bioengineering

Article
An Investigation into Spent Coffee Waste as
a Renewable Source of Bioactive Compounds
and Industrially Important Sugars
Damhan S. Scully, Amit K. Jaiswal and Nissreen Abu-Ghannam *
School of Food Science and Environmental Health, College of Sciences and Health, Dublin Institute of Technology,
Cathal Brugha Street, Dublin 1, Ireland; damhanscully@gmail.com (D.S.S.); amit.jaiswal@dit.ie (A.K.J.)
* Correspondence: nissreen.abughannam@dit.ie; Tel.: +353-1-402-7570; Fax: +353-1-878-8978.

Academic Editor: Gou-Jen Wang

Received: 28 September 2016; Accepted: 15 November 2016; Published: 21 November 2016

Abstract: Conventional coffee brewing techniques generate vast quantities of spent espresso grounds
(SEGs) rich in lignocellulose and valuable bioactives. These bioactive compounds can be exploited
as a nutraceutical or used in a range of food products, while breakdown of lignocellulose generates
metabolizable sugars that can be used for the production of various high-value products such as
biofuels, amino acids and enzymes. Response surface methodology (RSM) was used to optimize
the enzymatic saccharification of lignocellulose in SEGs following a hydrothermal pretreatment.
A maximum reducing sugar yield was obtained at the following optimized hydrolysis conditions:
4.97 g of pretreated SEGs, 120 h reaction time, and 1246 and 250 µL of cellulase and hemicellulase,
respectively. Industrially important sugars (glucose, galactose and mannose) were identified as
the principal hydrolysis products under the studied conditions. Total flavonoids (p = 0.0002), total
polyphenols (p = 0.03) and DPPH free-radical scavenging activity (p = 0.004) increased significantly
after processing. A 14-fold increase in caffeine levels was also observed. This study provides insight
into SEGs as a promising source of industrially important sugars and polyphenols.

Keywords: spent coffee waste; lignocellulose; enzymatic saccharification; reducing sugars; polyphenols

1. Introduction
Coffee is one of the most popular beverages in the world. The International Coffee Organization
(ICO) estimates that 8.5 billion kg of coffee was produced in 2014, the majority of which was consumed
in the EU, the USA, Brazil and Japan [1]. Due to extensive post-harvest processing of the coffee cherry
and conventional brewing techniques, vast quantities of co-products are generated in both producing
and consuming regions [2]. Legislation increasingly limits the amount of agri-food waste that can
enter landfills; therefore, the generation of waste and co-products represents a significant challenge to
the coffee industry [3].
The primary by-product from coffee production is spent coffee grounds (SCGs) [2]. SCGs are
the insoluble residue that remains after coffee beans are dehydrated, milled and brewed, and there
are two sources: those generated by the soluble coffee industry, which utilizes ~50% of the global
coffee harvest each year, and those generated by cafés and the public, accounting for the remaining
50% [4]. Previously, soluble or “instant” coffee producers dumped large quantities of SCGs in landfills,
a practice that might alter the local ecology. Incorporating SCGs into animal feed is also limited due to
the anti-nutritional activity of tannins [4,5].
SCGs are rich in lignocellulose, a covalently bonded network of lignin, cellulose and hemicellulosic
polysaccharides that gives structural stability to the plant cell wall [6]. The structure of lignocellulose is
such that covalently cross-linked lignin and hemicellulose sheath a crystalline cellulose core resulting in
a strong, recalcitrant network. Consequently, intensive treatment(s) of biomass is required to overcome

Bioengineering 2016, 3, 33; doi:10.3390/bioengineering3040033 www.mdpi.com/journal/bioengineering

Bioengineering 2016, 3, 33 2 of 13

the covalent linkages between the lignin and hemicellulose [7]. Disrupting lignocellulose in this way
improves hydrolysis of hemicellulose and cellulose into industrially significant monosaccharides.
Generally, enzymatic saccharification of pretreated lignocellulose is favored over other processing
methods as it is environmentally friendly, requires mild reaction conditions, is substrate specific,
and produces fewer degradation by-products [8,9]. Nevertheless, efficient enzymatic saccharification
of pretreated lignocellulose depends on optimizing experimental variables [10].
Response surface methodology (RSM) is a collection of mathematical and statistical techniques
for optimizing and analyzing the effect of multiple experimental variables, alone or in combination,
on a given process while maintaining a high degree of statistical significance [11]. RSM optimization is
useful because it not only expedites the experimental process, taking into account complex interactions
between the variables, but it also reduces operation costs [12]. For these reasons, RSM is routinely
used when optimizing enzymatic saccharification of lignocellulosic biomass [10–13].
To date, the majority of research into coffee by-products has focused on SCGs. SCGs are
generated through industrial-scale brewing of coffee beans to produce instant or soluble coffee
grounds. Relatively few studies have investigated spent espresso grounds (SEGs), generated through
conventional brewing of coffee beans by the foodservice industry (cafés, restaurants) and the public.
The present study investigates SEGs as a viable substrate for the production of industrially significant
sugars and polyphenols.

2. Materials and Methods

2.1. Chemicals and Reagents

The following chemicals and reagents were purchased from Sigma Aldrich, Germany: Hemicellulase
from A. niger, 2,2-Diphenyl-1-picrylhydrazyl (DPPH); 6-Hydroxy-2,5,7,8-tetramethylchroman-
2-carboxylic acid (Trolox), and Multi-element Standard Solution 1 for ICP-AES. Celluclast 1.5L
(700 U·mL−1 ) was purchased from Novozymes (Copenhagen, Denmark). All other chemicals were of
analytical grade.

2.2. Sample Acquisition and Preparation

SEGs were acquired from a local café in Dublin city centre and subsequently dehydrated (Rational
Combi-Dämpfer CCC 101, Rational, Landsberg, Germany) at 80 ± 1 ◦ C for 48 h. Dehydrated SEGs
were manually sieved to a particle size of 500 µm (Woven Wire Sieve, Endecotts Ltd., London, UK)
before being stored at −20 ◦ C until required.

