Significance of mineral elements characterization for anaerobic digestion substrates: The case of avocado oil processing by-products

doi:10.21203/rs.3.rs-5427414/v1

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Significance of mineral elements characterization for anaerobic digestion substrates: The case of avocado oil processing by-products

https://doi.org/10.21203/rs.3.rs-5427414/v1

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published 10 Apr, 2025

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Mineral elements effects on anaerobic biogas production (AD) were reviewed and inferences made for avocado oil processing by-products. Mineral elements in these by-products were measured using ICP-OES methods. It was noted that there is no consensus among researchers on absolute mineral requirements for optimal digester performance and only ranges stating lowest and highest mineral requirements could be found from different studies. These ranges vary for each element depending on analytical methods used, substrate type and digester operating conditions. Mineral elements exceeding certain limits in digesters can be toxic to the AD microorganisms, a situation which negatively affect the efficiency and stability of substrate biodegradation to biogas. Other mineral elements, such as arsenic, despite their concentration in the digester, are naturally toxic to microorganisms. These toxic minerals must therefore be removed or detoxified if detected in substrates. Researchers choose to perform substrate mineral elemental analysis depending on their pre-existing knowledge of the substrate, recommendations from standards adopted in the research and objective of their studies. The bioavailability of minerals in substrates require further investigation to accurately correlate mineral elements concentration in avocado oil processing by-products by ICP-EOS methods to the expected AD performance when using these substrates. The current study revealed mineral elements related to avocado oil processing by-products that may need possible attention for optimal biodegradation of these substrates to biogas.

Avocado

biogas

inhibition

minerals

nutrients

microorganisms

The broad spectrum of substrates that can be used in biogas digesters imply varying physico-chemical parameters across these potential substrates. Researchers normally perform proximate and ultimate characterization of substrates to understand substrate behaviour in AD performance (Holliger et al. 2016; Matheri et al. 2019). Recently, a study by Rashama et al (2021) outlined the importance of phytochemicals in ruminant and possibly digester microbial dynamics. This means that phytochemical characterization of substrates should also become a standard practice for previously untested biodigester substrates of plant/crop origin. Another interesting substrate characterization determines substrate inorganic mineral elements as opposed to ultimate analysis that focuses on organics only (Vintiloiu et al. 2012). However, other researchers omit this step for reasons not immediately known to us and other practitioners (Chala et al. 2018; Du et al. 2021). Mineral elements are also investigated as a pre-cursor to understanding the AD digestate as biofertilizer and for this application the slurry must meet certain heavy metal concentration standards (Demirel et al. 2013). Generally the role and effects of elements in digesters is poorly understood by many AD practitioners (Ezebuiro and Korner 2016). Therefore, a recollection of literature on this subject focusing on possible reasons of why substrate elemental characterization is sometimes omitted in other studies may be necessary to understand the current state of this issue in the AD industry. Coupling the finding from this review with elemental characterization of avocado oil processing by-products will be used to infer AD behaviour of these by-products. Utilization of avocado oil processing waste biomass in AD process addresses pollution, energy and fertilizer issues while freeing up productive land which is currently being used for landfilling and lagoon ponds in current methods of managing these wastes.

2.1 Literature survey

A literature search using keywords such as macro and micro elements, mineral and trace nutrients, inorganic elements, etc in biodigesters or anaerobic digestion was conducted. Some researchers classify mineral elements into macro and micro elements, with others preferring to name these elements as nutrients instead. Information pertaining to inorganic elements effects on digesters, the acceptable ranges and other important issues were populated for subsequent use in inferential studies involving avocado oil processing by-products’ mineral element analysis.

2.2 Materials sourcing and handling

Westfalia Fruit (Pvt) Ltd with operations in Tzaneen and Howick both in South Africa provided the avocado oil processing by-products. Two technologies are employed in the company’s operations. The hot process technology is employed at the Tzaneen factory where whole fruit avocados are crushed while in the cold process technology (Howick) the fruit is first peeled and destoned prior to crushing. After crushing, the pulp is malaxed at 55 -60^oC for hot process and 45-48^oC for the cold process. Oil is later squeezed out of the malaxed pulp using centrifugal separators. The hot process generates pomace (HDP) and wastewater (HDW) while the cold process generates seeds (AK), peels (AS), pomace (CDP) and wastewater (CDW). Samples were collected fresh during fruit processing then transported in cooler boxes (4^oC) to University of South Africa, Science campus laboratories. All samples were air dried until achieving constant weight within 5 days after collections. The dry samples were then characterized for macro and micro element profiles at Agricultural Research Council (ARC) laboratories in the Institute of Soil, Water and Climate unit. All chemicals sourced from Sigma Aldrich (South Africa) were of analytical grade.

