lower pH values. Therefore, complete inhibition of LAB in the synthetic culture medium could only be achieved for concentrations of 2984 ppm hydroxytyrosol at a pH of 3.95. These findings suggest that extracts derived from "alperujo" could be utilized as a natural preservative in acidified foods with a bitter flavor and antioxidant requirements.

The impact of metal transport processes on bioavailability of free and complex metal ions in methanogenic granular sludge.

Bioavailability of metals in anaerobic granular sludge has been extensively studied, because it can have a major effect on metal limitation and metal toxicity to microorganisms present in the sludge. Bioavailability of metals can be manipulated by bonding to complexing molecules such as ethylenediaminetetraacetate (EDTA) or diethylenetriaminepentaacetate (DTPA). It has been shown that although the stimulating effect of the complexed metal species (e.g. [CoEDTA](2-)) is very fast, it is not sustainable when applied to metal-limited continuously operated reactors. The present paper describes transport phenomena taking place inside single methanogenic granules when the granules are exposed to various metal species. This was done using magnetic resonance imaging (MRI). The MRI results were subsequently related to technological observations such as changes in methanogenic activity upon cobalt injection into cobalt-limited up-flow anaerobic sludge blanket (UASB) reactors. It was shown that transport of complexed metal species is fast (minutes to tens of minutes) and complexed metal can therefore quickly reach the entire volume of the granule. Free metal species tend to interact with the granular matrix resulting in slower transport (tens of minutes to hours) but higher final metal concentrations.

Effect of harvesting time in the methane production on the anaerobic digestion of microalgae.

Microalgae are being proposed as excellent substrates for different biorefinery processes. Anaerobic digestion process of microalgae is one of these interesting processes but has some limitations in deleting cell walls. For this reason, many studies proposed different types of pre-treatments, entailing energy, operation, and investment costs. This work aims to optimize the anaerobic digestion of the microalgae Chlorella sorokiniana and Chlorella sorokiniana (strain S12/S13/S16) without any pre-treatment by selecting the optimal harvesting time. The greatest influence is seen at 5:00 PM in methane production for both microalgae. For Chlorella sorokiniana, it is the most optimal moment for anaerobic digestion, whereas Chlorella sorokiniana (strain S12/S13/S16) is the least optimal. In the other harvesting times, both microalgae present a similar methane production, i.e. 173 ± 12 mL CH4/g of total volatile solids. The highest methane production rate values were obtained during peak sunlight, 1:00 PM and 8:00 AM, respectively, and lower overnight.

Assessment of different mechanical treatments for improving the anaerobic biodegradability of residual raspberry extrudate.

Mechanical treatments can be simple and feasible methods for enhancing the anaerobic digestion of lignocellulosic substrates. This work aims to relate the direct effect of five different mechanical treatments, i.e., variation in the size and number of particles, with the variations in the chemical composition and, subsequently, the effect over the anaerobic digestion of residual raspberry extrudate, which was used as a model substrate. A high variation in the number of particles and the particle size distribution was achieved depending on the mechanical treatment applied, reaching the highest number of particles for the treatments with knife mills and mortar (around 8000 particles per gram). The higher number of particles was related to higher solubilisation, including phenolic compounds and sugars. The combination of knife mills and mortar pretreatment, which presented the highest number of particles, resulted in a 66% more of polyphenols in comparison to the raw substrate. However, the presence of anthocyanins was higher in mechanical treatments with less effect. The enhancement of the anaerobic digestion was clearly related to the increment in the number of particles of small size after the mechanical treatments. The highest methane yield coefficient (236 ± 11 mL CH4/g volatile solids) was achieved for the raspberry extrudate treated with knife mills.

Transcription of fdh and hyd in Syntrophobacter spp. and Methanospirillum spp. as a diagnostic tool for monitoring anaerobic sludge deprived of molybdenum, tungsten and selenium.

Formate dehydrogenases and hydrogenases contain molybdenum or tungsten and/or selenium. These enzymes are crucial for interspecies formate and hydrogen transfer between propionate degrading Syntrophobacter spp. and methanogenic Methanospirillum spp. Here we used reverse transcription of total RNA followed by quantitative PCR (RT-qPCR) with specific primers to get insight into interspecies formate and hydrogen transfer. Transcriptional regulation of formate dehydrogenases and hydrogenases in Syntrophobacter and Methanospirillum spp. in a propionate-fed up-flow anaerobic sludge bed (UASB) reactor was examined. In both microorganisms formate dehydrogenase and hydrogenase coding genes (fdh and hyd respectively) were transcribed simultaneously. During 249 days in which molybdenum, tungsten and selenium were not supplied to the reactor feed, the microbial activity and transcription of fdh and hyd in Syntrophobacter spp. decreased. Transcription of fdh and hyd in Methanospirillum spp. did not decrease, but transcription of fdh increased when after 249 days molybdenum, tungsten and selenium were supplied to the reactor feed. The developed RT-qPCR is a technique that can give rapid information about active processes in methanogenic granular sludge and may contribute to predict metal limitation and failure in UASB reactors.

High-Value-Added Compound Recovery with High-Temperature Hydrothermal Treatment and Steam Explosion, and Subsequent Biomethanization of Residual Strawberry Extrudate.

This study was on the comparison of hydrothermal treatments at 170 °C (steam injection) and 220 °C (steam explosion) to solubilize the organic matter contained in residual strawberry extrudate, focusing on phenolic compounds that were susceptible to be extracted and on sugars. After the extraction step, the remaining strawberry extrudate phases were subjected to anaerobic digestion to generate biogas that would compensate the energy requirements of the suggested hydrothermal treatments and to stabilize the remaining waste. Hydrothermal treatment at 220 °C allowed the recovery of 2053 mg of gallic acid eq. per kg of residual strawberry extrudate. By contrast, after hydrothermal treatment at 170 °C, only 394 mg of gallic acid eq. per kg of residual strawberry extrudate was recovered. Anaerobic digestion processes were applied to the de-phenolized liquid phase and the solid phase together, which generated similar methane productions, i.e., around 430 mL CH4/g volatile solids, after both 170 °C and 220 °C hydrothermal treatments. Considering the latest observation, hydrothermal treatment at 220 °C is a preferable option for the valorization of residual strawberry extrudate (RSE) due to the high solubilization of valuable phenolic compounds that can be recovered.