2.3. Proximate Composition

2.3.1. Moisture Content

Moisture content was determined as per AOAC method 930.94 [14]. SEGs (5 g) were dehydrated
in an oven (Gallenhamp BS Oven 250, Weiss Gallenkamp, Loughborough, UK) for 48 h at 80 ± 2 ◦ C.
Weight difference was calculated before and after dehydration to determine the moisture content using
Equation (1):
Wet sample − Dry sample
 
Moisture content (%) = × 100 (1)
Wet sample

2.3.2. pH
pH was determined as per the method of Cruz et al. [4], with minor modifications. SEGs
(5 g) were boiled in 50 mL distilled water (dH2 O) for 5 min with continuous stirring (Bibby HB502,
Bibby-Scientific, Staffordshire, UK). The mixture was centrifuged (Sigma 2-16 K, Sigma Zentrifugen,
Osterode am Harz, Germany) at 7000 rpm for 10 min. The supernatant was decanted, allowed to cool,
then made up to 100 mL with dH2 O. The pH was measured (Thermo Scientific Orion 2 Star, Thermo
Fisher Scientific, Waltham, MA, USA) following calibration with pH 4 and 7 buffer solutions.
Bioengineering 2016, 3, 33 3 of 13

2.3.3. Ash Content

Ash content was determined as per AOAC method 920.93 [14]. SEGs (0.5 g) were added to
a crucible, previously washed and dried with deionized water, and dry ashed at 500 ± 10 ◦ C in
a muffle furnace (Carbolite AAF 11-3, Carbolite, Derbyshire, UK) for 24 h.

2.3.4. Minerals and Trace Elements

Sample preparation for ICP-AES analysis was performed as per the aqua regia method of
Szymczycha-Madeja, et al. [15]. Briefly, SEGs (0.5 g) and freshly prepared aqua regia (2 mL) were
added to a 30 mL polypropylene centrifuge tube. The mixture was ultrasonicated (Branson 3510
Ultrasonicator, Emerson Electronics, St. Louis, MO, USA) for 15 min and subsequently made up to
25 mL with dH2 O. Next, the solution was centrifuged at 7000 rpm for 10 min. The supernatant was
decanted and stored at −20 ◦ C until required.
ICP (Liberty 150 Series II, Varian, Palo Alto, CA, USA) radio frequency power, plasma flow rate
and sheath gas flow rate were 1.1 kW, 10 L·min−1 and 0.75 L·min−1 , respectively. Sample solutions
were introduced into the plasma using a V-groove nebulizer and a Scott type spray chamber at a flow
rate of 0.7 mL·min−1 .

2.3.5. Soluble Protein

Hydrolysate protein concentration was determined as per the method of Bradford [16]. Briefly,
boiled and filtered SEGs solution (200 µL) was mixed with Bradford reagent (800 µL) and allowed
to stand for 5 min at 25 ◦ C. Absorbance was measured (Shimadzu UV-1800, Shimadzu Corp., Kyoto,
Japan) at 595 nm.

2.3.6. Total Lipids

Lipids were extracted as per the method of De Melo et al. [17]. SEGs (10 g) were added to
a cellulose thimble and subsequently placed in a Soxhlet extraction chamber. Lipids were extracted
over 6 h by refluxing in 100 mL n-hexane. After 6 h extraction lipids were concentrated by evaporating
n-hexane (Büchi Rotovapor R-215, Büchi, Postfach, Switzerland).

2.4. Enzymatic Hydrolysis of Spent Espresso Coffee Lignocellulose

2.4.1. Preliminary Study

SEGs (5 g) and dH2 O (40 mL) were added to an Erlenmeyer flask (100 mL). Each mixture was
hydrothermally pretreated (autoclaved) at 120 ◦ C and 15 psi for 1 h and subsequently allowed to cool
to 25 ◦ C. Next, 50 mM sodium citrate buffer (5 mL) was added to each flask. A volume (0.5, 1.0, 1.5 or
2.0 mL) of cellulase (77.08 U·mL−1 ) or hemicellulase solution (7.23 U·mL−1 ) was then added to the
flasks. The final volume was made up to 50 mL with dH2 O. The flasks were incubated at 40 ◦ C for
24, 48, 72, 96 and 120 h, respectively. After incubation, each suspension was centrifuged at 7000 rpm
for 10 min. Hydrolysates were stored at −20 ◦ C until required. Table 1 shows the range of values
established from the preliminary study.

Table 1. Process variables and their coded levels in the Box-Wilson Central Composite Design.

Coded Values
Process Variables
−2 −1 0 1 2
X1: SEG Quantity (g) 1 2 3 4 5
X2: Cellulase (µL) 250 500 750 1000 1250
X3: Hemicellulase (µL) 250 500 750 1000 1250
X4: Incubation Time (h) 24 48 72 36 120
Bioengineering 2016, 3, 33 4 of 13

2.4.2. Central Composite Design

A 24 factorial Box-Wilson Central Composite Design (CCD) was used for experimental design.
A total of 28 experiments were generated by the software to determine the optimum levels of the
process variables SEGs quantity (X1 ), cellulase volume (X2 ), hemicellulase volume (X3 ) and reaction
time (X4 ). All variables were assumed to be measurable; therefore, the response surface was expressed
using Equation (2):
y = f (X1 , X2 , X3 , X4 ) (2)

where y is the response variable and Xi (i = 1, 2, 3, 4) represents the process variables.

To find the values that optimally describe the functional relationship between the process variables
and the response surface a second-order polynomial model (Equation (3)) was utilized:

k k k−1 k
y = β0 + ∑ βi Xi + ∑ βii X2i + ∑ ∑ βij Xi Xj + ε (3)
i =1 i =1 i =1 j =2

where X1 , . . . Xk are the process variables that influence the response y; β0 , βii (i = 1, 2, . . . k),
βij (i = 1, 2, . . . k; j = 1, 2, . . . k) are the unknown parameters of the variables; and ε the random error.