2.3 Substrate mineral and trace element analysis

The air-dried samples were milled, weighed (1g) and each was digested in two ways following guidelines from Zasoski and Burau (1977) and (Sailor et al. 2020). For mineral element analysis, the 1g sample was digested at 180^oC using 7ml of conc nitric acid and 3ml of conc perchloric acid while adding a few drops of hydrogen peroxide. Upon cooling, 10 ml of 1:1 hydrochloric acid was added to this mixture. Contents were topped up to 100 ml in a conical flask using distilled water. The resulting digest solution was ready for mineral elements analysis using Inductively Coupled Plasma - Optical Emission Spectrometry (ICP-EOS). For trace element analysis more concentrated solutions are required so a separate sample digestion was done in the same way as for mineral elements but with sample quantity increased to 5g, temperature reduced to 120^oC and final volume reduced to 50 ml. A total of 12 mineral elements and 9 trace elements were determined from the digest solutions using an Agilent 725 ICP-EOS Simultaneous instrument. An unpublished optimized ARC-ISWC procedure adapted from the original equipment supplier guidelines was used in running the instrument (Agilent Technologies 2018). The methods used for phosphorous, ammonium and ammoniacal nitrogen (ARC-ISWC 1977a, b) were adapted from Kaul and Sharma (1976), Krom (1980) and Bessinger (1985)

There are various reasons why some researchers in AD studies conduct elemental analysis while others do not. Some of these reasons and their implications are noted in Table 1. This discussion is linked to mineral profile results of avocado oil processing by-products which are displayed in Fig. 1 and Fig. 2. Metal requirements in digesters and biodigestion inferences made from avocado oil by-products mineral analysis are summarized in Table 2. Though digester mineral requirements reported in Table 2 are based on different substrates and operating conditions, they give an idea of the general ranges applicable in AD practice. This allows biodigestion inferences based on mineral element analysis of the substrates being considered in this study to be made. Heavy metals (Al, Pb, Hg, Cd, As, Cu) complex with thiols and disrupt enzyme activities ultimately negatively affecting AD microbial activities (Mudhoo and Kumar 2013). None of these metals are however detected in high enough concentrations across the avocado oil by-processing by-products to induce the anticipated microbial toxicity. However, attention must be given to Hg and Cd which are detected in the range of 0.002 to 0.032 mg/L across avocado oil processing by-products and are toxic to microbes despite their low concentrations. These metals including As pose health threats to humans as well if they enter the food chain through digestate use as biofertilizer in human consumed crops (Sturmer et al. 2020). Lower levels of certain heavy metals can however be tolerated and sometimes certain microorganisms at these low metal concentrations can biotransform these heavy metals helping in neutralizing their toxicities. Some metals (Fe, Zn, Ni, Co) when existing in acceptable concentrations have been reported to induce microbial growth and activity for certain species in AD consortia which ultimately improve biogas yields (Wall et al. 2014). Specifically, methanogens were found to contain metals in their bodies in the order Fe > > Zn > Ni > Cu ≈ Co ≈ Mo > Mn implying that these metals play a crucial role in these microbes (Choong et al. 2016). The minimum inhibitory concentrations for these metals are generally low calling for digester operators to exercise vigilance in monitoring these metals in digesters.

Table 1

Reasons for inclusion or non-inclusion of mineral characterization in studies.
Reason	Explanation and implication
No agreement among researchers on optimal metal concentrations for efficient anaerobic digestion.	Requirements recommended by different authors vary from one study to the other (Thanh et al. 2016; Schmidt et al. 2018). These variances tend to justify why other authors then choose not to analyse minerals as they cannot make informed deductions from those results.
No universal standard for biomethane potential (BMP) evaluation protocols.	For convenience, authors choose protocols that address their key research questions without looking at what other protocols emphasize on. Efforts to standardize substrate biomethanation evaluations are still in progress ((VDI 2006; Raposo et al. 2011; Holliger et al. 2016, 2021).
Cost (time and money) and equipment or expertise availability.	Analysis of metal bioavalabilities is important but it takes time to run the sequential extraction procedures. These procedures use lots of different extractants hence are costly to carry out (Choong et al. 2016).Other researchers can’t afford this then ultimately focus on other characterization that can be conducted with available resources.
Prior knowledge about the substrate from independent and other studies.	If a researcher wants to investigate certain aspects of a substrate and that substrate has been previously characterized for metals elsewhere and found to be within acceptable digester ranges, rather than repeating metal analysis they go straight into investigating issues of concern relating directly to their research and leave out the already known metal profile information.
Inconsistent nomenclature	There is a proliferation of classification terminologies such as mineral elements, macro nutrients, trace elements, inorganic elements, micronutrients without a consistent definition of these by researchers. To escape, these confusions, some researchers then opt not to characterize or include minerals in their substrates (Vintiloiu et al. 2012; Banks et al. 2012; Romero-Güiza et al. 2016; Roussel et al. 2018).