2.5. Analysis of Reducing Sugars

2.5.1. The DNS Assay

The dinitrosalicylic acid (DNS) assay was used to quantify total reducing sugars in the preliminary
study and RSM-optimized hydrolysates. Briefly, each hydrolysate (1 mL) was diluted 1:10 in dH2 O
before addition of dinitrosalicylic acid reagent (1 mL) and dH2 O (2 mL). The mixture was submerged
in a water bath for 5 min at 100 ◦ C. The volume was made up to 10 mL by the addition of dH2 O (6 mL).
Absorbance was measured at 540 nm.

2.5.2. HPLC-RI
Reducing sugars were analyzed using the method described by Jaiswal et al. [18]. An Alliance
High Performance Liquid Chromatography (HPLC) (Waters, e2695 Separation module, Waters, Milford,
MA, USA) equipped with an auto-sampler and controller with dual pump was used. The detection
system consisted of a Waters 486 UV detector and a Waters 410 Differential Refractometer (RI detector)
connected in series. Empower® software was used for data acquisition and analysis. An aliquot (20 µL)
was injected into a thermostatically controlled compartment set to 65 ◦ C, followed by elution through
a Rezex ROA-Organic acid H+ (8%) (350 mm × 7.8 mm, Phenomenex, Macclesfield, UK) column fitted
with a guard column (50 mm × 7.8 mm, Phenomenex, Macclesfield, UK), at a flow rate of 0.6 mL·min−1 .
The mobile phase was H2 SO4 (5 mM). All solutions were filtered through a 0.22 µm Millipore® filter
(Millipore, Merck-Millipore, Billerica, MA, USA) before being injected into the instrument.

2.6. Analysis of Caffeine, Polyphenols and Antioxidant Activities

2.6.1. Total Flavonoids Content (TFC) Assay

Total flavonoids content (TFC) was measured using the method described by Rajauria et al. [19].
Briefly, an aliquot (250 µL) was mixed with dH2 O (1.25 mL) and 5% NaNO2 (75 µL ). After 6 min,
10% AlCl3 (150 µL) was added. After 5 min, 0.1 M NaOH (0.5 mL) was added. The total volume
was made up to 2.5 mL with dH2 O. Absorbance was measured at 510 nm. Results are expressed as
quercetin equivalents (QE).

2.6.2. Total Polyphenol Content (TPC) Assay

Total polyphenol content (TPC) was measured using the FC method described by Rajauria et al. [18].
An aliquot (100 µL) was mixed with 2% Na2 CO3 (2 mL) and allowed to stand for 2 min at 25 ◦ C.
Bioengineering 2016, 3, 33 5 of 13

Next, 1 N FC reagent (100 µL) was added. The sample was incubated in darkness for 30 min at 25 ◦ C.
Absorbance was measured at 720 nm. Results are expressed as gallic acid equivalents (GAE).

2.6.3. DPPH Assay

The DPPH assay was used to determine the free-radical scavenging activity (EC50 ) of preliminary
study hydrolysates, but not in the RSM-optimized hydrolysates due to the inclusion of citric acid
as a buffer [20]. Briefly, aliquots (100 µL) were added to a 96-well plate followed by the addition of
0.16 mM methanolic DPPH solution (100 µL). The mixture was shaken and incubated in darkness
for 30 min at 25 ◦ C. Absorbance was monitored at 517 nm (Powerwave, Biotek, Winooski, VT, USA).
The ability to scavenge the DPPH radical was calculated using Equation (4):

As − Ab
  
Scavenging effect (%) = 1 − × 100 (4)
Ac

where As is the absorbance of the test sample; Ab is the absorbance of the sample blank; and Ac is the
absorbance of the control. Results are expressed as ascorbic acid equivalents (AAE).

2.6.4. FRAP Assay

The ferric reducing antioxidant potential (FRAP) of preliminary study hydrolysates was assessed
using the method of Benzie and Strain [21]. The assay was performed in a 96-well microtitre plate
reader at 37 ◦ C. An aliquot of FRAP reagent (100 µL) was preheated to 37 ◦ C and subsequently
dispensed in wells containing hydrolysates (50 µL). Absorbance was measured at 593 nm after 10 min.
Results are expressed as trolox equivalents (TE).

2.6.5. Analysis of Caffeine Using HPLC-DAD

Caffeine was identified as per the method of Jaiswal et al. [18]. The system consisted of
a reversed-phase column in an Alliance HPLC equipped with an auto-sampler and controller with dual
pump, a Waters 2998 photodiode array detector (DAD) set to 280 nm, and the Empower® software.
An Atlantis C18 column (250 mm × 4.6 mm, 5 µm particle size) maintained at 25 ◦ C was used.
A gradient solvent system of 2 mM sodium acetate with 6% acetic acid (Solvent A) and acetonitrile
(Solvent B) gave optimal peak separation and resolution.

2.7. Statistical Analysis

All samples were prepared in triplicate and results are reported as the mean ± the standard
deviation. Preliminary and RSM optimization experiments were conducted in triplicate. Experiment
design and statistical and regression analyses were carried out in STATGRAPHICS Centurion XV®
software (StatPoint Technologies, Warrenton, VA, USA). Lack-of-fit, the R2 statistic and analysis of
variance (ANOVA) p-values were analyzed to determine if the model was adequate.

3. Results and Discussions

3.1. Proximate Analysis of SEGs

Two major issues arise when agri-food by-products are not sufficiently dehydrated:
(i) contamination of the feedstock by spoilage microorganisms resulting in fermentation with
concomitant acidification; and (ii) decreased efficiency of the pretreatment [8]. In both instances the
hydrolytic activity of enzymes can be affected. Therefore, dehydrating biomass to a moisture content
of ≤10% is recommended [22]. The moisture content of SEGs used in this study was 65.3% ± 0.4%.
This compared favorably with values for SEGs (53.0%–69.8%) previously reported by Cruz et al. [4].
The ash content of roasted coffee is ~4.6%; however, conventional brewing can reduce this by as
much as 93%, owing to the hydrophilic nature of inorganic compounds and minerals [23,24]. The ash
Bioengineering 2016, 3, 33 6 of 13

content of SEGs used in our study was 0.7% ± 0.1%. This was lower than the range (0.8%–3.5%)
reported by Cruz et al. [4]. Mussatto et al. [24] conducted a similar analysis on SCGs and found a lower
ash content, suggesting that brewing conditions used in industrial coffee extraction result in greater
loss of ash and minerals. Examining the ash in greater detail, we identified and quantified eight
elements in SEGs, namely potassium, magnesium, calcium, manganese, copper, sodium, iron and
zinc (Table 2). Phosphorus was not included in our analysis; however, this element is present at high
concentration in both SCGs and SEGs [4,24]. The concentrations of potassium, magnesium and calcium
followed the same trend (potassium > magnesium > calcium) as values reported by Cruz et al. [4] and
Mussatto et al. [24], despite large variations between the values. The minor elements—manganese,
copper, sodium, iron and zinc—were lower than those previously reported.

Table 2. Trace elements in spent coffee grounds

Trace Elements Concentration (mg· (100 g)−1 )

Potassium 258.2 ± 23.66
Phosphorus ND
Magnesium 49.6 ± 3.34
Calcium 37.2 ± 2.89
Manganese 1.8 ± 0.06
Copper 1.2 ± 0.19
Sodium 1.1 ± 0.01
Iron 0.9 ± 0.02
Zinc 0.1 ± 0.03
ND = Not Determined.

The concentration of acids in the coffee bean is largely influenced by species, growth conditions,
processing method(s), degree of roasting and brewing method. The unroasted green coffee bean is
comprised of ~11% acids (w·w−1 ), including phenolic acids such as chlorogenic acid; non-volatile
aliphatic acids, such as citric and malic acids; and volatile acids such as acetic and propanoic acids,
all of which contribute to coffee’s complex flavor and aroma profile [4]. Roasting effectively reduces
coffee bean acidity to ~6% (w·w−1 ) and, once brewed, Arabica varieties typically are between pH 5.02
and 5.45, whereas Robusta varieties are between pH 5.32 and 5.49 [23,25]. The pH of brewed coffee
is an important indicator of quality; however, as mentioned above, low pH can reduce enzyme
activity and other saccharification and fermentation bioprocesses. The pH of a secondary extraction
was 5.1 ± 0.0. Compared with pH values reported for SCGs (pH 4.9 ± 0.9) by Kostenberg and
Marchaim [26], a secondary extraction had a negligible effect on brew acidity when compared with
industrial processes.
Despite comprising a modest percentage of the coffee bean (11%–20% w·w−1 ), lipids are a minor
constituent in the beverage because they are poorly extracted by hot water, making SEGs a good source.
Arabica beans have higher levels than Robusta (14%–20% vs. 11%–16%), with the principal lipids
being linoleic acid (~40%); palmitic acid (~30%); oleic acid (~10%); and stearic acid (~7%), and coffee
bean lipids contribute to the sensory profile of coffee [4,27]. Our analysis revealed that SEGs were
comprised of 13.1% ± 0.8% (dry w·w−1 ), making them a good source of lipids.

3.2. Optimization of Variables to Maximize Lignocellulose Saccharification

Following the preliminary study, a 24-factorial Box-Wilson CCD was used to design experiments.
A total of 28 experiments were generated by the software to determine the optimum levels of the
process variables SEGs (g), cellulase volume (µL), hemicellulase volume (µL) and reaction time (h)
(Table 3). A multiple regression analysis of the observed reducing sugar values (Table 3) revealed that
the variables were related by a second-order polynomial equation (Equation (5)):
Bioengineering 2016, 3, 33 7 of 13

y = −2.4775 + 2.80458X1 + 0.0023X2 + 0.00072X3 + 0.042X4 − 0.150X21

+0.00082X1 X2 + 0.00001X1 X3 + 0.029X1 X4 + 0.0000045X22 − 0.0000035X2 X3 (5)
−0.000057X2 X4 − 0.0000064X23 − 0.000063X3 X4 − 0.00095X24

where X1 , X2 , X3 and X4 are coded values for SEGs (g), cellulase volume (µL), hemicellulase volume
(µL) and reaction time (h), respectively.

Table 3. Experiment conditions as determined by STATGraphic Centurion XV software®

(STATGraphics Centurion, Warrenton, VA, USA). Also included are predicted and observed reducing
sugar values for each condition.

Process Variables * Concentration (mg·mL−1 )

Experiment
X1 (g) X2 (µL) X3 (µL) X4 (h) Predicted Observed
1 3 750 750 120 20.38 18.37
2 3 750 750 72 13.76 12.76
3 3 250 750 72 12.35 10.94
4 4 1000 500 96 23.69 26.05
5 4 1000 500 48 17.77 17.79
6 5 750 750 72 22.44 19.65
7 4 500 500 48 13.70 14.57
8 4 1000 1000 48 18.97 20.80
9 3 750 750 72 13.76 14.12
10 2 500 1000 48 8.32 7.76
11 4 1000 1000 96 23.38 23.24
12 4 500 1000 96 21.56 23.20
13 2 500 1000 96 11.27 12.14
14 3 1250 750 72 17.42 16.14
15 2 500 500 96 10.71 10.69
16 2 500 500 48 6.24 7.27
17 1 750 750 72 3.88 3.97
18 4 500 1000 72 15.79 16.55
19 3 750 750 96 13.76 13.91
20 3 750 250 72 14.48 13.09
21 2 1000 500 96 12.59 12.72
22 3 750 750 72 13.76 14.24
23 2 1000 1000 96 12.27 13.21
24 3 750 1250 72 16.25 14.94
25 3 750 750 24 11.51 10.82
26 2 1000 500 48 9.50 9.66
27 2 1000 1000 48 10.69 10.65
28 4 500 500 96 20.99 21.92
* X1 : Spent espresso grounds (SEG) quantity; X2 : Cellulase; X3 : Hemicellulase; X4 : Time.