Reported heavy metal toxicity in digesters is ranked as Ni > Cu > > Cr (IV) ≈ Cr(III) > Pb > Zn (Hayes and Theis 1978) and Hg > Cd > Cr(III) (Abdel-Shafy and Mansour 2014). Agro-processing residues usually lack the required minerals for optimal digestion as opposed to industrial based AD substrates (Demirel and Scherer 2011). The studied by-products had 11 minerals falling within acceptable range for optimal AD though Mo, Se, Ni and Cr were more on the lower end of this range implying that they may require supplementation in digesters. Eight (8) minerals across the avocado by-products were below optimal range and they must be supplemented in digesters. Potassium is within range in five by-products except in CDW where it is slightly higher than the toxicity level of K in digesters. Across all by-products P concentration was exceeded by more than 1000% of the max requirement for digesters though the toxicity level of this element in digesters could not be established from literature consulted. There are high chances of inhibition or toxicity from P across all by-products. Zinc also assayed above the toxicity level across all substrates and this calls for detoxification strategies for this element. The full mineral element profiles for the by-products are summarized in Table 2. Just like the mineral elements, ammonium and ammonium nitrogen analysis is also critical in AD studies. These parameters should be checked alongside mineral element analysis for the same reasons as those outlined in the latter’s case. Methanogens are more sensitive to ammonium and ammoniacal nitrogen than the other three distinct microbial groups found in digesters and they cease to grow at NH₄⁺ concentrations greater than 3000 mg/L (Niu et al. 2013).

Figure 1 Macro and micro element composition of substrates a) High b) Medium c) Low concentrations