Analysis of variance (ANOVA) was carried out and the results show that the SEGs’ quantity
(p = 0.000), reaction time (p = 0.000) and cellulase volume (p = 0.003) were significant variables with
p = 0.05. The effect of hemicellulase was not significant (p = 0.228). This is likely due to the activity
of this particular enzyme. For example, Jooste et al. [28] reported that mannanase gave the optimal
hydrolysis of spent coffee hemicellulose as compared with xylanase or cellulase. The significance of
the regression equation was checked by the F-test, and the R2 statistic showed that the fitted model
explained 94.897% of the variability between observed and predicted values. The adjusted R2 statistic
was 89.402%, meaning that the models could predict most of the observed variability. The standard
error of the estimates showed that the prediction errors of the residuals were 1.7069 (mg·g−1 ), while the
absolute mean error of the residuals was 0.9025 (mg·g−1 ). Finally, the Durbin-Watson statistic was
used to test if there was a significant correlation based on the order in which the residuals occurred.
This was 1.8821 (mg·g−1 ), or p = 0.235. Since p > 0.05, there was no indication of serial auto-correlation
in the residuals.
was 89.402%, meaning that the models could predict most of the observed variability. The standard
was 89.402%, meaning that the models could predict most of the observed variability. The standard
error of the estimates showed that the prediction errors of the residuals were 1.7069 (mg·g−1−1), while the
error of the estimates showed that the prediction errors of the residuals were 1.7069 (mg·g ), while the
absolute mean error of the residuals was 0.9025 (mg·g−1−1). Finally, the Durbin-Watson statistic was
absolute mean error of the residuals was 0.9025 (mg·g ). Finally, the Durbin-Watson statistic was
used to test if there was a significant correlation based on the order in which the residuals occurred.
used to test if there was a significant correlation based on the order in which the residuals occurred.
This was 1.8821 (mg·g−1−1), or p = 0.235. Since p > 0.05, there was no indication of serial auto-correlation
This was 1.8821
Bioengineering 2016, 3,(mg·
33 g ), or p = 0.235. Since p > 0.05, there was no indication of serial auto-correlation 8 of 13
in the residuals.
in the residuals.
The interaction of process variables is depicted in selected three-dimensional response surface
The interaction of process variables is depicted in selected three-dimensional response surface
plots The
(Figures 1–4.). As
interaction shown variables
of process in Figure is1,depicted
the cellulase volume
in selected and time produced
three-dimensional a non-linear
response surface
plots (Figures 1–4.). As shown in Figure 1, the cellulase volume and time produced a non-linear
response surface
plots (Figures 1–4). when the SEG
As shown quantity
in Figure (3 cellulase
1, the g) and hemicellulase
volume and time volume (750 µa L)
produced were fixed.
non-linear The
response
response surface when the SEG quantity (3 g) and hemicellulase volume (750 µ L) were fixed. The
reaction time had a large effect on the reaction under these conditions, while
surface when the SEG quantity (3 g) and hemicellulase volume (750 µL) were fixed. The reaction increasing the cellulase
reaction time had a large effect on the reaction under these conditions, while increasing the cellulase
volume
time hadalso increased
a large effect onthethe
reducing
reactionsugar
under yield.
theseThe synergy while
conditions, between SEGs and
increasing thecellulase
cellulasevolume
volume
volume also increased the reducing sugar yield. The synergy between SEGs and cellulase volume
when the reaction
also increased time and sugar
the reducing hemicellulase
yield. The volume
synergywere 72 h and
between SEGs750andµ Lcellulase
is shown in Figure
volume when 2.the
A
when the reaction time and hemicellulase volume were 72 h and 750 µ L is shown in Figure 2. A
concomitant
reaction timeincrease in SEGs andvolume
and hemicellulase cellulasewere
volume72 heffectively
and 750 µL doubled
is showntheinconcentration of reducing
Figure 2. A concomitant
concomitant increase in SEGs and cellulase volume effectively doubled the concentration of reducing
sugars.
increaseUnder
in SEGstheandconditions
cellulaseshown
volume ineffectively
Figures 3 and 4, as time
doubled increased from
the concentration 48 to 98 hsugars.
of reducing the reducing
Under
sugars. Under the conditions shown in Figures 3 and 4, as time increased from 48 to 98 h the reducing
sugar yield increased linearly. In Figure 4, however, it is clear that under optimized
the conditions shown in Figures 3 and 4, as time increased from 48 to 98 h the reducing sugar yield conditions the
sugar yield increased linearly. In Figure 4, however, it is clear that under optimized conditions the
hemicellulase volume
increased linearly. has a negligible
In Figure 4, however, response onthat
it is clear the reducing sugar concentration.
under optimized conditions the hemicellulase
hemicellulase volume has a negligible response on the reducing sugar concentration.
volume has a negligible response on the reducing sugar concentration.

Figure
Figure1.1.Three-dimensional
Three-dimensionalresponse
responsesurface
surfaceplot
plotshowing
showinginfluence
influence of
of cellulase
cellulase (uL)
(uL) and
and incubation
incubation
Figure 1. Three-dimensional response surface plot showing influence of cellulase (uL) and incubation
time
time(h)
(h) when
when the
the response
responsesurface
surfaceisisfixed
fixedatataa spent
spent espresso
espresso grounds
grounds (SEG)
(SEG) quantity
quantity ==33 gg and
and
time (h) when the response surface is fixed at a spent espresso grounds (SEG) quantity = 3 g and
hemicellulase
hemicellulase==750
750uL.
uL.
hemicellulase = 750 uL.

Figure 2. Three-dimensional response surface plot showing influence of SEG quantity and cellulase
Figure2.2.Three-dimensional
Figure Three-dimensional response
response surface
surface plot
plotshowing
showing influence
influence of
ofSEG
SEGquantity
quantityand
andcellulase
cellulase
loading
loadingwhen
whenthe
theresponse
response surface
surface is
is fixed
fixed at
at an
an incubation
incubation time
time ==72
72hhand
and hemicellulase
hemicellulase ==750
750uL.
uL.
loading when the response surface is fixed at an incubation time = 72 h and hemicellulase = 750 uL.
Bioengineering 2016, 3, 33 9 of 13
Bioengineering 2016, 3, 33 9 of 13
Bioengineering 2016, 3, 33 9 of 13

Figure 3. Three-dimensional response surface plot showing influence of SEG quantity (g) and
Figure
Figure 3. Three-dimensionalresponse
3. Three-dimensional response surface
surface plotplot showing
showinginfluence
influenceof of
SEG
SEGquantity
quantity(g) (g)
and and
incubation time (h) when the response surface is fixed at cellulase and hemicellulose loadings = 750 uL.
incubation
incubation timetime (h) when
(h) when thethe response
response surfaceisisfixed
surface fixedat
atcellulase
cellulase and
andhemicellulose
hemicelluloseloadings
loadings= 750 uL. uL.
= 750

Figure 4. Three-dimensional response surface plot showing influence of incubation time (h) and
Figure 4. Three-dimensionalresponse
4. Three-dimensional responsesurface
surface plot
plot showing influence ofofincubation time (h) (h)
and
Figure
hemicellulase (uL) loading when the response surface isshowing influence
fixed at SEG quantity (3 g)incubation
and cellulasetime
= 750 uL.and
hemicellulase (uL) loading when the response surface is fixed at SEG quantity (3 g) and cellulase
hemicellulase (uL) loading when the response surface is fixed at SEG quantity (3 g) and cellulase = 750 = 750 uL. uL.

3.3. Optimization of Reducing Sugar Yield

3.3. Optimization of Reducing Sugar Yield
3.3. Optimization
The aimofofReducing
optimizingSugar Yield variables was to find the combination of values that would
process
The aim of optimizing process variables was to find the combination of values that would
maximize
The aim of reducing sugar yield. Optimal
optimizing values were obtained by solving the regression equation
maximize reducing sugar process variables
yield. Optimal was
values to obtained
were find the bycombination of values equation
solving the regression that would
(Equation
maximize (5))
reducing using STATGRAPHICS
sugar yield. Optimal values Centurion XV ®
were obtained software (STATGraphics
by solving the regressionCenturion,
equation
(Equation (5)) using STATGRAPHICS Centurion XV® software (STATGraphics Centurion,
Warrenton, VA, USA). Table 4 shows the values required ® softwareto maximize reducing sugar yield. SEGs
(Equation (5)) using
Warrenton, VA, STATGRAPHICS
USA). Table 4 shows Centurion
the values XVrequired (STATGraphics
to maximize reducingCenturion,
sugar yield. Warrenton,
SEGs
and cellulase volume were rounded to 5 g and 1250 µ L, respectively. Based on the optimum values,
and cellulase volume were rounded to 5 g and
VA, USA). Table 4 shows the values required to maximize reducing1250 µ L, respectively. Based on the optimum values,
sugar yield. SEGs and cellulase
the predicted maximum reducing sugar yield was 379.0 mg·g−1−1. The observed maximum reducing sugar
the were
volume predicted maximum
rounded to 5 reducing
g and sugar
1250 µL,yield was 379.0 mg·
respectively. g . The
Based on observed
the maximum
optimum reducing
values, sugar
the predicted
yield was 325.6 mg·g−1−1. Our results show that optimizing the process variables effectively increased
yield
maximum was 325.6
reducing mg· g
sugar . Our results
yield show that optimizing
− 1
mg·g . sugars the process variables effectively increased
the total reducing sugars. Forwas 379.0 reducing
example, The observed maximum reducing
in the RSM-optimized sugar
hydrolysate yield
were 4.4was
the total− 1reducing sugars. For example, reducing sugars in the RSM-optimized hydrolysate were 4.4
mg·gmore
325.6times . Our results show
concentrated than that optimizing
the equivalent the process
pretreated variables
hydrolysate (50 effectively
mg·g−1) and increased the total
11.7 times more
times more concentrated than the equivalent pretreated hydrolysate (50 mg·g−1) and 11.7 times more
reducing sugars. For
concentrated thanexample,
the controlreducing
(18.5 mg·sugars
g−1
−1). in the RSM-optimized hydrolysate were 4.4 times more
concentrated than the control (18.5 mg·g ). −1
concentrated than the equivalent pretreated hydrolysate (50 mg·g ) and 11.7 times more concentrated
Table
than the controlTable 4.
(18.5 4.
mg ·g−high,
Low,
Low,
high,
1 ). and optimum values for the process variables used in this study.
and optimum values for the process variables used in this study.
Limit Values
Process Variables Limit Values Optimum Values
Table 4. Low, high, and optimum
Process Variablesvalues
Lowfor the
Highprocess variables
Optimum used in this study.
Values
Low High
SEG Quantity (g) 1 5 4.97
SEG Quantity (g) 1 5 4.97
Cellulase (µ L) 250 Values
Limit 1250 1246.07
Cellulase (µ L) 250 1250 1246.07 Values
Optimum
Process Variables(µ L)
Hemicellulase 250 1250 250
Hemicellulase (µ L) Low
250 1250
High 250
Incubation Time (h) 24 120 120
Incubation Time (h) 24 120 120
SEG Quantity (g) 1 5 4.97
Cellulase (µL) 250 1250 1246.07
Hemicellulase (µL) 250 1250 250
Incubation Time (h) 24 120 120
Bioengineering 2016, 3, 33 10 of 13
Bioengineering 2016, 3, 33 10 of 13
Bioengineering 2016, 3, 33 10 of 13
3.4.
3.4.Identification
IdentificationofofReducing
ReducingSugars
Sugars
3.4. Identification of Reducing Sugars
Liberated
Liberatedsugars sugars ininthe
thecontrol,
control, pretreated
pretreated and
and RSM-optimized
RSM-optimized hydrolysates
hydrolysates are are shown
shown in in
Figures Liberated
Figures 5–7.
5–7.Peaks sugars
Peaks in the control,
atatapproximately
approximately 8.0pretreated
8.0 min,
min, 10.5 and and
10.5 min,
min, RSM-optimized
and 22.7
22.7 min in allhydrolysates
chromatograms
chromatogramsare shown in
represent
represent
HFigures
H2 SO
2SO
5–7. Peaks
4 ,4,citric
citric acid, at approximately
acid, and
and MeOH, 8.0 min, Sodium
MeOH, respectively. 10.5 min,citrate
Sodium and 22.7
citrate was
was min in all
used
used as achromatograms
as a buffer
buffer in
in all represent
all the
the sample
sample
H 2SO4, citric acid, and MeOH, respectively. Sodium citrate was used as a buffer in all the sample
solutions,
solutions,0.0050.005MMHH 2 SO
2SO4 4was
wasused
used asas the
the mobile phase throughout all analyses, and and MeOH
MeOH was wasused
used
tosolutions,
toequilibrate
equilibrate0.005
theMcolumn
the H2SO4 was
column used
prior
prior as the mobile
totorunning
running phase throughout
thehydrolysates.
the hydrolysates. Mostall
Most analyses,sugars
six-carbon and MeOH
sugars eluted
elutedwas used
around
around
12to equilibrate
12minmin duedue thestructural
to column
to structural prior to running
similarities.
similarities. As the
As shown inhydrolysates.
shown 5, onlyMost
in Figure
Figure 5, six-carbon
only
mannose mannose sugars eluted
and galactose
and galactose around
were
were identified
12 min
inidentified due
the control. to structural
in the control. similarities. As shown in Figure 5, only mannose and galactose were
identified in the control.