Table 2

Digester mineral requirements/limits and inferences from studied substrates’ characterisation
Metal	Low needs	High needs	Toxic levels	Avocado oil by-products digestion inferences and strategies (Assume 8% TS in digester slurries)(Rashama et al. 2019)	References
Metal	mg/L				References
K	-	1423.8	3000	All by-products give digester slurry K concentrations in the range 656 mg/L (seed) to 2035 mg/L (HDW) which is adequate and below toxic levels except for 3424 mg/L (CDW) which is lightly above the toxic limit.	(Bozym et al. 2015; Wu et al. 2016)
P	0.1	1	-	Lowest P in seeds at 109 mg/L while highest is 359 mg/L (CDW). P is more than adequate in all substrates and toxicity levels are however not known.	(Alphenaar et al. 1993)
Ca	100	1035	> 2500	Lowest Ca in peels slurry is 55 mg/L while highest value is 68 mg/L (CDW). Ca needs to be supplemented in all substrates. No threats of Ca toxicity are presented by these substrates	(Bozym et al. 2015; Romero-Güiza et al. 2016)
Mg	360	4800	> 2400	Mg concentration in digesters would be lowest 69 mg/L (seeds) and highest 184 mg/L (CDW). Mg supplementation to meet minimum requirements is necessary in all substrates.	(Bozym et al. 2015)
Zn	0.15	0.32	1	Lowest Zn levels in slurry are 1.7 mg/L (seeds) while highest value is 4.8 mg/L (CDW). All byproducts assay Zn concentrations above the toxicity limit. Strategies of detoxification (codigestion, removal, chelation) may need to be explored.	(Patidar and Tare 2008; Bozym et al. 2015; Schmidt et al. 2018)
Na	100	350	6000–30000	Lowest Na assay in the by-products gives slurry concentration of 4.9 mg/L(seeds) and highest value is 52 mg/L (CDW). These values are way below Na toxicity levels though information on requirements for optimal substrate degradation to biogas are also unknown.	(Bozym et al. 2015; Romero-Güiza et al. 2016)
Fe	0.43	400	10	Within the acceptable range, the lowest Fe concentration in digester slurry is 5.5 mg/L (HDW) while highest value is 9.6 mg/L (HDP). Potential inhibition may be observed in CDP where slurries will have Fe concentrations of 22 mg/L.	(Thanh et al. 2016; Schmidt et al. 2018)
Al	-	-	1000	All substrates produce slurries with Al assays (1.1–11.8 mg/L) which are below the toxic level of 1000 mg/L. Though CDP and CDW could not be characterized, it is also expected that these two will also be safe from reaching toxic levels since they are derived from same fruits where the other by-products were way far below toxic limit.
Mn	8	120	-	Lowest Mn concentration in digester slurries is 0.7 mg/L(seeds) and highest value is 1.6 mg/L (CDW). This means Mn needs to be supplemented in digesters that use avocado oil processing by-products as substrates.	(Schmidt et al. 2018)
Cu	0.06	0.512	3.2	Slurry concentration of Cu were lowest 0.5 mg/L(seeds) and highest 2.3 mg/L (peels). These values are more than adequate for optimal digestion and are still below toxicity levels.	(Bozym et al. 2015)
B	-	-	-	No informative AD studies addressing digester requirements for this element were found. The element has been found in methanogens so Practitioners normally add it in metal supplementation media for digesters. In the by-products boron assays were lowest 26 mg/kgDM (seeds) and highest 119 mg/kgDM (HDW)	(Azbar et al. 2001; Kim et al. 2002; Choong et al. 2018)
Co	0.032	8	-	Cobalt (Co) in the by-products gives slurry concentration of 0.008 mg/L(seeds) and highest value is 0.05 mg/L (CDW). Only CDW capable of slightly exceed the minimum Co requirements of digesters. Co supplementation is therefore recommended.	(Thanh et al. 2016; Schmidt et al. 2018)
As	-	-	-	Sludges from by-products measured low As concentration of 1.0 mg/kgDM and highest 1.6 mg/kgDM. These values are below the ceiling values permissible in biofertilisers (< 40 mg/kgDM) implying that digestates from these by-products can be safely used as biofertilizer	(Sturmer et al. 2020)
Mo	0.0024	1.28	-	Lowest Mo assay in the by-products gives slurry concentration of 0.004 mg/L (peels, HDW and HDP) and highest value is 0.012 mg/L (CDW and CDP). Though these values meet minimal requirements, they are way below the max requirements stated in literature. Supplementation of Mo in all the by-products may be necessary to meet high demands.	(Schmidt et al. 2018)
Cd	-	1.6	14.4	All by-product substrates result in low Cd assays in digester slurries between 0.0024 mg/L(HDW) and highest value is 0.032 mg/L (CDP). These values are way below Cd toxicity levels and below Cd requirements for optimal substrate degradation to biogas. All by-products require supplementation but taking cognizance of not exceeding the safe Cd limit of < 0.3 mg/kgDM for biofertilizer use.	(Bozym et al. 2015; Romero-Güiza et al. 2016; Sturmer et al. 2020)
Pb	0.02	200	340	Low Pb assays in digester slurries between 0.01 mg/L (HDW) and highest value is 0.09 mg/L (CDP) across by-products are inadequate to support biodigestion or cause toxicity in digesters. Supplementation may be necessary.	(Hayes and Theis 1978; Bozym et al. 2015; Matheri et al. 2016)
Hg	-	-	-	Sludges from by-products assay Hg concentrations lowest 0.25 mg/kgDM and highest 0.3 mg/kgDM. These values are below the ceiling values permissible in biofertilisers (< 1.0 mg/kgDM) implying that digestates from these by-products can be safely used as biofertilizer.	(Sturmer et al. 2020)
Se	0.004	0.8	-	Selenium (Se) assays in digester slurries across by-products are lowest 0.06 mg/L(CDW) and highest 0.112 mg/L (HDW). These values are within recommended range for Se requirements in digesters though on the low end of the range. Supplementation may therefore be necessary.	(Thanh et al. 2016; Schmidt et al. 2018)
Cr	0.005	50	130	All by-product substrates result in low Cr assays in digester slurries between 0.025 mg/L (HDP) and highest value is 0.13 mg/L (CDW). These values are in the recommended range of Cr requirements though on the lower end. There is no risk of toxicity and supplementation is necessary reach optimal substrate degradation to biogas.	(Bozym et al. 2015)
Ni	0.029	8	10	Nickel in byproducts will give digester slurries concentrations ranging lowest 0.07 mg/L(seeds) and highest 0.8 mg/L (CDW). These values are way below Ni toxicity levels though they meet minimum requirements in digesters. Supplementation is required to meet maximum demands.	(Bozym et al. 2015; Thanh et al. 2016)