Figure5.5.HPLC
Figure HPLC(High
(HighPerformance
PerformanceLiquid
LiquidChromatography)
Chromatography) chromatogram
chromatogram of
of spent
spent coffee
coffee waste
waste
Figure 5. HPLC
(control)sample. (High
sample. Performance Liquid Chromatography) chromatogram of spent coffee waste
(control)
(control) sample.
The effects of hydrothermal pretreatment conditions on SEGs were evident as mannose,
The
Theeffects of of
effects hydrothermal
hydrothermalpretreatment conditions
pretreatment on SEGs
conditions on were
SEGs evident
were 6). as mannose,
evident galactose
as mannose,
galactose and arabinose were identified in the pretreated hydrolysate (Figure It was expected that
and arabinose
galactose and were identified
arabinose were in the pretreated
identified in the hydrolysate
pretreated (Figure(Figure
hydrolysate 6). It was
6). expected
It was that these
expected that
these sugars would be identified during the course of the analysis as they comprise the hemicellulosic
sugars
these wouldwould
sugars be identified during
be identified the course
during the of the
course analysis
of the analysis asasthey
theycomprise
comprise the the hemicellulosic
arabinogalactans and galactomannans in spent coffee [29]. The concentrations of hemicellulosic
mannose and
arabinogalactans
arabinogalactans and
and galactomannans
galactomannans in spent coffee [29]. The concentrations
concentrations of mannose
mannose andand
arabinose in the pretreated hydrolysate werespent
0.087 coffee
mg·mL[29].
−1 andThe
0.108 mg· mL of
−1, respectively.
arabinose
arabinosein
inthe
thepretreated
pretreated hydrolysate werewere 0.087
0.087mg·
mgmL·mL−1−and
1 and 0.108 mg·mL −1 , respectively.
0.108 mg·mL−1, respectively.

Figure 6. HPLC chromatogram of pretreated spent coffee waste sample.

Figure 6. HPLC chromatogram of pretreated spent coffee waste sample.
Figure 6. HPLC chromatogram of pretreated spent coffee waste sample.
As shown in Figure 7, the variety of sugars was significantly higher in the RSM-optimized
As shown
hydrolysate. in Figure
Glucose, 7, theand
mannose variety of sugars
galactose was significantly
were present, in additionhigher in the RSM-optimized
to cellobiose. The presence of
hydrolysate. Glucose, mannose and galactose were present, in addition to cellobiose.
cellobiose and glucose was indicative of cellulase activity. The hydrothermal pretreatmentThe presence of
effectively
cellobiose and glucose was indicative of cellulase activity. The hydrothermal pretreatment effectively
Bioengineering 2016, 3, 33 11 of 13

As shown in Figure 7, the variety of sugars was significantly higher in the RSM-optimized
hydrolysate. Glucose, mannose and galactose were present, in addition to cellobiose. The presence of
Bioengineering 2016, 3, 33 11 of 13
cellobiose and glucose was indicative of cellulase activity. The hydrothermal pretreatment effectively
disrupted
disrupted the the SEGs’
SEGs’ lignocellulosic structure to
lignocellulosic structure to the
the extent
extent that
that cellulase
cellulase could
couldeffectively
effectivelydegrade
degrade
cellulose. Interestingly, arabinose was not identified in the RSM-optimized hydrolysate.
cellulose. Interestingly, arabinose was not identified in the RSM-optimized hydrolysate. Retention Retention
time
timedata
data (not
(not shown)
shown) supports
supports the
the explanation that arabinose
explanation that arabinoseco-eluted
co-elutedwith
withgalactose
galactoseand andthat
thatitsits
peak was engulfed by the galactose peak. The concentrations of cellobiose, glucose
peak was engulfed by the galactose peak. The concentrations of cellobiose, glucose and mannose and mannose in in
the
RSM-optimized hydrolysate were 3.258 mg·mg· − 1
mLmL, −16.501 mg ·mL − 1 mg·mL − 1
the RSM-optimized hydrolysate were 3.258 , 6.501 mg· mL−1 and
and 5.660
5.660 mg· mL−1, respectively.
, respectively.
The concentrations of cellobiose and glucose were high considering that
The concentrations of cellobiose and glucose were high considering that these sugars these sugars were undetectable
were
in the control and SA hydrolysates. The concentration of mannose was 65 times
undetectable in the control and SA hydrolysates. The concentration of mannose was 65 times greater greater in the
RSM-optimized
in the RSM-optimized hydrolysate when compared
hydrolysate with the
when compared with SAthe
hydrolysate.
SA hydrolysate.

Figure
Figure 7. HPLC chromatogram
7. HPLC chromatogram of
of pretreated
pretreated spent
spentcoffee
coffeewaste
wastesample.
sample.