Therefore, in many cases ammonia inhibition is compounded by VFA inhibition as well as acidogenesis overtakes methanogenesis. Niu et al (2013) noted that during AD studies with high protein containing chicken manure substrate, when NH₄⁺ concentration reached 3000 mg/L the VFA levels also fast accumulated to 200 mg/L and methane production rate was observed decreasing with all this reflecting an imbalanced methanogen/acidogen ratios. Figure 2 depicts the NH₄⁺ and NH₄N results for four of the avocado oil processing by-products. Assays for the other two by-products could not be completed because of equipment failure. These results translate to a maximum of 3.6 mg/L ammonium ions in digesters slurry for HDP with the other substrates registering even less concentrations. This implies that that there is no risk of ammonium toxicity from these substrates. Ammonium and free ammonia requirements have been estimated at 1100 mg/L and less than 47 mg/L respectively for optimal methanogens growth (Steinhaus et al. 2007). This indicates the need for ammonium supplementation in digesting avocado oil processing by-products. In the overall analysis of mineral elements and ammonia in substrates it is however important to highlight the shortcomings of recalcitrance hence concepts of bioavailability (Thanh et al. 2016). Lignocellulosic material in substrates lock up minerals so that they may not be accessed by microorganisms. Minerals interact among themselves to form non-active, more active or toxic intermediates. Minerals also tend to adsorb to carbonaceous materials which renders the minerals ineffective to participate in microbial pathways. All these issues require further investigation for better understanding of this topic in AD. Despite these shortcomings in direct inferences made based on direct substrate digestion and ICP-OES based results. This method though not ideal, sheds some light for AD Practitioners to at least consider mineral effects in digestion rather than ignore them completely at the detriment of running poorly optimized processes or failures to troubleshoot digester poor performances.

It is important to carry out mineral elemental analysis of AD substrates especially for new and unknown substrates. This analysis helps in explaining substrate digestion behaviour and devising possible optimization of the AD process by adjusting these elements where necessary. Elements promote microbial growth and activities although this is dependent on concentration and elemental bioavailability. Lower concentrations may impede microbial growth while high assays may be toxic to the same microbes. Some elements such as Hg, As and Cd are toxic even at low concentrations and must therefore be checked for possible removal or de-activation in digesters. South African avocado oil processing byproducts from Westfalia Fruit factories contain inorganic elements in different proportions. Only zinc across all by-products and potassium in CDW may need corrective action to avoid potential inhibition to the AD process. The other minerals that are in range and may not need supplementation or detoxification are K, P, Fe, Cu, Se and Cr. With the rest requiring supplementation for optimal digester performances. This last conclusion may be affected by issues of mineral bioavailability which is a substrate dependent parameter. Further studies are recommended to confirm and address bioavailability concerns. Going forward, substrate evaluation standard protocol review committees/bodies must consider incorporating universally debated adequate and standard scientific guidance on substrate mineral element characterization, mineral requirements and toxicities in digesters. This in combination with recommended bioavailability studies will be key in developing mineral supplementation strategies.

Acknowledgements

Authors are thankful to the University of South Africa (UNISA) and the Agricultural Research Council (ARC) for providing the laboratory space to carry out this research. The permission from Westfalia Fruit (RSA) management allowing us to use by-products from their factory operations is gratefully acknowledged.

Funding

This work was financially supported by the Department of Science and Innovation South Africa (DSI) and the Technology Innovation Agency (TIA) grant number 2022/FUN252/AA awarded to Prof. T. Matambo. Additional funding was sourced from the National Research Foundation (NRF) of South Africa (NRF Grant Number: 138093).

Conflict of interest/Competing interests

The authors declare no conflict of interest in this work.

Ethics approval

An ethical clearance certificate number 2019/CSET/SOE_CR/001 was granted by the University of South Africa’s School of Engineering Ethics Review Comittee on the execution of this work

Availability of data and material

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20 Dec, 2024
Reviewers agreed at journal
24 Nov, 2024
Reviewers agreed at journal
23 Nov, 2024
Reviewers invited by journal
12 Nov, 2024
Editor assigned by journal
12 Nov, 2024
Submission checks completed at journal
12 Nov, 2024
First submitted to journal
10 Nov, 2024

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Significance of mineral elements characterization for anaerobic digestion substrates: The case of avocado oil processing by-products

Status:

Journal Publication

Version 1

Abstract

Figures

1 Introduction

2 Materials and Methods

2.1 Literature survey

2.2 Materials sourcing and handling

2.3 Substrate mineral and trace element analysis

3 Results and Discussion

4 Conclusions

Declarations

References

Additional Declarations

Status:

Journal Publication

Version 1