3.5.
3.5.Caffeine,
Caffeine, Polyphenols
Polyphenols and Antioxidant Activities
Hydrothermal
Hydrothermal pretreatment
pretreatment of SEGs increased increased thethe concentration
concentrationof of flavonoids
flavonoidsand andphenolic
phenolic
acids
acids inin the
the hydrolysates.
hydrolysates. For example, example, when when compared
compared with with thethe control,
control,pretreatment
pretreatmentincreased
increased
flavonoids by
flavonoids by 24.0% (495.8 ±
24.0% (495.8 ± 0.2
0.2 µµg QE·gg−1−vs.
g··QE· 1 vs.
651.4 ± 1.1
± 1.1
651.4 µ g·QE·
µg g−1·),g−while
·QE 1 ), while
phenolics increased
phenolics by
increased
by33.9%
33.9%(417.6 ± 0.3±µ g·
(417.6 0.3 µg·gGAE
GAE· −1 and ·g− 1 and
631.4 ± 0.8 ± 0.8g−1µg
µ g·GAE·
631.4 GAE·g−1t-test
). ·Student’s was usedt-test
). Student’s to determine
was used if ato
significantifdifference
determine existed
a significant between
difference the concentration
existed between the of flavonoids and
concentration phenolics in
of flavonoids and thephenolics
control
inandthepretreated
control and hydrolysate.
pretreated Thehydrolysate.
increase in flavonoids (p = 0.03)
The increase and phenolics
in flavonoids (p =(p 0.03)
= 0.00002)
and resulting
phenolics
(pfrom pretreatment
= 0.00002) resultingwas significant
from pretreatment when was p = 0.05. Pretreatment
significant when peffectively disrupted complexes
= 0.05. Pretreatment effectively
between phenolic
disrupted complexes compounds
between and lignin,
phenolic polysaccharides
compounds and proteins.
and lignin, When compared
polysaccharides with values
and proteins. When
for unbrewed
compared withcoffee,
valuesthefordata also highlighted
unbrewed coffee, the thedata
efficacy
also of conventional
highlighted thebrewing
efficacyon of polyphenols
conventional
extraction.
brewing on For example, extraction.
polyphenols Cheong et al. For[30] found total
example, Cheongphenolics in roasted
et al. [30] found total coffee to be 33.67–43.13
phenolics in roasted
mg GAE·
coffee to beg 33.67–43.13
−1, while Hečimović
mg·GAE·et −
g al., while
1 [31] reported
Hečimovićtheettotal flavanoid
al. [31] reportedcontent
the total offlavanoid
coffee ascontent
17.29
ofmg· GAE·
coffee asg−117.29
–20.58 mgmg· GAE·
·GAE ·g−g−1 .
1 –20.58 mg·GAE·g−1 .
Antioxidant activities
Antioxidant activities ofof the
the control
control andand pretreated
pretreated hydrolysate
hydrolysatewere weredetermined
determinedby bymeasuring
measuring
the DPPH free-radical scavenging activity and FRAP. The DPPH
the DPPH free-radical scavenging activity and FRAP. The DPPH free-radical scavenging activity free-radical scavenging activity
(EC50 )
(EC 50) was greater for the pretreated hydrolysate (59.9%) than the control (47.4%). FRAP results
was greater for the pretreated hydrolysate (59.9%) than the control (47.4%). FRAP results showed that
showed
there wasthatno there was nodifference
significant significantbetween
difference between hydrolysate
pretreated pretreated hydrolysate
(82.8 ± 2.1(82.8µg·TE± 2.1
·g−μg·
1 ) TE·
andg the
−1)

and the(82.0
control control
± 2.1(82.0
µg·±TE
2.1·gμg·
−1 ).TE· g−1). Student’s
Student’s t-test
t-test was wastoused
used to determine
determine if a significant
if a significant difference
difference existed
existed between the DPPH free-radical scavenging activities and FRAP
between the DPPH free-radical scavenging activities and FRAP values of the control and pretreated values of the control and
pretreated hydrolysate. When p = 0.05, there was no significant difference
hydrolysate. When p = 0.05, there was no significant difference between the FRAP values (p = 0.403); between the FRAP values
(p = 0.403);
however, however,
differences differences
between the DPPH between
valuesthe DPPH
were values(pwere
significant significant
= 0.004). (p = 0.004).
One explanation Oneis
for this
explanation for this is the difference between the electron transfer mechanism(s) in the assays. For
example, the FRAP assay measures antioxidants capable only of a single electron transfer, whereas
the DPPH assay measures antioxidants that transfer both a single electron and a hydrogen atom.
Hence, obtaining a good agreement between the assays is difficult [32].
Bioengineering 2016, 3, 33 12 of 13

the difference between the electron transfer mechanism(s) in the assays. For example, the FRAP assay
measures antioxidants capable only of a single electron transfer, whereas the DPPH assay measures
antioxidants that transfer both a single electron and a hydrogen atom. Hence, obtaining a good
agreement between the assays is difficult [32].
We identified and quantified caffeine in the control, pretreated and RSM-optimized hydrolysates.
Our analysis revealed that pretreatment resulted in a 14-fold increase in caffeine when compared with
the control (110.8 µg·mL−1 vs. 7.8 µg·mL−1 ). When compared against the pretreated hydrolysate,
however, enzymatic saccharification only slightly increased the caffeine concentration (118.2 µg·mL−1 ).

4. Conclusions
This study highlighted SEGs, an underutilized source of biomass, as a promising source of
industrially important reducing sugars and extractable bioactives, while also revealing the importance
of optimizing experimental parameters and selecting appropriate enzyme(s) when hydrolyzing or
processing lignocellulosic biomass. A deeper analysis and more in-depth studies of SEGs and the
process chain are required. SEGs can be systematically exploited as biomass for the production
of fermentable sugars glucose, mannose, galactose and arabinose, in addition to extractable lipids,
polyphenols and caffeine.

Acknowledgments: The authors would like to gratefully acknowledge Swarna Jaiswal, Centre for Research in
Engineering Surface Technology (CREST), FOCAS Institute, Dublin Institute of Technology, Dublin 8, Ireland,
for assistance in elemental analysis. The authors would also like to acknowledge the support and facilities
provided by Dublin Institute of Technology, Dublin, Ireland, to carry out this research.
Author Contributions: Damhan S. Scully completed experiments, data analysis, collated all tables, and prepared
the manuscript for publication. Amit K. Jaiswal conceived of the research, designed RSM experiments, formatted
graphs, and assisted in drafting sections of the manuscript. Nissreen Abu-Ghannam supervised the research and
contributed in the analysis of the results.
Conflicts of Interest: The authors declare no conflict of interest.

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article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).